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Sample records for protein crystallography performance

  1. Protein structures by spallation neutron crystallography

    PubMed Central

    Langan, Paul; Fisher, Zoë; Kovalevsky, Andrii; Mustyakimov, Marat; Sutcliffe Valone, Amanda; Unkefer, Cliff; Waltman, Mary Jo; Coates, Leighton; Adams, Paul D.; Afonine, Pavel V.; Bennett, Brad; Dealwis, Chris; Schoenborn, Benno P.

    2008-01-01

    The Protein Crystallography Station at Los Alamos Neutron Science Center is a high-performance beamline that forms the core of a capability for neutron macromolecular structure and function determination. This capability also includes the Macromolecular Neutron Crystallography (MNC) consortium between Los Alamos (LANL) and Lawrence Berkeley National Laboratories for developing computational tools for neutron protein crystallography, a biological deuteration laboratory, the National Stable Isotope Production Facility, and an MNC drug design consortium between LANL and Case Western Reserve University. PMID:18421142

  2. Infrared Protein Crystallography

    SciTech Connect

    J Sage; Y Zhang; J McGeehan; R Ravelli; M Weik; J van Thor

    2011-12-31

    We consider the application of infrared spectroscopy to protein crystals, with particular emphasis on exploiting molecular orientation through polarization measurements on oriented single crystals. Infrared microscopes enable transmission measurements on individual crystals using either thermal or nonthermal sources, and can accommodate flow cells, used to measure spectral changes induced by exposure to soluble ligands, and cryostreams, used for measurements of flash-cooled crystals. Comparison of unpolarized infrared measurements on crystals and solutions probes the effects of crystallization and can enhance the value of the structural models refined from X-ray diffraction data by establishing solution conditions under which they are most relevant. Results on several proteins are consistent with similar equilibrium conformational distributions in crystal and solutions. However, the rates of conformational change are often perturbed. Infrared measurements also detect products generated by X-ray exposure, including CO{sub 2}. Crystals with favorable symmetry exhibit infrared dichroism that enhances the synergy with X-ray crystallography. Polarized infrared measurements on crystals can distinguish spectral contributions from chemically similar sites, identify hydrogen bonding partners, and, in opportune situations, determine three-dimensional orientations of molecular groups. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

  3. Protein crystallography prescreen kit

    DOEpatents

    Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

    2007-10-02

    A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

  4. Protein crystallography prescreen kit

    DOEpatents

    Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

    2005-07-12

    A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

  5. HRTEM in protein crystallography

    NASA Astrophysics Data System (ADS)

    Dyson, P. W.; Spargo, A. E. C.; Tulloch, P. A.; Johnson, A. W. S.

    Electron microscopy/diffraction (ED/D) using spot-scan and low-dose imaging has been successfully applied to investigate microcrystals of an alpha-helical coiled-coil protein extracted from ootheca of the praying mantis. Fourier transforms of the images show resolution out to 4 A and can be used to phase the corresponding ED data which shows reflections out to 2 A.

  6. Macromolecular neutron crystallography at the Protein Crystallography Station (PCS)

    PubMed Central

    Kovalevsky, Andrey; Fisher, Zoe; Johnson, Hannah; Mustyakimov, Marat; Waltman, Mary Jo; Langan, Paul

    2010-01-01

    The Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center is a high-performance beamline that forms the core of a capability for neutron macromolecular structure and function determination. Neutron diffraction is a powerful technique for locating H atoms and can therefore provide unique information about how biological macro­molecules function and interact with each other and smaller molecules. Users of the PCS have access to neutron beam time, deuteration facilities, the expression of proteins and the synthesis of substrates with stable isotopes and also support for data reduction and structure analysis. The beamline exploits the pulsed nature of spallation neutrons and a large electronic detector in order to collect wavelength-resolved Laue patterns using all available neutrons in the white beam. The PCS user facility is described and highlights from the user program are presented. PMID:21041938

  7. Microfluidic Tools for Protein Crystallography

    NASA Astrophysics Data System (ADS)

    Abdallah, Bahige G.

    X-ray crystallography is the most widely used method to determine the structure of proteins, providing an understanding of their functions in all aspects of life to advance applications in fields such as drug development and renewable energy. New techniques, namely serial femtosecond crystallography (SFX), have unlocked the ability to unravel the structures of complex proteins with vital biological functions. A key step and major bottleneck of structure determination is protein crystallization, which is very arduous due to the complexity of proteins and their natural environments. Furthermore, crystal characteristics govern data quality, thus need to be optimized to attain the most accurate reconstruction of the protein structure. Crystal size is one such characteristic in which narrowed distributions with a small modal size can significantly reduce the amount of protein needed for SFX. A novel microfluidic sorting platform was developed to isolate viable ~200 nm -- ~600 nm photosystem I (PSI) membrane protein crystals from ~200 nm -- ~20 ?m crystal samples using dielectrophoresis, as confirmed by fluorescence microscopy, second-order nonlinear imaging of chiral crystals (SONICC), and dynamic light scattering. The platform was scaled-up to rapidly provide 100s of microliters of sorted crystals necessary for SFX, in which similar crystal size distributions were attained. Transmission electron microscopy was used to view the PSI crystal lattice, which remained well-ordered postsorting, and SFX diffraction data was obtained, confirming a high-quality, viable crystal sample. Simulations indicated sorted samples provided accurate, complete SFX datasets with 3500-fold less protein than unsorted samples. Microfluidic devices were also developed for versatile, rapid protein crystallization screening using nanovolumes of sample. Concentration gradients of protein and precipitant were generated to crystallize PSI, phycocyanin, and lysozyme using modified counterdiffusion

  8. Protein Crystallography in Vaccine Research and Development

    PubMed Central

    Malito, Enrico; Carfi, Andrea; Bottomley, Matthew J.

    2015-01-01

    The use of protein X-ray crystallography for structure-based design of small-molecule drugs is well-documented and includes several notable success stories. However, it is less well-known that structural biology has emerged as a major tool for the design of novel vaccine antigens. Here, we review the important contributions that protein crystallography has made so far to vaccine research and development. We discuss several examples of the crystallographic characterization of vaccine antigen structures, alone or in complexes with ligands or receptors. We cover the critical role of high-resolution epitope mapping by reviewing structures of complexes between antigens and their cognate neutralizing, or protective, antibody fragments. Most importantly, we provide recent examples where structural insights obtained via protein crystallography have been used to design novel optimized vaccine antigens. This review aims to illustrate the value of protein crystallography in the emerging discipline of structural vaccinology and its impact on the rational design of vaccines. PMID:26068237

  9. E-Science and Protein Crystallography

    SciTech Connect

    Miller, Laniece E.; Powell, James E. Jr.

    2012-08-09

    techniques. Many of the major funders as well as the major journals dealing with protein crystallography require deposition of the structural data in the Protein Data Bank (PDB). Files formatted for the PDB are automatically generated when the data is compressed. The header files in the PDB included experimental conditions of the experiment as well as experimental methods. Depending on the completeness and how 'hot' of a topic, it may not be needed to contact the original experimenter about using the data. Having said that, not all of the data is accurate and does requires some back and forth with the creators of the data. The RCSB PDB staff at Rutgers University goes through all submissions and works with the submitters to verify that the data meets their minimum standards of completeness and robustness. The Protein Data Bank (PDB) was initially created by Walter Hamilton at Brookhaven National Laboratory in 1971 after discussions about the value of scientists having access to structural biology data. Originally a partnership between Brookhaven and the Cambridge Crystallographic Data Center, the idea was conceived as a global initiative, which is certainly has become with partner sites in the US, Europe, and Japan. The PDB now contains structures determined from many different experimental techniques (Berman et al. 2012). Deposited structures are assigned a unique ID, and the structures are embargoed until the publication that references and describes them is published. The PDB staff often monitors these publications and takes the initiative to release protein structures when papers describing them are published. Dr. Fisher records setup and experimental details in word documents and inserts printed copies into paper lab notebooks. These details appear in the final published papers and the header files for structures in the PDB. Analysis of data collected at the PCS is performed with a combination of locally developed tools and commercial products which are capable of

  10. Serial Femtosecond Crystallography of Membrane Proteins.

    PubMed

    Zhu, Lan; Weierstall, Uwe; Cherezov, Vadim; Liu, Wei

    2016-01-01

    Membrane proteins, including G protein-coupled receptors (GPCRs), constitute the most important drug targets. The increasing number of targets requires new structural information, which has proven tremendously challenging due to the difficulties in growing diffraction-quality crystals. Recent developments of serial femtosecond crystallography at X-ray free electron lasers combined with the use of membrane-mimetic gel-like matrix of lipidic cubic phase (LCP-SFX) for crystal growth and delivery hold significant promise to accelerate structural studies of membrane proteins. This chapter describes the development and current status of the LCP-SFX technology and elaborates its future role in structural biology of membrane proteins. PMID:27553241

  11. Serial Millisecond Crystallography of Membrane Proteins.

    PubMed

    Jaeger, Kathrin; Dworkowski, Florian; Nogly, Przemyslaw; Milne, Christopher; Wang, Meitian; Standfuss, Joerg

    2016-01-01

    Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) is a powerful method to determine high-resolution structures of pharmaceutically relevant membrane proteins. Recently, the technology has been adapted to carry out serial millisecond crystallography (SMX) at synchrotron sources, where beamtime is more abundant. In an injector-based approach, crystals grown in lipidic cubic phase (LCP) or embedded in viscous medium are delivered directly into the unattenuated beam of a microfocus beamline. Pilot experiments show the application of microjet-based SMX for solving the structure of a membrane protein and compatibility of the method with de novo phasing. Planned synchrotron upgrades, faster detectors and software developments will go hand-in-hand with developments at free-electron lasers to provide a powerful methodology for solving structures from microcrystals at room temperature, ligand screening or crystal optimization for time-resolved studies with minimal or no radiation damage. PMID:27553240

  12. Protein Crystallization for X-ray Crystallography

    PubMed Central

    Dessau, Moshe A.; Modis, Yorgo

    2011-01-01

    Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography. To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999). Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin

  13. Lipidic phase membrane protein serial femtosecond crystallography

    PubMed Central

    Johansson, Linda C; Arnlund, David; White, Thomas A; Katona, Gergely; DePonte, Daniel P; Weierstall, Uwe; Doak, R Bruce; Shoeman, Robert L; Lomb, Lukas; Malmerberg, Erik; Davidsson, Jan; Nass, Karol; Liang, Mengning; Andreasson, Jakob; Aquila, Andrew; Bajt, Sasa; Barthelmess, Miriam; Barty, Anton; Bogan, Michael J; Bostedt, Christoph; Bozek, John D; Caleman, Carl; Coffee, Ryan; Coppola, Nicola; Ekeberg, Tomas; Epp, Sascha W; Erk, Benjamin; Fleckenstein, Holger; Foucar, Lutz; Graafsma, Heinz; Gumprecht, Lars; Hajdu, Janos; Hampton, Christina Y; Hartmann, Robert; Hartmann, Andreas; Hauser, Günter; Hirsemann, Helmut; Holl, Peter; Hunter, Mark S; Kassemeyer, Stephan; Kimmel, Nils; Kirian, Richard A; Maia, Filipe R N C; Marchesini, Stefano; Martin, Andrew V; Reich, Christian; Rolles, Daniel; Rudek, Benedikt; Rudenko, Artem; Schlichting, Ilme; Schulz, Joachim; Seibert, M Marvin; Sierra, Raymond G; Soltau, Heike; Starodub, Dmitri; Stellato, Francesco; Stern, Stephan; Strüder, Lothar; Timneanu, Nicusor; Ullrich, Joachim; Wahlgren, Weixiao Y; Wang, Xiaoyu; Weidenspointner, Georg; Wunderer, Cornelia; Fromme, Petra; Chapman, Henry N; Spence, John C H; Neutze, Richard

    2012-01-01

    X-ray free electron laser (X-feL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-feL beam using a sponge phase micro-jet. PMID:22286383

  14. Neutron protein crystallography in JAERI

    NASA Astrophysics Data System (ADS)

    Tanaka, I.

    2004-07-01

    Neutron diffraction provides an experimental method of directly locating hy- drogen atoms in proteins. After developing an original neutron detector (neutron imaging plate) and a novel practical neutron monochromator (elastically bent perfect Si monochro- mator), BIX-type diffractometers which were equipped with these tools were e+/-ciently constructed at JRR-3 in Japan Atomic Energy Research Institute (JAERI), Japan and they have finished many protein crystallographic measurements and interesting results have come one after another. At the same time a method of growing large protein single crystals and a database of hydrogen and hydration have also been developed. In the near future, a pulsed neutron diffractometer for biological macromolecules has been proposed at J-PARC in JAERI.

  15. Time-resolved protein crystallography.

    PubMed Central

    Johnson, L. N.

    1992-01-01

    Advances in synchrotron radiation technology have allowed exposure times from protein crystals of the order of milliseconds to be used routinely, and in exceptional circumstances exposure times of 100 ps have been obtained. However, many data sets take seconds to record because of the slow time scale of film change or crystal reorientation or translation when more than one exposure is required. This problem has been addressed by Amemiya et al. (1989). There has been considerable progress in methods to initiate reactions in protein crystals, especially the development of photolabile caged compounds but also temperature jump, pH jump, and diffusion. Although flash lamps deliver pulses of 100 mJ/ms, often several pulses are required to release sufficient product, and reaction initiation can take several seconds. Laser illumination can provide more powerful input, but the laser must be accommodated within the restricted space at the synchrotron station. The requirement to maintain synchrony among the molecules in the crystal lattice as the reaction proceeds and to ensure that the lifetime of intermediates is longer than data collection rates emphasizes the need for chemical characterization of the reaction under study. As Ringe advocated in the studies with chymotrypsin, it may be more profitable to devise conditions under which certain intermediates along the reaction pathway accumulate in the crystal and to record these in a series of discrete steps rather than continuous monitoring of the reaction. The Laue method is limited to those proteins that give well-ordered crystals and problems of transient disorder on initiation of reaction and problems of radiation damage need to be overcome or avoided by suitable experimental protocols.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1303743

  16. Membrane protein structure determination by electron crystallography

    PubMed Central

    Ubarretxena-Belandia, Iban; Stokes, David L.

    2012-01-01

    During the past year, electron crystallography of membrane proteins has provided structural insights into the mechanism of several different transporters and into their interactions with lipid molecules within the bilayer. From a technical perspective there have been important advances in high-throughput screening of crystallization trials and in automated imaging of membrane crystals with the electron microscope. There have also been key developments in software, and in molecular replacement and phase extension methods designed to facilitate the process of structure determination. PMID:22572457

  17. Proline: Mother Nature’s cryoprotectant applied to protein crystallography

    PubMed Central

    Pemberton, Travis A.; Still, Brady R.; Christensen, Emily M.; Singh, Harkewal; Srivastava, Dhiraj; Tanner, John J.

    2012-01-01

    l-Proline is one of Mother Nature’s cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that l-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included the commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6–8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration needed for cryoprotection of these crystals is in the range 2.0–3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that l-proline is an effective cryoprotectant for protein crystallography. PMID:22868767

  18. Proline: Mother Nature;s cryoprotectant applied to protein crystallography

    SciTech Connect

    Pemberton, Travis A.; Still, Brady R.; Christensen, Emily M.; Singh, Harkewal; Srivastava, Dhiraj; Tanner, John J.

    2012-09-05

    L-Proline is one of Mother Nature's cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that L-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included the commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6-8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration needed for cryoprotection of these crystals is in the range 2.0-3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that L-proline is an effective cryoprotectant for protein crystallography.

  19. Protein Crystallography from the Perspective of Technology Developments

    PubMed Central

    Su, Xiao-Dong; Zhang, Heng; Terwilliger, Thomas C.; Liljas, Anders; Xiao, Junyu; Dong, Yuhui

    2015-01-01

    Early on, crystallography was a domain of mineralogy and mathematics and dealt mostly with symmetry properties and imaginary crystal lattices. This changed when Wilhelm Conrad Röntgen discovered X-rays in 1895, and in 1912 Max von Laue and his associates discovered X-ray irradiated salt crystals would produce diffraction patterns that could reveal the internal atomic periodicity of the crystals. In the same year the father-and-son team, Henry and Lawrence Bragg successfully solved the first crystal structure of sodium chloride and the era of modern crystallography began. Protein crystallography (PX) started some 20 years later with the pioneering work of British crystallographers. In the past 50-60 years, the achievements of modern crystallography and particularly those in protein crystallography have been due to breakthroughs in theoretical and technical advancements such as phasing and direct methods; to more powerful X-ray sources such as synchrotron radiation (SR); to more sensitive and efficient X-ray detectors; to ever faster computers and to improvements in software. The exponential development of protein crystallography has been accelerated by the invention and applications of recombinant DNA technology that can yield nearly any protein of interest in large amounts and with relative ease. Novel methods, informatics platforms, and technologies for automation and high-throughput have allowed the development of large-scale, high efficiency macromolecular crystallography efforts in the field of structural genomics (SG). Very recently, the X-ray free-electron laser (XFEL) sources and its applications in protein crystallography have shown great potential for revolutionizing the whole field again in the near future. PMID:25983389

  20. Proline: Mother Nature’s cryoprotectant applied to protein crystallography

    SciTech Connect

    Pemberton, Travis A.; Still, Brady R.; Christensen, Emily M.; Singh, Harkewal; Srivastava, Dhiraj; Tanner, John J.

    2012-08-01

    The amino acid l-proline is shown to be a good cryoprotectant for protein crystals. Four examples are provided; the range of proline used for cryoprotection is 2.0–3.0 M. l-Proline is one of Mother Nature’s cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that l-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included the commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6–8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration needed for cryoprotection of these crystals is in the range 2.0–3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that l-proline is an effective cryoprotectant for protein crystallography.

  1. High resolution electron crystallography of protein molecules

    SciTech Connect

    Glaeser, R.M. |; Downing, K.H.

    1993-06-01

    Electron diffraction data and high resolution images can now be used to obtain accurate, three-dimensional density maps of biological macromolecules. These density maps can be interpreted by building an atomic-resolution model of the structure into the experimental density. The Cowley-Moodie formalism of dynamical diffraction theory has been used to validate the use of kinematic diffraction theory, strictly the weak phase object approximation, in producing such 3-D density maps. Further improvements in the preparation of very flat specimens and in the retention of diffraction to a resolution of 0.2 nm or better could result in electron crystallography becoming as important a technique as x-ray crystallography currently is for the field of structural molecular biology.

  2. The spherical drift chamber used at LURE for protein crystallography; last developments, performances, and results (invited) (abstract)

    NASA Astrophysics Data System (ADS)

    Kahn, R.; Fourme, R.; Bosshard, R.; Lewit-Bentley, A.; Prangé, T.

    1989-07-01

    We have developed at LURE a multiwire proportional chamber with a spherical drift space. The wire chamber consists of two cathode planes, comprising 512 wires with a spacing of 1 mm, set on both sides of the anodic plane. The drift space, a gas filled region bounded by two spherically curved electrodes 144 mm apart, offers several advantages: high quantum efficiency, no parallax effect, equivalent spatial resolution in both directions, and smoothing of the pulsed structure of the synchrotron radiation. The gaseous mixture of argon-xenon (58%), ethane (40%), and ethyl alcohol (2%) is circulated in a closed circuit and is continuously purified. Ethyl alcohol, which avoids electrical discharges and sparks, is essential to operate the instrument at high counting rates (>300 000 events/s). The signal processing, which makes use of one amplifier per cathode wire and of fast priority encoders, determines both coordinates with a resolution of 1 mm and a dead time of 240 ns. Each encoded event is stored into a 512×512 16 bit CAMAC histogramming memory. The experiment is controlled by a PDP11/34 linked to a VAX by a direct memory access channel. A complete set of programs, which performs the data collection and an off line data reduction, is operational. The adaptation of madnes, a general software package which performs an on line data reduction, is under way. Data, collected on a lysozyme crystal to 3.4-Å resolution, give a reliability factor based on intensities of equivalent reflections of 4.7% without absorption corrections; the variation of the detector efficiency is <2.7%. A new version of the instrument is under realization. The position encoder, which uses flash ADCs and signal processors, has a resolution of 0.5 mm. Data are stored into a 1024×1024 16 bit VME histogramming memory linked to a micro VAX.

  3. Two-dimensional pixel array image sensor for protein crystallography

    SciTech Connect

    Beuville, E.; Beche, J.-F.; Cork, C.

    1996-07-01

    A 2D pixel array image sensor module has been designed for time resolved Protein Crystallography. This smart pixels detector significantly enhances time resolved Laue Protein crystallography by two to three orders of magnitude compared to existing sensors like films or phosphor screens coupled to CCDs. The resolution in time and dynamic range of this type of detector will allow one to study the evolution of structural changes that occur within the protein as a function of time. This detector will also considerably accelerate data collection in static Laue or monochromatic crystallography and make better use of the intense beam delivered by synchrotron light sources. The event driven pixel array detectors, based on the column Architecture, can provide multiparameter information (energy discrimination, time), with sparse and frameless readout without significant dead time. The prototype module consists of a 16x16 pixel diode array bump-bonded to the integrated circuit. The detection area is 150x150 square microns.

  4. Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography

    PubMed Central

    Weierstall, Uwe; James, Daniel; Wang, Chong; White, Thomas A.; Wang, Dingjie; Liu, Wei; Spence, John C.H.; Doak, R. Bruce; Nelson, Garrett; Fromme, Petra; Fromme, Raimund; Grotjohann, Ingo; Kupitz, Christopher; Zatsepin, Nadia A.; Liu, Haiguang; Basu, Shibom; Wacker, Daniel; Han, Gye Won; Katritch, Vsevolod; Boutet, Sébastien; Messerschmidt, Marc; Williams, Garth J.; Koglin, Jason E.; Seibert, M. Marvin; Klinker, Markus; Gati, Cornelius; Shoeman, Robert L.; Barty, Anton; Chapman, Henry N.; Kirian, Richard A.; Beyerlein, Kenneth R.; Stevens, Raymond C.; Li, Dianfan; Shah, Syed T.A.; Howe, Nicole; Caffrey, Martin; Cherezov, Vadim

    2014-01-01

    Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals, providing a continuously-renewed source of material for serial femtosecond crystallography. Data collected from sub-10 μm-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor. PMID:24525480

  5. Crystal Dehydration in Membrane Protein Crystallography.

    PubMed

    Sanchez-Weatherby, Juan; Moraes, Isabel

    2016-01-01

    Crystal dehydration has been successfully implemented to facilitate the structural solution of a number of soluble and membrane protein structures over the years. This chapter will present the currently available tools to undertake controlled crystal dehydration, focusing on some successful membrane protein cases. Also discussed here will be some practical considerations regarding membrane protein crystals and the relationship between different techniques in order to help researchers to select the most suitable technique for their projects. PMID:27553236

  6. Membrane-Protein Crystallography and Potentiality for Drug Design

    NASA Astrophysics Data System (ADS)

    Yamashita, Atsuko

    Structure-based drug design for membrane proteins is far behind that for soluble proteins due to difficulty in crystallographic structure determination, despite the fact that about 60% of FDA-approved drugs target membrane proteins located at the cell surface. Stable homologs for a membrane protein of interest, such as prokaryotic neurotransmitter transporter homolog LeuT, might enable cooperative analyses by crystallography and functional assays, provide useful information for functional mechanisms, and thus serve as important probes for drug design based on mechanisms as well as structures.

  7. Breaking the barriers in membrane protein crystallography.

    PubMed

    Kang, Hae Joo; Lee, Chiara; Drew, David

    2013-03-01

    As we appreciate the importance of stabilising membrane proteins, the barriers towards their structure determination are being broken down. This change in mindset comes hand-in-hand with more effort placed on developing methods focused at screening for membrane proteins which are naturally stable in detergent solution or improving those that are not so. In practice, however, it is not easy to decide the best strategy to monitor and improve detergent stability, requiring a decision-making process that can be even more difficult for those new to the field. In this review we outline the importance of membrane protein stability with discussions of the stabilisation strategies applied in context with the use of crystallisation scaffolds and the different types of crystallisation methods themselves. Where possible we also highlight areas that we think could push this field forward with emerging technologies, such as X-ray free electron lasers (X-feL), which could have a big impact on the membrane protein structural biology community. We hope this review will serve as a useful guide for those striving to solve structures of both pro- and eukaryotic membrane proteins. PMID:23291355

  8. Design and feasibility of active matrix flat panel detector using avalanche amorphous selenium for protein crystallography.

    PubMed

    Sultana, Afrin; Reznik, Alla; Karim, Karim S; Rowlands, J A

    2008-10-01

    Protein crystallography is the most important technique for resolving the three-dimensional atomic structure of protein by measuring the intensity of its x-ray diffraction pattern. This work proposes a large area flat panel detector for protein crystallography based on direct conversion x-ray detection technique using avalanche amorphous selenium (a-Se) as the high gain photoconductor, and active matrix readout using amorphous silicon (a-Si:H) thin film transistors. The detector employs avalanche multiplication phenomenon of a-Se to make the detector sensitive to each incident x ray. The advantages of the proposed detector over the existing imaging plate and charge coupled device detectors are large area, high dynamic range coupled to single x-ray detection capability, fast readout, high spatial resolution, and inexpensive manufacturing process. The optimal detector design parameters (such as detector size, pixel size, and thickness of a-Se layer), and operating parameters (such as electric field across the a-Se layer) are determined based on the requirements for protein crystallography application. The performance of the detector is evaluated in terms of readout time (<1 s), dynamic range (approximately 10(5)), and sensitivity (approximately 1 x-ray photon), thus validating the detector's efficacy for protein crystallography. PMID:18975678

  9. A new thermal neutron detector for protein crystallography

    SciTech Connect

    Mahler, G.J.; Radeka, V.; Schaknowski, N.A.; Smith, G.C.; Yu, B.; Zojceski, Z.

    1999-12-01

    A new position-sensitive detector is being developed for protein crystallography studies at a spallation source. Based on eight, independent, wire proportional chamber segments housed in a curved pressure vessel, the device covers a scattering angle of 120 degrees, and has a collecting area of 1.5m by 20cm. The position resolution will be about 1.3 mm FWHM, with a total counting rate in excess of one million per second. Timing resolution, essential for a spallation source application, is of order 1{micro}s and provides neutron energy determination that is well suited for crystallography. Advanced features of this device include a digital centroid finding scheme, a seamless readout between segments, and a wire array design that minimizes anode modulation. Details of the mechanical design are given, together with digital centroid measurements that illustrate accurate, uniform response.

  10. High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography

    PubMed Central

    Boutet, Sébastien; Lomb, Lukas; Williams, Garth J.; Barends, Thomas R. M.; Aquila, Andrew; Doak, R. Bruce; Weierstall, Uwe; DePonte, Daniel P.; Steinbrener, Jan; Shoeman, Robert L.; Messerschmidt, Marc; Barty, Anton; White, Thomas A.; Kassemeyer, Stephan; Kirian, Richard A.; Seibert, M. Marvin; Montanez, Paul A.; Kenney, Chris; Herbst, Ryan; Hart, Philip; Pines, Jack; Haller, Gunther; Gruner, Sol M.; Philipp, Hugh T.; Tate, Mark W.; Hromalik, Marianne; Koerner, Lucas J.; van Bakel, Niels; Morse, John; Ghonsalves, Wilfred; Arnlund, David; Bogan, Michael J.; Caleman, Carl; Fromme, Raimund; Hampton, Christina Y.; Hunter, Mark S.; Johansson, Linda C.; Katona, Gergely; Kupitz, Christopher; Liang, Mengning; Martin, Andrew V.; Nass, Karol; Redecke, Lars; Stellato, Francesco; Timneanu, Nicusor; Wang, Dingjie; Zatsepin, Nadia A.; Schafer, Donald; Defever, James; Neutze, Richard; Fromme, Petra; Spence, John C. H.; Chapman, Henry N.; Schlichting, Ilme

    2013-01-01

    Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules. PMID:22653729

  11. Serial femtosecond crystallography of soluble proteins in lipidic cubic phase

    DOE PAGESBeta

    Fromme, Raimund; Ishchenko, Andrii; Metz, Markus; Chowdhury, Shatabdi Roy; Basu, Shibom; Boutet, Sébastien; Fromme, Petra; White, Thomas A.; Barty, Anton; Spence, John C. H.; et al

    2015-08-04

    Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP–SFX method has recently been introduced in which microcrystals of membrane proteins are grown and delivered for SFX data collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic phase (LCP), using a special LCP microextrusion injector. Here, it is shown enabling a dramatic reduction in the amount of crystallized protein required for data collection compared with crystals deliveredmore » by liquid injectors. High-quality LCP–SFX data sets were collected for two soluble proteins, lysozyme and phycocyanin, using less than 0.1 mg of each protein.« less

  12. Serial femtosecond crystallography of soluble proteins in lipidic cubic phase

    SciTech Connect

    Fromme, Raimund; Ishchenko, Andrii; Metz, Markus; Chowdhury, Shatabdi Roy; Basu, Shibom; Boutet, Sébastien; Fromme, Petra; White, Thomas A.; Barty, Anton; Spence, John C. H.; Weierstall, Uwe; Liu, Wei; Cherezov, Vadim

    2015-08-04

    Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP–SFX method has recently been introduced in which microcrystals of membrane proteins are grown and delivered for SFX data collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic phase (LCP), using a special LCP microextrusion injector. Here, it is shown enabling a dramatic reduction in the amount of crystallized protein required for data collection compared with crystals delivered by liquid injectors. High-quality LCP–SFX data sets were collected for two soluble proteins, lysozyme and phycocyanin, using less than 0.1 mg of each protein.

  13. Advances in structural and functional analysis of membrane proteins by electron crystallography

    PubMed Central

    Wisedchaisri, Goragot; Reichow, Steve L.; Gonen, Tamir

    2011-01-01

    Summary Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane proteins by electron crystallography. PMID:22000511

  14. A prototype direct-detection CCD for protein crystallography

    PubMed Central

    Green, Katherine S.; Szebenyi, Doletha M. E.; Boggs, Kasey; Bredthauer, Richard; Tate, Mark W.; Gruner, Sol M.

    2013-01-01

    The fabrication and testing of a prototype deep-depletion direct-conversion X-ray CCD detector are described. The device is fabricated on 600 µm-thick high-resistivity silicon, with 24 × 24 µm pixels in a 4k × 4k pixel format. Calibration measurements and the results of initial protein crystallography experiments at the Cornell High Energy Synchrotron Source (CHESS) F1 beamline are described, as well as suggested improvements for future versions of the detector. PMID:24046505

  15. Integrated Controlling System and Unified Database for High Throughput Protein Crystallography Experiments

    SciTech Connect

    Gaponov, Yu.A.; Igarashi, N.; Hiraki, M.; Sasajima, K.; Matsugaki, N.; Suzuki, M.; Kosuge, T.; Wakatsuki, S.

    2004-05-12

    An integrated controlling system and a unified database for high throughput protein crystallography experiments have been developed. Main features of protein crystallography experiments (purification, crystallization, crystal harvesting, data collection, data processing) were integrated into the software under development. All information necessary to perform protein crystallography experiments is stored (except raw X-ray data that are stored in a central data server) in a MySQL relational database. The database contains four mutually linked hierarchical trees describing protein crystals, data collection of protein crystal and experimental data processing. A database editor was designed and developed. The editor supports basic database functions to view, create, modify and delete user records in the database. Two search engines were realized: direct search of necessary information in the database and object oriented search. The system is based on TCP/IP secure UNIX sockets with four predefined sending and receiving behaviors, which support communications between all connected servers and clients with remote control functions (creating and modifying data for experimental conditions, data acquisition, viewing experimental data, and performing data processing). Two secure login schemes were designed and developed: a direct method (using the developed Linux clients with secure connection) and an indirect method (using the secure SSL connection using secure X11 support from any operating system with X-terminal and SSH support). A part of the system has been implemented on a new MAD beam line, NW12, at the Photon Factory Advanced Ring for general user experiments.

  16. Integrated Controlling System and Unified Database for High Throughput Protein Crystallography Experiments

    NASA Astrophysics Data System (ADS)

    Gaponov, Yu. A.; Igarashi, N.; Hiraki, M.; Sasajima, K.; Matsugaki, N.; Suzuki, M.; Kosuge, T.; Wakatsuki, S.

    2004-05-01

    An integrated controlling system and a unified database for high throughput protein crystallography experiments have been developed. Main features of protein crystallography experiments (purification, crystallization, crystal harvesting, data collection, data processing) were integrated into the software under development. All information necessary to perform protein crystallography experiments is stored (except raw X-ray data that are stored in a central data server) in a MySQL relational database. The database contains four mutually linked hierarchical trees describing protein crystals, data collection of protein crystal and experimental data processing. A database editor was designed and developed. The editor supports basic database functions to view, create, modify and delete user records in the database. Two search engines were realized: direct search of necessary information in the database and object oriented search. The system is based on TCP/IP secure UNIX sockets with four predefined sending and receiving behaviors, which support communications between all connected servers and clients with remote control functions (creating and modifying data for experimental conditions, data acquisition, viewing experimental data, and performing data processing). Two secure login schemes were designed and developed: a direct method (using the developed Linux clients with secure connection) and an indirect method (using the secure SSL connection using secure X11 support from any operating system with X-terminal and SSH support). A part of the system has been implemented on a new MAD beam line, NW12, at the Photon Factory Advanced Ring for general user experiments.

  17. Smarter Drugs: How Protein Crystallography Revolutionizes Drug Design

    SciTech Connect

    Smith, Clyde

    2005-04-26

    According to Smith, protein crystallography allows scientists to design drugs in a much more efficient way than the standard methods traditionally used by large drug companies, which can cost close to a billion dollars and take 10 to 15 years. 'A lot of the work can be compressed down,' Smith said. Protein crystallography enables researchers to learn the structure of molecules involved in disease and health. Seeing the loops, folds and placement of atoms in anything from a virus to a healthy cell membrane gives important information about how these things work - and how to encourage, sidestep or stop their functions. Drug design can be much faster when the relationship between structure and function tells you what area of a molecule to target. Smith will use a timeline to illustrate the traditional methods of drug development and the new ways it can be done now. 'It is very exciting work. There have been some failures, but many successes too.' A new drug to combat the flu was developed in a year or so. Smith will tell us how. He will also highlight drugs developed to combat HIV, Tuberculosis, hypertension and Anthrax.

  18. The Protein Micro-Crystallography Beamlines for Targeted Protein Research Program

    NASA Astrophysics Data System (ADS)

    Hirata, Kunio; Yamamoto, Masaki; Matsugaki, Naohiro; Wakatsuki, Soichi

    In order to collect proper diffraction data from outstanding micro-crystals, a brand-new data collection system should be designed to provide high signal-to noise ratio in diffraction images. SPring-8 and KEK-PF are currently developing two micro-beam beamlines for Targeted Proteins Research Program by MEXT of Japan. The program aims to reveal the structure and function of proteins that are difficult to solve but have great importance in both academic research and industrial application. At SPring-8, a new 1-micron beam beamline for protein micro-crystallography, RIKEN Targeted Proteins Beamline (BL32XU), is developed. At KEK-PF a new low energy micro-beam beamline, BL-1A, is dedicated for SAD micro-crystallography. The two beamlines will start operation in the end of 2010. The present status of the research and development for protein micro-crystallography will be presented.

  19. Large-volume protein crystal growth for neutron macromolecular crystallography

    SciTech Connect

    Ng, Joseph D.; Baird, James K.; Coates, Leighton; Garcia-Ruiz, Juan M.; Hodge, Teresa A.; Huang, Sijay

    2015-03-30

    Neutron macromolecular crystallography (NMC) is the prevailing method for the accurate determination of the positions of H atoms in macromolecules. As neutron sources are becoming more available to general users, finding means to optimize the growth of protein crystals to sizes suitable for NMC is extremely important. Historically, much has been learned about growing crystals for X-ray diffraction. However, owing to new-generation synchrotron X-ray facilities and sensitive detectors, protein crystal sizes as small as in the nano-range have become adequate for structure determination, lessening the necessity to grow large crystals. Here, some of the approaches, techniques and considerations for the growth of crystals to significant dimensions that are now relevant to NMC are revisited. We report that these include experimental strategies utilizing solubility diagrams, ripening effects, classical crystallization techniques, microgravity and theoretical considerations.

  20. Large-volume protein crystal growth for neutron macromolecular crystallography.

    PubMed

    Ng, Joseph D; Baird, James K; Coates, Leighton; Garcia-Ruiz, Juan M; Hodge, Teresa A; Huang, Sijay

    2015-04-01

    Neutron macromolecular crystallography (NMC) is the prevailing method for the accurate determination of the positions of H atoms in macromolecules. As neutron sources are becoming more available to general users, finding means to optimize the growth of protein crystals to sizes suitable for NMC is extremely important. Historically, much has been learned about growing crystals for X-ray diffraction. However, owing to new-generation synchrotron X-ray facilities and sensitive detectors, protein crystal sizes as small as in the nano-range have become adequate for structure determination, lessening the necessity to grow large crystals. Here, some of the approaches, techniques and considerations for the growth of crystals to significant dimensions that are now relevant to NMC are revisited. These include experimental strategies utilizing solubility diagrams, ripening effects, classical crystallization techniques, microgravity and theoretical considerations. PMID:25849493

  1. Serial Femtosecond Crystallography of G Protein-Coupled Receptors

    PubMed Central

    Liu, Wei; Wacker, Daniel; Gati, Cornelius; Han, Gye Won; James, Daniel; Wang, Dingjie; Nelson, Garrett; Weierstall, Uwe; Katritch, Vsevolod; Barty, Anton; Zatsepin, Nadia A.; Li, Dianfan; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Koglin, Jason E.; Seibert, M. Marvin; Wang, Chong; Shah, Syed T.A.; Basu, Shibom; Fromme, Raimund; Kupitz, Christopher; Rendek, Kimberley N.; Grotjohann, Ingo; Fromme, Petra; Kirian, Richard A.; Beyerlein, Kenneth R.; White, Thomas A.; Chapman, Henry N.; Caffrey, Martin; Spence, John C.H.; Stevens, Raymond C.; Cherezov, Vadim

    2014-01-01

    X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. Here we used an x-ray free-electron laser (XFEL) with individual 50-fs duration x-ray pulses to minimize radiation damage and obtained a high-resolution room temperature structure of a human serotonin receptor using sub-10 µm microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared to the structure solved by traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment. PMID:24357322

  2. Large-volume protein crystal growth for neutron macromolecular crystallography

    DOE PAGESBeta

    Ng, Joseph D.; Baird, James K.; Coates, Leighton; Garcia-Ruiz, Juan M.; Hodge, Teresa A.; Huang, Sijay

    2015-03-30

    Neutron macromolecular crystallography (NMC) is the prevailing method for the accurate determination of the positions of H atoms in macromolecules. As neutron sources are becoming more available to general users, finding means to optimize the growth of protein crystals to sizes suitable for NMC is extremely important. Historically, much has been learned about growing crystals for X-ray diffraction. However, owing to new-generation synchrotron X-ray facilities and sensitive detectors, protein crystal sizes as small as in the nano-range have become adequate for structure determination, lessening the necessity to grow large crystals. Here, some of the approaches, techniques and considerations for themore » growth of crystals to significant dimensions that are now relevant to NMC are revisited. We report that these include experimental strategies utilizing solubility diagrams, ripening effects, classical crystallization techniques, microgravity and theoretical considerations.« less

  3. Protein energy landscapes determined by five-dimensional crystallography

    SciTech Connect

    Schmidt, Marius; Srajer, Vukica; Henning, Robert; Ihee, Hyotcherl; Purwar, Namrta; Tenboer, Jason; Tripathi, Shailesh

    2013-12-01

    Barriers of activation within the photocycle of a photoactive protein were extracted from comprehensive time courses of time resolved crystallographic data collected at multiple temperature settings. Free-energy landscapes decisively determine the progress of enzymatically catalyzed reactions [Cornish-Bowden (2012 ▶), Fundamentals of Enzyme Kinetics, 4th ed.]. Time-resolved macromolecular crystallography unifies transient-state kinetics with structure determination [Moffat (2001 ▶), Chem. Rev.101, 1569–1581; Schmidt et al. (2005 ▶), Methods Mol. Biol.305, 115–154; Schmidt (2008 ▶), Ultrashort Laser Pulses in Medicine and Biology] because both can be determined from the same set of X-ray data. Here, it is demonstrated how barriers of activation can be determined solely from five-dimensional crystallography, where in addition to space and time, temperature is a variable as well [Schmidt et al. (2010 ▶), Acta Cryst. A66, 198–206]. Directly linking molecular structures with barriers of activation between them allows insight into the structural nature of the barrier to be gained. Comprehensive time series of crystallographic data at 14 different temperature settings were analyzed and the entropy and enthalpy contributions to the barriers of activation were determined. One hundred years after the discovery of X-ray scattering, these results advance X-ray structure determination to a new frontier: the determination of energy landscapes.

  4. Development of Control Applications for High-Throughput Protein Crystallography Experiments

    SciTech Connect

    Gaponov, Yurii A.; Matsugaki, Naohiro; Honda, Nobuo; Sasajima, Kumiko; Igarashi, Noriyuki; Hiraki, Masahiko; Yamada, Yusuke; Wakatsuki, Soichi

    2007-01-19

    An integrated client-server control system (PCCS) with a unified relational database (PCDB) has been developed for high-throughput protein crystallography experiments on synchrotron beamlines. The major steps in protein crystallographic experiments (purification, crystallization, crystal harvesting, data collection, and data processing) are integrated into the software. All information necessary for performing protein crystallography experiments is stored in the PCDB database (except raw X-ray diffraction data, which is stored in the Network File Server). To allow all members of a protein crystallography group to participate in experiments, the system was developed as a multi-user system with secure network access based on TCP/IP secure UNIX sockets. Secure remote access to the system is possible from any operating system with X-terminal and SSH/X11 (Secure Shell with graphical user interface) support. Currently, the system covers the high-throughput X-ray data collection stages and is being commissioned at BL5A and NW12A (PF, PF-AR, KEK, Tsukuba, Japan)

  5. Towards long-wavelength protein crystallography: keeping a protein crystal frozen in vacuum

    NASA Astrophysics Data System (ADS)

    Mykhaylyk, Vitaliy; Wagner, Armin

    2013-03-01

    There is growing interest to explore the long-wavelength X-ray domain for macromolecular crystallography (MX) experiments but there are a number of practical issues that make these experiments difficult to perform. In this article we study several aspects related to cooling a protein crystal in a vacuum environment. We investigated thermal contact conductance (TCC) of copper-copper joints and designed a demountable sample holder assembly with a magnetic joint that facilitates good thermal conductivity and reliability over a long time period. The thermal conductivity of amorphous ice formed by a 20% solution of ethylene glycol was studied. It is concluded that the ice thickness is the factor that can compromise the cooling of protein crystals and therefore it should be carefully controlled.

  6. Serial femtosecond crystallography of soluble proteins in lipidic cubic phase

    SciTech Connect

    Fromme, Raimund; Ishchenko, Andrii; Metz, Markus; Chowdhury, Shatabdi Roy; Basu, Shibom; Boutet, Sébastien; Fromme, Petra; White, Thomas A.; Barty, Anton; Spence, John C. H.; Weierstall, Uwe; Liu, Wei; Cherezov, Vadim

    2015-08-04

    Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP–SFX method has recently been introduced in which microcrystals of membrane proteins are grown and delivered for SFX data collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic phase (LCP), using a special LCP microextrusion injector. Here, it is demonstrated that LCP can also be used as a suitable carrier medium for microcrystals of soluble proteins, enabling a dramatic reduction in the amount of crystallized protein required for data collection compared with crystals delivered by liquid injectors. High-quality LCP–SFX data sets were collected for two soluble proteins, lysozyme and phycocyanin, using less than 0.1 mg of each protein.

  7. Serial femtosecond crystallography of soluble proteins in lipidic cubic phase

    PubMed Central

    Fromme, Raimund; Ishchenko, Andrii; Metz, Markus; Chowdhury, Shatabdi Roy; Basu, Shibom; Boutet, Sébastien; Fromme, Petra; White, Thomas A.; Barty, Anton; Spence, John C. H.; Weierstall, Uwe; Liu, Wei; Cherezov, Vadim

    2015-01-01

    Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP–SFX method has recently been introduced in which microcrystals of membrane proteins are grown and delivered for SFX data collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic phase (LCP), using a special LCP microextrusion injector. Here, it is demonstrated that LCP can also be used as a suitable carrier medium for microcrystals of soluble proteins, enabling a dramatic reduction in the amount of crystallized protein required for data collection compared with crystals delivered by liquid injectors. High-quality LCP–SFX data sets were collected for two soluble proteins, lysozyme and phycocyanin, using less than 0.1 mg of each protein. PMID:26306196

  8. Serial femtosecond crystallography of soluble proteins in lipidic cubic phase.

    PubMed

    Fromme, Raimund; Ishchenko, Andrii; Metz, Markus; Chowdhury, Shatabdi Roy; Basu, Shibom; Boutet, Sébastien; Fromme, Petra; White, Thomas A; Barty, Anton; Spence, John C H; Weierstall, Uwe; Liu, Wei; Cherezov, Vadim

    2015-09-01

    Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP-SFX method has recently been introduced in which microcrystals of membrane proteins are grown and delivered for SFX data collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic phase (LCP), using a special LCP microextrusion injector. Here, it is demonstrated that LCP can also be used as a suitable carrier medium for microcrystals of soluble proteins, enabling a dramatic reduction in the amount of crystallized protein required for data collection compared with crystals delivered by liquid injectors. High-quality LCP-SFX data sets were collected for two soluble proteins, lysozyme and phycocyanin, using less than 0.1 mg of each protein. PMID:26306196

  9. Cryogenic Neutron Protein Crystallography: routine methods and potential benefits

    SciTech Connect

    Weiss, Kevin L; Tomanicek, Stephen J; NG, Joseph D

    2014-01-01

    The use of cryocooling in neutron diffraction has been hampered by several technical challenges such as the need for specialized equipment and techniques. Recently we have developed and deployed equipment and strategies that allow for routine neutron data collection on cryocooled crystals using off the shelf components. This system has several advantages, compared to a closed displex cooling system such as fast cooling coupled with easier crystal mounting and centering. The ability to routinely collect cryogenic neutron data for analysis will significantly broaden the range of scientific questions that can be examined by neutron protein crystallography. Cryogenic neutron data collection for macromolecules has recently become available at the new Biological Diffractometer BIODIFF at FRM II and the Macromolecular Diffractometer (MaNDi) at the Spallation Neutron Source, Oak Ridge National Laboratory. To evaluate the benefits of a cryocooled neutron structure we collected a full neutron data set on the BIODIFF instrument on a Toho-1 lactamase structure at 100K.

  10. Serial femtosecond crystallography datasets from G protein-coupled receptors.

    PubMed

    White, Thomas A; Barty, Anton; Liu, Wei; Ishchenko, Andrii; Zhang, Haitao; Gati, Cornelius; Zatsepin, Nadia A; Basu, Shibom; Oberthür, Dominik; Metz, Markus; Beyerlein, Kenneth R; Yoon, Chun Hong; Yefanov, Oleksandr M; James, Daniel; Wang, Dingjie; Messerschmidt, Marc; Koglin, Jason E; Boutet, Sébastien; Weierstall, Uwe; Cherezov, Vadim

    2016-01-01

    We describe the deposition of four datasets consisting of X-ray diffraction images acquired using serial femtosecond crystallography experiments on microcrystals of human G protein-coupled receptors, grown and delivered in lipidic cubic phase, at the Linac Coherent Light Source. The receptors are: the human serotonin receptor 2B in complex with an agonist ergotamine, the human δ-opioid receptor in complex with a bi-functional peptide ligand DIPP-NH2, the human smoothened receptor in complex with an antagonist cyclopamine, and finally the human angiotensin II type 1 receptor in complex with the selective antagonist ZD7155. All four datasets have been deposited, with minimal processing, in an HDF5-based file format, which can be used directly for crystallographic processing with CrystFEL or other software. We have provided processing scripts and supporting files for recent versions of CrystFEL, which can be used to validate the data. PMID:27479354

  11. Serial femtosecond crystallography datasets from G protein-coupled receptors

    PubMed Central

    White, Thomas A.; Barty, Anton; Liu, Wei; Ishchenko, Andrii; Zhang, Haitao; Gati, Cornelius; Zatsepin, Nadia A.; Basu, Shibom; Oberthür, Dominik; Metz, Markus; Beyerlein, Kenneth R.; Yoon, Chun Hong; Yefanov, Oleksandr M.; James, Daniel; Wang, Dingjie; Messerschmidt, Marc; Koglin, Jason E.; Boutet, Sébastien; Weierstall, Uwe; Cherezov, Vadim

    2016-01-01

    We describe the deposition of four datasets consisting of X-ray diffraction images acquired using serial femtosecond crystallography experiments on microcrystals of human G protein-coupled receptors, grown and delivered in lipidic cubic phase, at the Linac Coherent Light Source. The receptors are: the human serotonin receptor 2B in complex with an agonist ergotamine, the human δ-opioid receptor in complex with a bi-functional peptide ligand DIPP-NH2, the human smoothened receptor in complex with an antagonist cyclopamine, and finally the human angiotensin II type 1 receptor in complex with the selective antagonist ZD7155. All four datasets have been deposited, with minimal processing, in an HDF5-based file format, which can be used directly for crystallographic processing with CrystFEL or other software. We have provided processing scripts and supporting files for recent versions of CrystFEL, which can be used to validate the data. PMID:27479354

  12. Femtosecond crystallography of membrane proteins in the lipidic cubic phase

    PubMed Central

    Liu, Wei; Wacker, Daniel; Wang, Chong; Abola, Enrique; Cherezov, Vadim

    2014-01-01

    Despite recent technological advances in heterologous expression, stabilization and crystallization of membrane proteins (MPs), their structural studies remain difficult and require new transformative approaches. During the past two years, crystallization in lipidic cubic phase (LCP) has started gaining a widespread acceptance, owing to the spectacular success in high-resolution structure determination of G protein-coupled receptors (GPCRs) and to the introduction of commercial instrumentation, tools and protocols. The recent appearance of X-ray free-electron lasers (XFELs) has enabled structure determination from substantially smaller crystals than previously possible with minimal effects of radiation damage, offering new exciting opportunities in structural biology. The unique properties of LCP material have been exploited to develop special protocols and devices that have established a new method of serial femtosecond crystallography of MPs in LCP (LCP-SFX). In this method, microcrystals are generated in LCP and streamed continuously inside the same media across the intersection with a pulsed XFEL beam at a flow rate that can be adjusted to minimize sample consumption. Pioneering studies that yielded the first room temperature GPCR structures, using a few hundred micrograms of purified protein, validate the LCP-SFX approach and make it attractive for structure determination of difficult-to-crystallize MPs and their complexes with interacting partners. Together with the potential of femtosecond data acquisition to interrogate unstable intermediate functional states of MPs, LCP-SFX holds promise to advance our understanding of this biomedically important class of proteins. PMID:24914147

  13. Fragment-based screening by protein crystallography: successes and pitfalls.

    PubMed

    Chilingaryan, Zorik; Yin, Zhou; Oakley, Aaron J

    2012-01-01

    Fragment-based drug discovery (FBDD) concerns the screening of low-molecular weight compounds against macromolecular targets of clinical relevance. These compounds act as starting points for the development of drugs. FBDD has evolved and grown in popularity over the past 15 years. In this paper, the rationale and technology behind the use of X-ray crystallography in fragment based screening (FBS) will be described, including fragment library design and use of synchrotron radiation and robotics for high-throughput X-ray data collection. Some recent uses of crystallography in FBS will be described in detail, including interrogation of the drug targets β-secretase, phenylethanolamine N-methyltransferase, phosphodiesterase 4A and Hsp90. These examples provide illustrations of projects where crystallography is straightforward or difficult, and where other screening methods can help overcome the limitations of crystallography necessitated by diffraction quality. PMID:23202926

  14. Fragment-Based Screening by Protein Crystallography: Successes and Pitfalls

    PubMed Central

    Chilingaryan, Zorik; Yin, Zhou; Oakley, Aaron J.

    2012-01-01

    Fragment-based drug discovery (FBDD) concerns the screening of low-molecular weight compounds against macromolecular targets of clinical relevance. These compounds act as starting points for the development of drugs. FBDD has evolved and grown in popularity over the past 15 years. In this paper, the rationale and technology behind the use of X-ray crystallography in fragment based screening (FBS) will be described, including fragment library design and use of synchrotron radiation and robotics for high-throughput X-ray data collection. Some recent uses of crystallography in FBS will be described in detail, including interrogation of the drug targets β-secretase, phenylethanolamine N-methyltransferase, phosphodiesterase 4A and Hsp90. These examples provide illustrations of projects where crystallography is straightforward or difficult, and where other screening methods can help overcome the limitations of crystallography necessitated by diffraction quality. PMID:23202926

  15. Secure UNIX socket-based controlling system for high-throughput protein crystallography experiments.

    PubMed

    Gaponov, Yurii; Igarashi, Noriyuki; Hiraki, Masahiko; Sasajima, Kumiko; Matsugaki, Naohiro; Suzuki, Mamoru; Kosuge, Takashi; Wakatsuki, Soichi

    2004-01-01

    A control system for high-throughput protein crystallography experiments has been developed based on a multilevel secure (SSL v2/v3) UNIX socket under the Linux operating system. Main features of protein crystallography experiments (purification, crystallization, loop preparation, data collecting, data processing) are dealt with by the software. All information necessary to perform protein crystallography experiments is stored (except raw X-ray data, that are stored in Network File Server) in a relational database (MySQL). The system consists of several servers and clients. TCP/IP secure UNIX sockets with four predefined behaviors [(a) listening to a request followed by a reply, (b) sending a request and waiting for a reply, (c) listening to a broadcast message, and (d) sending a broadcast message] support communications between all servers and clients allowing one to control experiments, view data, edit experimental conditions and perform data processing remotely. The usage of the interface software is well suited for developing well organized control software with a hierarchical structure of different software units (Gaponov et al., 1998), which will pass and receive different types of information. All communication is divided into two parts: low and top levels. Large and complicated control tasks are split into several smaller ones, which can be processed by control clients independently. For communicating with experimental equipment (beamline optical elements, robots, and specialized experimental equipment etc.), the STARS server, developed at the Photon Factory, is used (Kosuge et al., 2002). The STARS server allows any application with an open socket to be connected with any other clients that control experimental equipment. Majority of the source code is written in C/C++. GUI modules of the system were built mainly using Glade user interface builder for GTK+ and Gnome under Red Hat Linux 7.1 operating system. PMID:14646123

  16. Fundamental studies for the proton polarization technique in neutron protein crystallography.

    PubMed

    Tanaka, Ichiro; Kusaka, Katsuhiro; Chatake, Toshiyuki; Niimura, Nobuo

    2013-11-01

    The isotope effect in conventional neutron protein crystallography (NPC) can be eliminated by the proton polarization technique (ppt). Furthermore, the ppt can improve detection sensitivity of hydrogen (relative neutron scattering length of hydrogen) by approximately eight times in comparison with conventional NPC. Several technical difficulties, however, should be overcome in order to perform the ppt. In this paper, two fundamental studies to realise ppt are presented: preliminary trials using high-pressure flash freezing has shown the advantage of making bulk water amorphous without destroying the single crystal; and X-ray diffraction and liquid-chromatography/mass-spectrometry analyses of standard proteins after introducing radical molecules into protein crystals have shown that radical molecules could be distributed non-specifically around proteins, which is essential for better proton polarization. PMID:24121348

  17. Fundamental studies for the proton polarization technique in neutron protein crystallography

    PubMed Central

    Tanaka, Ichiro; Kusaka, Katsuhiro; Chatake, Toshiyuki; Niimura, Nobuo

    2013-01-01

    The isotope effect in conventional neutron protein crystallography (NPC) can be eliminated by the proton polarization technique (ppt). Furthermore, the ppt can improve detection sensitivity of hydrogen (relative neutron scattering length of hydrogen) by approximately eight times in comparison with conventional NPC. Several technical difficulties, however, should be overcome in order to perform the ppt. In this paper, two fundamental studies to realise ppt are presented: preliminary trials using high-pressure flash freezing has shown the advantage of making bulk water amorphous without destroying the single crystal; and X-ray diffraction and liquid-chromatography/mass-spectrometry analyses of standard proteins after introducing radical molecules into protein crystals have shown that radical molecules could be distributed non-specifically around proteins, which is essential for better proton polarization. PMID:24121348

  18. Suite of three protein crystallography beamlines with single superconducting bend magnet as the source

    SciTech Connect

    MacDowell, Alastair A.; Celestre, Richard S.; Howells, Malcolm; McKinney, Wayne; Krupnick, James; Cambie, Daniella; Domning, Edward E; Duarte, Robert M.; Kelez, Nicholas; Plate, David W.; Cork, Carl W.; Earnest, Thomas N.; Dickert, Jeffery; Meigs, George; Ralston, Corie; Holton, James M.; Alber, Thomas; Berger, James M.; Agard, David A.; Padmore, Howard A.

    2004-08-01

    At the Advanced Light Source (ALS), three protein crystallography (PX) beamlines have been built that use as a source one of the three 6 Tesla single pole superconducting bending magnets (superbends) that were recently installed in the ring. The use of such single pole superconducting bend magnets enables the development of a hard x-ray program on a relatively low energy 1.9 GeV ring without taking up insertion device straight sections. The source is of relatively low power, but due to the small electron beam emittance, it has high brightness. X-ray optics are required to preserve the brightness and to match the illumination requirements for protein crystallography. This was achieved by means of a collimating premirror bent to a plane parabola, a double crystal monochromator followed by a toroidal mirror that focuses in the horizontal direction with a 2:1 demagnification. This optical arrangement partially balances aberrations from the collimating and toroidal mirrors such that a tight focused spot size is achieved. The optical properties of the beamline are an excellent match to those required by the small protein crystals that are typically measured. The design and performance of these new beamlines are described.

  19. Protein-detergent interactions in single crystals of membrane proteins studied by neutron crystallography

    SciTech Connect

    Timmins, P.A.; Pebay-Peyroula, E.

    1994-12-31

    The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H{sub 2}O/D{sub 2}O contrast variation. If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering. The application of various constraints allows the resulting scattering length density map to be realistically modeled. The method has been applied to two different forms of the membrane protein porin. In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished.

  20. In meso in situ serial X-ray crystallography of soluble and membrane proteins

    SciTech Connect

    Huang, Chia-Ying; Olieric, Vincent; Ma, Pikyee; Panepucci, Ezequiel; Diederichs, Kay; Wang, Meitian; Caffrey, Martin

    2015-05-14

    A method for performing high-throughput in situ serial X-ray crystallography with soluble and membrane proteins in the lipid cubic phase is described. It works with microgram quantities of protein and lipid (and ligand when present) and is compatible with the most demanding sulfur SAD phasing. The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high-resolution X-ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP or in meso method. Among the listings are some of the highest profile membrane proteins, including the β{sub 2}-adrenoreceptor–G{sub s} protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful in meso protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing in situ X-ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high-throughput in situ measurements. The novel in meso in situ serial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 Å using single-digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high-throughput in situ serial data collection at

  1. Neutron protein crystallography: A complementary tool for locating hydrogens in proteins.

    PubMed

    O'Dell, William B; Bodenheimer, Annette M; Meilleur, Flora

    2016-07-15

    Neutron protein crystallography is a powerful tool for investigating protein chemistry because it directly locates hydrogen atom positions in a protein structure. The visibility of hydrogen and deuterium atoms arises from the strong interaction of neutrons with the nuclei of these isotopes. Positions can be unambiguously assigned from diffraction at resolutions typical of protein crystals. Neutrons have the additional benefit to structural biology of not inducing radiation damage in protein crystals. The same crystal could be measured multiple times for parametric studies. Here, we review the basic principles of neutron protein crystallography. The information that can be gained from a neutron structure is presented in balance with practical considerations. Methods to produce isotopically-substituted proteins and to grow large crystals are provided in the context of neutron structures reported in the literature. Available instruments for data collection and software for data processing and structure refinement are described along with technique-specific strategies including joint X-ray/neutron structure refinement. Examples are given to illustrate, ultimately, the unique scientific value of neutron protein crystal structures. PMID:26592456

  2. In meso in situ serial X-ray crystallography of soluble and membrane proteins

    PubMed Central

    Huang, Chia-Ying; Olieric, Vincent; Ma, Pikyee; Panepucci, Ezequiel; Diederichs, Kay; Wang, Meitian; Caffrey, Martin

    2015-01-01

    The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high-resolution X-ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP or in meso method. Among the listings are some of the highest profile membrane proteins, including the β2-adrenoreceptor–Gs protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful in meso protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing in situ X-ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high-throughput in situ measurements. The novel in meso in situ serial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 Å using single-digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high-throughput in situ serial data collection at macromolecular crystallography synchrotron beamlines worldwide. Because of its simplicity and effectiveness, the IMISX approach is likely to supplant existing in meso crystallization protocols. It should prove particularly attractive in the area of ligand screening for drug discovery and development. PMID

  3. In meso in situ serial X-ray crystallography of soluble and membrane proteins.

    PubMed

    Huang, Chia Ying; Olieric, Vincent; Ma, Pikyee; Panepucci, Ezequiel; Diederichs, Kay; Wang, Meitian; Caffrey, Martin

    2015-06-01

    The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high-resolution X-ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP or in meso method. Among the listings are some of the highest profile membrane proteins, including the β2-adrenoreceptor-Gs protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful in meso protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing in situ X-ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high-throughput in situ measurements. The novel in meso in situ serial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 Å using single-digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high-throughput in situ serial data collection at macromolecular crystallography synchrotron beamlines worldwide. Because of its simplicity and effectiveness, the IMISX approach is likely to supplant existing in meso crystallization protocols. It should prove particularly attractive in the area of ligand screening for drug discovery and development. PMID:26057665

  4. A neutron image plate quasi-Laue diffractometer for protein crystallography

    SciTech Connect

    Cipriani, F.; Castagna, J.C.; Wilkinson, C.

    1994-12-31

    An instrument which is based on image plate technology has been constructed to perform cold neutron Laue crystallography on protein structures. The crystal is mounted at the center of a cylindrical detector which is 400mm long and has a circumference of 1000mm, with gadolinium oxide-containing image plates mounted on its exterior surface. Laue images registered on the plate are read out by rotating the drum and translating a laser read head parallel to the cylinder axis, giving a pixel size of 200{mu}m x 200{mu}m and a total read time of 5 minutes. Preliminary results indicate that it should be possible to obtain a complete data set from a protein crystal to atomic resolution in about two weeks.

  5. Rapid visualization of hydrogen positions in neutron protein crystallography structures

    SciTech Connect

    Blakeley, Matthew P.; Meilleur, Flora; Myles, Dean A A; Weiss, Kevin L; Munshi, Parthapratim; Shang-Lin, Chung

    2012-01-01

    Neutron crystallography is a powerful technique to visualize experimentally the position of light atoms, including hydrogen and its isotope deuterium. Over the last several years, structural biologists have shown an increasing interest for the technique as it uniquely complements X-ray crystallographic data by revealing the position of hydrogen/deuterium atoms in macromolecules. With this regained interest, access to macromolecule neutron crystallography beam lines is becoming a limiting step. In this report we show that rapid data collection could be a valuable alternative to long data collection time when appropriate. Comparison of perdeuterated Rubredoxin structures refined against neutron data sets collected over hours and up to five days shows that rapid neutron data collection in just 14 hours is sufficient to provide the position of 262 hydrogen positions atoms without ambiguity.

  6. A 2D smart pixel detector for time-resolved protein crystallography

    SciTech Connect

    Beuville, E.; Cork, C.; Earnest, T.

    1995-10-01

    A smart pixel detector is being developed for Time Resolved Crystallography for biological and material science applications. Using the Pixel detector presented here, the Laue method will enable the study of the evolution of structural changes that occur within the protein as a function of time. The x-ray pixellated detector is assembled to the integrated circuit through a bump bonding process. Within a pixel size of 150 x 150 {mu}m{sup 2}, a low noise preamplifier-shaper, a discriminator, a 3 bit counter and the readout logic are integrated. The readout, based on the Column Architecture principle, will accept hit rates above 5x10{sup 8}/cm{sup 2}/s with a maximum hit rate per pixel of 1 MHz. This detector will allow time resolved Laue crystallography to be performed in a frameless operation mode, without dead time. Target specifications, architecture, and preliminary results on the 8 x 8 front-end prototype and column readout are presented.

  7. Ink-jet printer heads for ultra-small-drop protein crystallography.

    PubMed

    Howard, E I; Cachau, R E

    2002-12-01

    Mass-produced automated piezoelectric driven picoliter delivery systems (printer heads) are fast, inexpensive, and reliable devices that are capable of delivering a very large range of volumes and are ideally suited for high-throughput protein crystallography studies. We used this technology to set up under-oil crystallization experiments with drop sizes from the 200-nL to 3-microL volume range, commonly used in protein crystallography, and show its application in setting ultra-small (2 nL) drops, the smallest drop volume reported to date for this type of assay. PMID:12503316

  8. System and method for forming synthetic protein crystals to determine the conformational structure by crystallography

    DOEpatents

    Craig, G.D.; Glass, R.; Rupp, B.

    1997-01-28

    A method is disclosed for forming synthetic crystals of proteins in a carrier fluid by use of the dipole moments of protein macromolecules that self-align in the Helmholtz layer adjacent to an electrode. The voltage gradients of such layers easily exceed 10{sup 6}V/m. The synthetic protein crystals are subjected to x-ray crystallography to determine the conformational structure of the protein involved. 2 figs.

  9. System and method for forming synthetic protein crystals to determine the conformational structure by crystallography

    DOEpatents

    Craig, George D.; Glass, Robert; Rupp, Bernhard

    1997-01-01

    A method for forming synthetic crystals of proteins in a carrier fluid by use of the dipole moments of protein macromolecules that self-align in the Helmholtz layer adjacent to an electrode. The voltage gradients of such layers easily exceed 10.sup.6 V/m. The synthetic protein crystals are subjected to x-ray crystallography to determine the conformational structure of the protein involved.

  10. Development of a shutterless continuous rotation method using an X-ray CMOS detector for protein crystallography

    PubMed Central

    Hasegawa, Kazuya; Hirata, Kunio; Shimizu, Tetsuya; Shimizu, Nobutaka; Hikima, Takaaki; Baba, Seiki; Kumasaka, Takashi; Yamamoto, Masaki

    2009-01-01

    A new shutterless continuous rotation method using an X-ray complementary metal-oxide semiconductor (CMOS) detector has been developed for high-speed, precise data collection in protein crystallography. The principle of operation and the basic performance of the X-ray CMOS detector (Hamamatsu Photonics KK C10158DK) have been shown to be appropriate to the shutterless continuous rotation method. The data quality of the continuous rotation method is comparable to that of the conventional oscillation method using a CCD detector and, furthermore, the combination with fine ϕ slicing improves the data accuracy without increasing the data-collection time. The new method is more sensitive to diffraction intensity because of the narrow dynamic range of the CMOS detector. However, the strong diffraction spots were found to be precisely measured by recording them on successive multiple images by selecting an adequate rotation step. The new method has been used to successfully determine three protein structures by multi- and single-wavelength anomalous diffraction phasing and has thereby been proved applicable in protein crystallography. The apparatus and method may become a powerful tool at synchrotron protein crystallography beamlines with important potential across a wide range of X-ray wavelengths. PMID:22477775

  11. Protein-ligand interactions probed by time-resolved crystallography

    SciTech Connect

    Schmidt, M.; Ihee, H.; Pahl, R.; Srajer, V.

    2005-03-09

    Time-resolved (TR) crystallography is a unique method for determining the structures of intermediates in biomolecular reactions. The technique reached its mature stage with the development of the powerful third-generation synchrotron X-ray sources, and the advances in data processing and analysis of time-resolved Laue crystallographic data. A time resolution of 100 ps has been achieved and relatively small structural changes can be detected even from only partial reaction initiation. The remaining challenge facing the application of this technique to a broad range of biological systems is to find an efficient and rapid, system-specific method for the reaction initiation in the crystal. Other frontiers for the technique involve the continued improvement in time resolution and further advances in methods for determining intermediate structures and reaction mechanisms. The time-resolved technique, combined with trapping methods and computational approaches, holds the promise for a complete structure-based description of biomolecular reactions.

  12. A Maltose-Binding Protein Fusion Construct Yields a Robust Crystallography Platform for MCL1

    PubMed Central

    Clifton, Matthew C.; Dranow, David M.; Leed, Alison; Fulroth, Ben; Fairman, James W.; Abendroth, Jan; Atkins, Kateri A.; Wallace, Ellen; Fan, Dazhong; Xu, Guoping; Ni, Z. J.; Daniels, Doug; Van Drie, John; Wei, Guo; Burgin, Alex B.; Golub, Todd R.; Hubbard, Brian K.; Serrano-Wu, Michael H.

    2015-01-01

    Crystallization of a maltose-binding protein MCL1 fusion has yielded a robust crystallography platform that generated the first apo MCL1 crystal structure, as well as five ligand-bound structures. The ability to obtain fragment-bound structures advances structure-based drug design efforts that, despite considerable effort, had previously been intractable by crystallography. In the ligand-independent crystal form we identify inhibitor binding modes not observed in earlier crystallographic systems. This MBP-MCL1 construct dramatically improves the structural understanding of well-validated MCL1 ligands, and will likely catalyze the structure-based optimization of high affinity MCL1 inhibitors. PMID:25909780

  13. Time-resolved structural studies with serial crystallography: A new light on retinal proteins

    PubMed Central

    Panneels, Valérie; Wu, Wenting; Tsai, Ching-Ju; Nogly, Przemek; Rheinberger, Jan; Jaeger, Kathrin; Cicchetti, Gregor; Gati, Cornelius; Kick, Leonhard M.; Sala, Leonardo; Capitani, Guido; Milne, Chris; Padeste, Celestino; Pedrini, Bill; Li, Xiao-Dan; Standfuss, Jörg; Abela, Rafael; Schertler, Gebhard

    2015-01-01

    Structural information of the different conformational states of the two prototypical light-sensitive membrane proteins, bacteriorhodopsin and rhodopsin, has been obtained in the past by X-ray cryo-crystallography and cryo-electron microscopy. However, these methods do not allow for the structure determination of most intermediate conformations. Recently, the potential of X-Ray Free Electron Lasers (X-FELs) for tracking the dynamics of light-triggered processes by pump-probe serial femtosecond crystallography has been demonstrated using 3D-micron-sized crystals. In addition, X-FELs provide new opportunities for protein 2D-crystal diffraction, which would allow to observe the course of conformational changes of membrane proteins in a close-to-physiological lipid bilayer environment. Here, we describe the strategies towards structural dynamic studies of retinal proteins at room temperature, using injector or fixed-target based serial femtosecond crystallography at X-FELs. Thanks to recent progress especially in sample delivery methods, serial crystallography is now also feasible at synchrotron X-ray sources, thus expanding the possibilities for time-resolved structure determination. PMID:26798817

  14. Apparatus and method for nanoflow liquid jet and serial femtosecond x-ray protein crystallography

    DOEpatents

    Bogan, Michael J.; Laksmono, Hartawan; Sierra, Raymond G.

    2016-03-01

    Techniques for nanoflow serial femtosecond x-ray protein crystallography include providing a sample fluid by mixing a plurality of a first target of interest with a carrier fluid and injecting the sample fluid into a vacuum chamber at a rate less than about 4 microliters per minute. In some embodiments, the carrier fluid has a viscosity greater than about 3 centipoise.

  15. Time-resolved structural studies with serial crystallography: A new light on retinal proteins.

    PubMed

    Panneels, Valérie; Wu, Wenting; Tsai, Ching-Ju; Nogly, Przemek; Rheinberger, Jan; Jaeger, Kathrin; Cicchetti, Gregor; Gati, Cornelius; Kick, Leonhard M; Sala, Leonardo; Capitani, Guido; Milne, Chris; Padeste, Celestino; Pedrini, Bill; Li, Xiao-Dan; Standfuss, Jörg; Abela, Rafael; Schertler, Gebhard

    2015-07-01

    Structural information of the different conformational states of the two prototypical light-sensitive membrane proteins, bacteriorhodopsin and rhodopsin, has been obtained in the past by X-ray cryo-crystallography and cryo-electron microscopy. However, these methods do not allow for the structure determination of most intermediate conformations. Recently, the potential of X-Ray Free Electron Lasers (X-FELs) for tracking the dynamics of light-triggered processes by pump-probe serial femtosecond crystallography has been demonstrated using 3D-micron-sized crystals. In addition, X-FELs provide new opportunities for protein 2D-crystal diffraction, which would allow to observe the course of conformational changes of membrane proteins in a close-to-physiological lipid bilayer environment. Here, we describe the strategies towards structural dynamic studies of retinal proteins at room temperature, using injector or fixed-target based serial femtosecond crystallography at X-FELs. Thanks to recent progress especially in sample delivery methods, serial crystallography is now also feasible at synchrotron X-ray sources, thus expanding the possibilities for time-resolved structure determination. PMID:26798817

  16. Protein energy landscapes determined by five-dimensional crystallography

    PubMed Central

    Schmidt, Marius; Srajer, Vukica; Henning, Robert; Ihee, Hyotcherl; Purwar, Namrta; Tenboer, Jason; Tripathi, Shailesh

    2013-01-01

    Free-energy landscapes decisively determine the progress of enzymatically catalyzed reactions [Cornish-Bowden (2012 ▶), Fundamentals of Enzyme Kinetics, 4th ed.]. Time-resolved macromolecular crystallography unifies transient-state kinetics with structure determination [Moffat (2001 ▶), Chem. Rev. 101, 1569–1581; Schmidt et al. (2005 ▶), Methods Mol. Biol. 305, 115–154; Schmidt (2008 ▶), Ultrashort Laser Pulses in Medicine and Biology] because both can be determined from the same set of X-ray data. Here, it is demonstrated how barriers of activation can be determined solely from five-dimensional crystallo­graphy, where in addition to space and time, temperature is a variable as well [Schmidt et al. (2010 ▶), Acta Cryst. A66, 198–206]. Directly linking molecular structures with barriers of activation between them allows insight into the structural nature of the barrier to be gained. Comprehensive time series of crystallo­graphic data at 14 different temperature settings were analyzed and the entropy and enthalpy contributions to the barriers of activation were determined. One hundred years after the discovery of X-ray scattering, these results advance X-ray structure determination to a new frontier: the determination of energy landscapes. PMID:24311594

  17. A NASA Recipe for Protein Crystallography. Educational Brief.

    ERIC Educational Resources Information Center

    National Aeronautics and Space Administration, Washington, DC.

    This brief discusses growing protein crystals. Protein crystals can be very difficult to grow. This activity for grades 9-12 provides a simple recipe for growing protein crystals from Brazil nuts. Included are a history of protein crystals, a discussion of microgravity effects on growth, connections to academic standards, and lab sheets. (MVL)

  18. Distributed control of protein crystallography beamline 5.0 using CORBA

    SciTech Connect

    Timossi, Chris

    1999-09-24

    The Protein Crystallography Beamline at Berkeley Lab's Advanced Light Source is a facility that is being used to solve the structure of proteins. The software that is being used to control this beamline uses Java for user interface applications which communicate via CORBA with workstations that control the beamline hardware. We describe the software architecture for the beamline and our experiences after two years of operation.

  19. Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

    PubMed

    Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

    2015-10-01

    Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters. PMID:26371748

  20. Laue diffraction protein crystallography at the National Synchrotron Light Source

    SciTech Connect

    Getzoff, E.D.; McRee, D.; Jones, K.W.; Spanne, P.; Sweet, R.M.; Moffat, K.; Ng, K.; Rivers, M.L.; Schildkamp, W.; Teng, T.Y.; Singer, P.T.; Westbrook, E.M.

    1992-12-31

    A new facility for the study of protein crystal structure using Laue diffraction has been established at the X26 beam line of the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory. The characteristics of the beam line and diffraction apparatus are described. Selected results of some of the initial experiments are discussed briefly by beam line users to illustrate the scope of the experimental program. Because the Laue method permits the recording of large data sets in a single shot, one goal in establishing this facility has been to develop the means to study time-resolved structures within protein crystals. Systems being studied include: the reactions catalyzed by trypsin; photolysis of carbonmonoxy myoglobin; and the photocycle of photoactive yellow protein.

  1. Laue diffraction protein crystallography at the National Synchrotron Light Source

    SciTech Connect

    Getzoff, E.D.; McRee, D. ); Jones, K.W.; Spanne, P.; Sweet, R.M. ); Moffat, K.; Ng, K.; Rivers, M.L.; Schildkamp, W.; Teng, T.Y. ); Singer, P.T.; Westbrook, E.M. )

    1992-01-01

    A new facility for the study of protein crystal structure using Laue diffraction has been established at the X26 beam line of the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory. The characteristics of the beam line and diffraction apparatus are described. Selected results of some of the initial experiments are discussed briefly by beam line users to illustrate the scope of the experimental program. Because the Laue method permits the recording of large data sets in a single shot, one goal in establishing this facility has been to develop the means to study time-resolved structures within protein crystals. Systems being studied include: the reactions catalyzed by trypsin; photolysis of carbonmonoxy myoglobin; and the photocycle of photoactive yellow protein.

  2. Electron crystallography of ultrathin 3D protein crystals: atomic model with charges.

    PubMed

    Yonekura, Koji; Kato, Kazuyuki; Ogasawara, Mitsuo; Tomita, Masahiro; Toyoshima, Chikashi

    2015-03-17

    Membrane proteins and macromolecular complexes often yield crystals too small or too thin for even the modern synchrotron X-ray beam. Electron crystallography could provide a powerful means for structure determination with such undersized crystals, as protein atoms diffract electrons four to five orders of magnitude more strongly than they do X-rays. Furthermore, as electron crystallography yields Coulomb potential maps rather than electron density maps, it could provide a unique method to visualize the charged states of amino acid residues and metals. Here we describe an attempt to develop a methodology for electron crystallography of ultrathin (only a few layers thick) 3D protein crystals and present the Coulomb potential maps at 3.4-Å and 3.2-Å resolution, respectively, obtained from Ca(2+)-ATPase and catalase crystals. These maps demonstrate that it is indeed possible to build atomic models from such crystals and even to determine the charged states of amino acid residues in the Ca(2+)-binding sites of Ca(2+)-ATPase and that of the iron atom in the heme in catalase. PMID:25730881

  3. Electron crystallography of ultrathin 3D protein crystals: Atomic model with charges

    PubMed Central

    Yonekura, Koji; Kato, Kazuyuki; Ogasawara, Mitsuo; Tomita, Masahiro; Toyoshima, Chikashi

    2015-01-01

    Membrane proteins and macromolecular complexes often yield crystals too small or too thin for even the modern synchrotron X-ray beam. Electron crystallography could provide a powerful means for structure determination with such undersized crystals, as protein atoms diffract electrons four to five orders of magnitude more strongly than they do X-rays. Furthermore, as electron crystallography yields Coulomb potential maps rather than electron density maps, it could provide a unique method to visualize the charged states of amino acid residues and metals. Here we describe an attempt to develop a methodology for electron crystallography of ultrathin (only a few layers thick) 3D protein crystals and present the Coulomb potential maps at 3.4-Å and 3.2-Å resolution, respectively, obtained from Ca2+-ATPase and catalase crystals. These maps demonstrate that it is indeed possible to build atomic models from such crystals and even to determine the charged states of amino acid residues in the Ca2+-binding sites of Ca2+-ATPase and that of the iron atom in the heme in catalase. PMID:25730881

  4. Raster-scanning serial protein crystallography using micro- and nano-focused synchrotron beams

    SciTech Connect

    Coquelle, Nicolas; Brewster, Aaron S.; Kapp, Ulrike; Shilova, Anastasya; Weinhausen, Britta; Burghammer, Manfred; Colletier, Jacques-Philippe

    2015-05-01

    A raster scanning serial protein crystallography approach is presented, that consumes as low ∼200–700 nl of sedimented crystals. New serial data pre-analysis software, NanoPeakCell, is introduced. High-resolution structural information was obtained from lysozyme microcrystals (20 µm in the largest dimension) using raster-scanning serial protein crystallography on micro- and nano-focused beamlines at the ESRF. Data were collected at room temperature (RT) from crystals sandwiched between two silicon nitride wafers, thereby preventing their drying, while limiting background scattering and sample consumption. In order to identify crystal hits, new multi-processing and GUI-driven Python-based pre-analysis software was developed, named NanoPeakCell, that was able to read data from a variety of crystallographic image formats. Further data processing was carried out using CrystFEL, and the resultant structures were refined to 1.7 Å resolution. The data demonstrate the feasibility of RT raster-scanning serial micro- and nano-protein crystallography at synchrotrons and validate it as an alternative approach for the collection of high-resolution structural data from micro-sized crystals. Advantages of the proposed approach are its thriftiness, its handling-free nature, the reduced amount of sample required, the adjustable hit rate, the high indexing rate and the minimization of background scattering.

  5. Time-Resolved X-Ray Crystallography of Heme Proteins

    SciTech Connect

    Srajer, Vukica; Royer, Jr., William E.

    2008-04-29

    Heme proteins, with their natural photosensitivity, are excellent systems for the application of time-resolved crystallographic methods. Ligand dissociation can be readily initiated by a short laser pulse with global structural changes probed at the atomic level by X-rays in real time. Third-generation synchrotrons provide 100-ps X-ray pulses of sufficient intensity for monitoring very fast processes. Successful application of such time-resolved crystallographic experiments requires that the structural changes being monitored are compatible with the crystal lattice. These techniques have recently permitted observing for the first time allosteric transitions in real time for a cooperative dimeric hemoglobin.

  6. Time-resolved x-ray crystallography of heme proteins

    PubMed Central

    Royer, William E.

    2012-01-01

    Heme proteins, with their natural photosensitivity, are excellent systems for the application of time-resolved crystallographic methods. Ligand dissociation can be readily initiated by a short laser pulse with global structural changes probed at the atomic level by X-rays in real time. Third generation synchrotrons provide 100ps X-ray pulses of sufficient intensity for monitoring very fast processes. Successful application of such time-resolved crystallographic experiments requires that the structural changes being monitored are compatible with the crystal lattice. These techniques have permitted observing allosteric transitions in real time for a cooperative dimeric hemoglobin. PMID:18433638

  7. Automated Technologies and Novel Techniques to Accelerate Protein Crystallography for Structrual Genomics

    SciTech Connect

    Manjasetty,B.; Turnbull, A.; Panjikar, S.; Bussow, K.; Chance, M.

    2008-01-01

    The sequence infrastructure that has arisen through large-scale genomic projects dedicated to protein analysis, has provided a wealth of information and brought together scientists and institutions from all over the world. As a consequence, the development of novel technologies and methodologies in proteomics research is helping to unravel the biochemical and physiological mechanisms of complex multivariate diseases at both a functional and molecular level. In the late sixties, when X-ray crystallography had just been established, the idea of determining protein structure on an almost universal basis was akin to an impossible dream or a miracle. Yet only forty years after, automated protein structure determination platforms have been established. The widespread use of robotics in protein crystallography has had a huge impact at every stage of the pipeline from protein cloning, over-expression, purification, crystallization, data collection, structure solution, refinement, validation and data management- all of which have become more or less automated with minimal human intervention necessary. Here, recent advances in protein crystal structure analysis in the context of structural genomics will be discussed. In addition, this review aims to give an overview of recent developments in high throughput instrumentation, and technologies and strategies to accelerate protein structure/function analysis.

  8. Anomalous Diffraction at Ultra-High Energy for Protein Crystallography

    SciTech Connect

    Jakoncic,J.; Di Michiel, M.; Zhong, Z.; Honkimaki, V.; Jouanneau, Y.; Stojanoff, V.

    2006-01-01

    Single-wavelength anomalous diffraction (SAD), multiwavelength anomalous diffraction (MAD) and single isomorphous replacement with anomalous scattering (SIRAS) phasing at ultra-high X-ray energy, 55 keV, are used successfully to determine a high-quality and high-resolution experimental electronic density map of hen egg-white lysozyme, a model protein. Several combinations, between single- and three-wavelength, with native data were exploited to demonstrate that standard phasing procedures with standard equipment and software can successfully be applied to three-dimensional crystal structure determination of a macromolecule, even at these very short wavelengths. For the first time, a high-quality three-dimensional molecular structure is reported from SAD phasing with ultra-high-energy X-rays. The quality of the crystallographic data and the experimental electron density maps meet current standards. The 2.7% anomalous signal from three Ho atoms, at the Ho K edge, was sufficient to obtain a remarkable electron density and build the first lanthanide structure for HEWL in its entirety.

  9. When Protein Crystallography Won't Show You the Membranes (446th Brookhaven Lecture)

    SciTech Connect

    Yan, Lin

    2009-02-18

    High fever, stomach ache, coughing, sneezing, and fatigue -- these are all painful signs that you may have caught the flu virus. But how does your body actually 'catch' a virus? Somewhere along the way, the virus infected your body by penetrating the membranes, or surfaces, of some of your body's cells. And then it spreads. Cell membranes are permeable surfaces made of proteins and lipids that allow vital materials to enter and exit cells. Many proteins and cell structures are studied at Brookhaven's National Synchrotron Light Source (NSLS) using a procedure called protein crystallography. But they sometimes have unique characteristics that do not allow them to be easily studied using this widely adopted method. These characteristics make it difficult to understand the cell membrane structure and its ability to both welcome and refuse certain materials and viruses, such as the flu, on behalf of the cell's internal components. Yang will explain the protein crystallography procedure, the simple structure of the cell membrane, and the unusual characteristics of its proteins and lipids. He will also discuss a new, unique method being developed at the NSLS to study proteins and lipids within their native environment as they form the essential permeable surface of a cell membrane.

  10. A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography

    DOE PAGESBeta

    Bunker, Richard D.; Mandal, Kalyaneswar; Bashiri, Ghader; Chaston, Jessica J.; Pentelute, Bradley L.; Lott, J. Shaun; Kent, Stephen B. H.; Baker, Edward N.

    2015-04-07

    Racemic protein crystallography was used to determine the X-ray structure of the predicted Mycobacterium tuberculosis protein Rv1738, which had been completely recalcitrant to crystallization in its natural L-form. Native chemical ligation was used to synthesize both L-protein and D-protein enantiomers of Rv1738. Crystallization of the racemic {D-protein + L-protein} mixture was immediately successful. The resulting crystals diffracted to high resolution and also enabled facile structure determination because of the quantized phases of the data from centrosymmetric crystals. The X-ray structure of Rv1738 revealed striking similarity with bacterial hibernation factors, despite minimal sequence similarity. As a result, we predict that Rv1738,more » which is highly up-regulated in conditions that mimic the onset of persistence, helps trigger dormancy by association with the bacterial ribosome.« less

  11. Protein crystallography for aspiring crystallographers or how to avoid pitfalls and traps in macromolecular structure determination

    PubMed Central

    Wlodawer, Alexander; Minor, Wladek; Dauter, Zbigniew; Jaskolski, Mariusz

    2014-01-01

    The number of macromolecular structures deposited in the Protein Data Bank now approaches 100 000, with the vast majority of them determined by crystallographic methods. Thousands of papers describing such structures have been published in the scientific literature, and 20 Nobel Prizes in chemistry or medicine have been awarded for discoveries based on macromolecular crystallography. New hardware and software tools have made crystallography appear to be an almost routine (but still far from being analytical) technique and many structures are now being determined by scientists with very limited experience in the practical aspects of the field. However, this apparent ease is sometimes illusory and proper procedures need to be followed to maintain high standards of structure quality. In addition, many noncrystallographers may have problems with the critical evaluation and interpretation of structural results published in the scientific literature. The present review provides an outline of the technical aspects of crystallography for less experienced practitioners, as well as information that might be useful for users of macromolecular structures, aiming to show them how to interpret (but not overinterpret) the information present in the coordinate files and in their description. A discussion of the extent of information that can be gleaned from the atomic coordinates of structures solved at different resolution is provided, as well as problems and pitfalls encountered in structure determination and interpretation. PMID:24034303

  12. Life in the fast lane for protein crystallization and X-ray crystallography

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Liu, Zhi-Jie; Tempel, Wolfram; Praissman, Jeremy; Lin, Dawei; Wang, Bi-Cheng; Gavira, Jose A.; Ng, Joseph D.

    2005-01-01

    The common goal for structural genomic centers and consortiums is to decipher as quickly as possible the three-dimensional structures for a multitude of recombinant proteins derived from known genomic sequences. Since X-ray crystallography is the foremost method to acquire atomic resolution for macromolecules, the limiting step is obtaining protein crystals that can be useful of structure determination. High-throughput methods have been developed in recent years to clone, express, purify, crystallize and determine the three-dimensional structure of a protein gene product rapidly using automated devices, commercialized kits and consolidated protocols. However, the average number of protein structures obtained for most structural genomic groups has been very low compared to the total number of proteins purified. As more entire genomic sequences are obtained for different organisms from the three kingdoms of life, only the proteins that can be crystallized and whose structures can be obtained easily are studied. Consequently, an astonishing number of genomic proteins remain unexamined. In the era of high-throughput processes, traditional methods in molecular biology, protein chemistry and crystallization are eclipsed by automation and pipeline practices. The necessity for high-rate production of protein crystals and structures has prevented the usage of more intellectual strategies and creative approaches in experimental executions. Fundamental principles and personal experiences in protein chemistry and crystallization are minimally exploited only to obtain "low-hanging fruit" protein structures. We review the practical aspects of today's high-throughput manipulations and discuss the challenges in fast pace protein crystallization and tools for crystallography. Structural genomic pipelines can be improved with information gained from low-throughput tactics that may help us reach the higher-bearing fruits. Examples of recent developments in this area are reported from

  13. Life in the Fast Lane for Protein Crystallization and X-Ray Crystallography

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Liu, Zhi-Jie; Tempel, Wolfram; Praissman, Jeremy; Lin, Dawei; Wang, Bi-Cheng; Gavira, Jose A.; Ng, Joseph D.

    2004-01-01

    The common goal for structural genomic centers and consortiums is to decipher as quickly as possible the three-dimensional structures for a multitude of recombinant proteins derived from known genomic sequences. Since X-ray crystallography is the foremost method to acquire atomic resolution for macromolecules, the limiting step is obtaining protein crystals that can be useful of structure determination. High-throughput methods have been developed in recent years to clone, express, purify, crystallize and determine the three-dimensional structure of a protein gene product rapidly using automated devices, commercialized kits and consolidated protocols. However, the average number of protein structures obtained for most structural genomic groups has been very low compared to the total number of proteins purified. As more entire genomic sequences are obtained for different organisms from the three kingdoms of life, only the proteins that can be crystallized and whose structures can be obtained easily are studied. Consequently, an astonishing number of genomic proteins remain unexamined. In the era of high-throughput processes, traditional methods in molecular biology, protein chemistry and crystallization are eclipsed by automation and pipeline practices. The necessity for high rate production of protein crystals and structures has prevented the usage of more intellectual strategies and creative approaches in experimental executions. Fundamental principles and personal experiences in protein chemistry and crystallization are minimally exploited only to obtain "low-hanging fruit" protein structures. We review the practical aspects of today s high-throughput manipulations and discuss the challenges in fast pace protein crystallization and tools for crystallography. Structural genomic pipelines can be improved with information gained from low-throughput tactics that may help us reach the higher-bearing fruits. Examples of recent developments in this area are reported from

  14. Life in the fast lane for protein crystallization and X-ray crystallography

    SciTech Connect

    Pusey, Marc L.; Liu, Zhi-Jie; Tempel, Wolfram; Praissman, Jeremy; Lin, Dawei; Wang, Bi-Cheng; Gavira, Jose A.; Ng, Joseph D.

    2010-07-20

    The common goal for structural genomic centers and consortiums is to decipher as quickly as possible the three-dimensional structures for a multitude of recombinant proteins derived from known genomic sequences. Since X-ray crystallography is the foremost method to acquire atomic resolution for macromolecules, the limiting step is obtaining protein crystals that can be useful of structure determination. High-throughput methods have been developed in recent years to clone, express, purify, crystallize and determine the three-dimensional structure of a protein gene product rapidly using automated devices, commercialized kits and consolidated protocols. However, the average number of protein structures obtained for most structural genomic groups has been very low compared to the total number of proteins purified. As more entire genomic sequences are obtained for different organisms from the three kingdoms of life, only the proteins that can be crystallized and whose structures can be obtained easily are studied. Consequently, an astonishing number of genomic proteins remain unexamined. In the era of high-throughput processes, traditional methods in molecular biology, protein chemistry and crystallization are eclipsed by automation and pipeline practices. The necessity for high-rate production of protein crystals and structures has prevented the usage of more intellectual strategies and creative approaches in experimental executions. Fundamental principles and personal experiences in protein chemistry and crystallization are minimally exploited only to obtain 'low-hanging fruit' protein structures. We review the practical aspects of today's high-throughput manipulations and discuss the challenges in fast pace protein crystallization and tools for crystallography. Structural genomic pipelines can be improved with information gained from low-throughput tactics that may help us reach the higher-bearing fruits. Examples of recent developments in this area are reported from

  15. In meso in situ serial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures.

    PubMed

    Huang, Chia Ying; Olieric, Vincent; Ma, Pikyee; Howe, Nicole; Vogeley, Lutz; Liu, Xiangyu; Warshamanage, Rangana; Weinert, Tobias; Panepucci, Ezequiel; Kobilka, Brian; Diederichs, Kay; Wang, Meitian; Caffrey, Martin

    2016-01-01

    Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase for in situ diffraction data collection at cryogenic temperatures is introduced. The method dispenses with the need for the technically demanding and inefficient crystal-harvesting step that is an integral part of the lipid cubic phase or in meso method of growing crystals. Crystals are dispersed in a bolus of mesophase sandwiched between thin plastic windows. The bolus contains tens to hundreds of crystals, visible with an in-line microscope at macromolecular crystallography synchrotron beamlines and suitably disposed for conventional or serial crystallographic data collection. Wells containing the crystal-laden boluses are removed individually from hermetically sealed glass plates in which crystallization occurs, affixed to pins on goniometer bases and excess precipitant is removed from around the mesophase. The wells are snap-cooled in liquid nitrogen, stored and shipped in Dewars, and manually or robotically mounted on a goniometer in a cryostream for diffraction data collection at 100 K, as is performed routinely with standard, loop-harvested crystals. The method is a variant on the recently introduced in meso in situ serial crystallography (IMISX) method that enables crystallographic measurements at cryogenic temperatures where crystal lifetimes are enormously enhanced whilst reducing protein consumption dramatically. The new approach has been used to generate high-resolution crystal structures of a G-protein-coupled receptor, α-helical and β-barrel transporters and an enzyme as model integral membrane proteins. Insulin and lysozyme were used as test soluble proteins. The quality of the data that can be generated by this method was attested to by performing sulfur and bromine SAD phasing with two of the test proteins. PMID:26894538

  16. In meso in situ serial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures

    PubMed Central

    Huang, Chia-Ying; Olieric, Vincent; Ma, Pikyee; Howe, Nicole; Vogeley, Lutz; Liu, Xiangyu; Warshamanage, Rangana; Weinert, Tobias; Panepucci, Ezequiel; Kobilka, Brian; Diederichs, Kay; Wang, Meitian; Caffrey, Martin

    2016-01-01

    Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase for in situ diffraction data collection at cryogenic temperatures is introduced. The method dispenses with the need for the technically demanding and inefficient crystal-harvesting step that is an integral part of the lipid cubic phase or in meso method of growing crystals. Crystals are dispersed in a bolus of mesophase sandwiched between thin plastic windows. The bolus contains tens to hundreds of crystals, visible with an in-line microscope at macromolecular crystallography synchrotron beamlines and suitably disposed for conventional or serial crystallographic data collection. Wells containing the crystal-laden boluses are removed individually from hermetically sealed glass plates in which crystallization occurs, affixed to pins on goniometer bases and excess precipitant is removed from around the mesophase. The wells are snap-cooled in liquid nitrogen, stored and shipped in Dewars, and manually or robotically mounted on a goniometer in a cryostream for diffraction data collection at 100 K, as is performed routinely with standard, loop-harvested crystals. The method is a variant on the recently introduced in meso in situ serial crystallography (IMISX) method that enables crystallo­graphic measurements at cryogenic temperatures where crystal lifetimes are enormously enhanced whilst reducing protein consumption dramatically. The new approach has been used to generate high-resolution crystal structures of a G-protein-coupled receptor, α-helical and β-barrel transporters and an enzyme as model integral membrane proteins. Insulin and lysozyme were used as test soluble proteins. The quality of the data that can be generated by this method was attested to by performing sulfur and bromine SAD phasing with two of the test proteins. PMID:26894538

  17. Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein

    DOE PAGESBeta

    Tenboer, Jason; Basu, Shibom; Zatsepin, Nadia; Pande, Kanupriya; Milathianaki, Despina; Frank, Matthias; Hunter, Mark; Boutet, Sebastien; Williams, Garth J.; Koglin, Jason E.; et al

    2014-12-05

    We report that serial femtosecond crystallography using ultrashort pulses from X-ray Free Electron Lasers (XFELs) offers the possibility to study light-triggered dynamics of biomolecules. Using microcrystals of the blue light photoreceptor, photoactive yellow protein, as a model system, we present high resolution, time-resolved difference electron density maps of excellent quality with strong features, which allow the determination of structures of reaction intermediates to 1.6 Å resolution. These results open the way to the study of reversible and non-reversible biological reactions on time scales as short as femtoseconds under conditions which maximize the extent of reaction initiation throughout the crystal.

  18. A Molecular Pharmacologist's Guide to G Protein-Coupled Receptor Crystallography.

    PubMed

    Piscitelli, Chayne L; Kean, James; de Graaf, Chris; Deupi, Xavier

    2015-09-01

    G protein-coupled receptor (GPCR) structural biology has progressed dramatically in the last decade. There are now over 120 GPCR crystal structures deposited in the Protein Data Bank of 32 different receptors from families scattered across the phylogenetic tree, including class B, C, and Frizzled GPCRs. These structures have been obtained in combination with a wide variety of ligands and captured in a range of conformational states. This surge in structural knowledge has enlightened research into the molecular recognition of biologically active molecules, the mechanisms of receptor activation, the dynamics of functional selectivity, and fueled structure-based drug design efforts for GPCRs. Here we summarize the innovations in both protein engineering/molecular biology and crystallography techniques that have led to these advances in GPCR structural biology and discuss how they may influence the resulting structural models. We also provide a brief molecular pharmacologist's guide to GPCR X-ray crystallography, outlining some key aspects in the process of structure determination, with the goal to encourage noncrystallographers to interrogate structures at the molecular level. Finally, we show how chemogenomics approaches can be used to marry the wealth of existing receptor pharmacology data with the expanding repertoire of structures, providing a deeper understanding of the mechanistic details of GPCR function. PMID:26152196

  19. Serial Femtosecond Crystallography and Ultrafast Absorption Spectroscopy of the Photoswitchable Fluorescent Protein IrisFP.

    PubMed

    Colletier, Jacques-Philippe; Sliwa, Michel; Gallat, François-Xavier; Sugahara, Michihiro; Guillon, Virginia; Schirò, Giorgio; Coquelle, Nicolas; Woodhouse, Joyce; Roux, Laure; Gotthard, Guillaume; Royant, Antoine; Uriarte, Lucas Martinez; Ruckebusch, Cyril; Joti, Yasumasa; Byrdin, Martin; Mizohata, Eiichi; Nango, Eriko; Tanaka, Tomoyuki; Tono, Kensuke; Yabashi, Makina; Adam, Virgile; Cammarata, Marco; Schlichting, Ilme; Bourgeois, Dominique; Weik, Martin

    2016-03-01

    Reversibly photoswitchable fluorescent proteins find growing applications in cell biology, yet mechanistic details, in particular on the ultrafast photochemical time scale, remain unknown. We employed time-resolved pump-probe absorption spectroscopy on the reversibly photoswitchable fluorescent protein IrisFP in solution to study photoswitching from the nonfluorescent (off) to the fluorescent (on) state. Evidence is provided for the existence of several intermediate states on the pico- and microsecond time scales that are attributed to chromophore isomerization and proton transfer, respectively. Kinetic modeling favors a sequential mechanism with the existence of two excited state intermediates with lifetimes of 2 and 15 ps, the second of which controls the photoswitching quantum yield. In order to support that IrisFP is suited for time-resolved experiments aiming at a structural characterization of these ps intermediates, we used serial femtosecond crystallography at an X-ray free electron laser and solved the structure of IrisFP in its on state. Sample consumption was minimized by embedding crystals in mineral grease, in which they remain photoswitchable. Our spectroscopic and structural results pave the way for time-resolved serial femtosecond crystallography aiming at characterizing the structure of ultrafast intermediates in reversibly photoswitchable fluorescent proteins. PMID:26866390

  20. Raster-scanning serial protein crystallography using micro- and nano-focused synchrotron beams.

    PubMed

    Coquelle, Nicolas; Brewster, Aaron S; Kapp, Ulrike; Shilova, Anastasya; Weinhausen, Britta; Burghammer, Manfred; Colletier, Jacques Philippe

    2015-05-01

    High-resolution structural information was obtained from lysozyme microcrystals (20 µm in the largest dimension) using raster-scanning serial protein crystallography on micro- and nano-focused beamlines at the ESRF. Data were collected at room temperature (RT) from crystals sandwiched between two silicon nitride wafers, thereby preventing their drying, while limiting background scattering and sample consumption. In order to identify crystal hits, new multi-processing and GUI-driven Python-based pre-analysis software was developed, named NanoPeakCell, that was able to read data from a variety of crystallographic image formats. Further data processing was carried out using CrystFEL, and the resultant structures were refined to 1.7 Å resolution. The data demonstrate the feasibility of RT raster-scanning serial micro- and nano-protein crystallography at synchrotrons and validate it as an alternative approach for the collection of high-resolution structural data from micro-sized crystals. Advantages of the proposed approach are its thriftiness, its handling-free nature, the reduced amount of sample required, the adjustable hit rate, the high indexing rate and the minimization of background scattering. PMID:25945583

  1. Ultrasonic acoustic levitation for fast frame rate X-ray protein crystallography at room temperature

    PubMed Central

    Tsujino, Soichiro; Tomizaki, Takashi

    2016-01-01

    Increasing the data acquisition rate of X-ray diffraction images for macromolecular crystals at room temperature at synchrotrons has the potential to significantly accelerate both structural analysis of biomolecules and structure-based drug developments. Using lysozyme model crystals, we demonstrated the rapid acquisition of X-ray diffraction datasets by combining a high frame rate pixel array detector with ultrasonic acoustic levitation of protein crystals in liquid droplets. The rapid spinning of the crystal within a levitating droplet ensured an efficient sampling of the reciprocal space. The datasets were processed with a program suite developed for serial femtosecond crystallography (SFX). The structure, which was solved by molecular replacement, was found to be identical to the structure obtained by the conventional oscillation method for up to a 1.8-Å resolution limit. In particular, the absence of protein crystal damage resulting from the acoustic levitation was carefully established. These results represent a key step towards a fully automated sample handling and measurement pipeline, which has promising prospects for a high acquisition rate and high sample efficiency for room temperature X-ray crystallography. PMID:27150272

  2. Raster-scanning serial protein crystallography using micro- and nano-focused synchrotron beams

    PubMed Central

    Coquelle, Nicolas; Brewster, Aaron S.; Kapp, Ulrike; Shilova, Anastasya; Weinhausen, Britta; Burghammer, Manfred; Colletier, Jacques-Philippe

    2015-01-01

    High-resolution structural information was obtained from lysozyme microcrystals (20 µm in the largest dimension) using raster-scanning serial protein crystallography on micro- and nano-focused beamlines at the ESRF. Data were collected at room temperature (RT) from crystals sandwiched between two silicon nitride wafers, thereby preventing their drying, while limiting background scattering and sample consumption. In order to identify crystal hits, new multi-processing and GUI-driven Python-based pre-analysis software was developed, named NanoPeakCell, that was able to read data from a variety of crystallographic image formats. Further data processing was carried out using CrystFEL, and the resultant structures were refined to 1.7 Å resolution. The data demonstrate the feasibility of RT raster-scanning serial micro- and nano-protein crystallography at synchrotrons and validate it as an alternative approach for the collection of high-resolution structural data from micro-sized crystals. Advantages of the proposed approach are its thriftiness, its handling-free nature, the reduced amount of sample required, the adjustable hit rate, the high indexing rate and the minimization of background scattering. PMID:25945583

  3. Raster-scanning serial protein crystallography using micro- and nano-focused synchrotron beams

    DOE PAGESBeta

    Coquelle, Nicolas; Brewster, Aaron S.; Kapp, Ulrike; Shilova, Anastasya; Weinhausen, Britta; Burghammer, Manfred; Colletier, Jacques -Philippe

    2015-04-25

    High-resolution structural information was obtained from lysozyme microcrystals (20 µm in the largest dimension) using raster-scanning serial protein crystallography on micro- and nano-focused beamlines at the ESRF. Data were collected at room temperature (RT) from crystals sandwiched between two silicon nitride wafers, thereby preventing their drying, while limiting background scattering and sample consumption. In order to identify crystal hits, new multi-processing and GUI-driven Python-based pre-analysis software was developed, named NanoPeakCell, that was able to read data from a variety of crystallographic image formats. Further data processing was carried out using CrystFEL, and the resultant structures were refined to 1.7 Åmore » resolution. The data demonstrate the feasibility of RT raster-scanning serial micro- and nano-protein crystallography at synchrotrons and validate it as an alternative approach for the collection of high-resolution structural data from micro-sized crystals. Advantages of the proposed approach are its thriftiness, its handling-free nature, the reduced amount of sample required, the adjustable hit rate, the high indexing rate and the minimization of background scattering.« less

  4. Raster-scanning serial protein crystallography using micro- and nano-focused synchrotron beams

    SciTech Connect

    Coquelle, Nicolas; Brewster, Aaron S.; Kapp, Ulrike; Shilova, Anastasya; Weinhausen, Britta; Burghammer, Manfred; Colletier, Jacques -Philippe

    2015-04-25

    High-resolution structural information was obtained from lysozyme microcrystals (20 µm in the largest dimension) using raster-scanning serial protein crystallography on micro- and nano-focused beamlines at the ESRF. Data were collected at room temperature (RT) from crystals sandwiched between two silicon nitride wafers, thereby preventing their drying, while limiting background scattering and sample consumption. In order to identify crystal hits, new multi-processing and GUI-driven Python-based pre-analysis software was developed, named NanoPeakCell, that was able to read data from a variety of crystallographic image formats. Further data processing was carried out using CrystFEL, and the resultant structures were refined to 1.7 Å resolution. The data demonstrate the feasibility of RT raster-scanning serial micro- and nano-protein crystallography at synchrotrons and validate it as an alternative approach for the collection of high-resolution structural data from micro-sized crystals. Advantages of the proposed approach are its thriftiness, its handling-free nature, the reduced amount of sample required, the adjustable hit rate, the high indexing rate and the minimization of background scattering.

  5. Ultrasonic acoustic levitation for fast frame rate X-ray protein crystallography at room temperature.

    PubMed

    Tsujino, Soichiro; Tomizaki, Takashi

    2016-01-01

    Increasing the data acquisition rate of X-ray diffraction images for macromolecular crystals at room temperature at synchrotrons has the potential to significantly accelerate both structural analysis of biomolecules and structure-based drug developments. Using lysozyme model crystals, we demonstrated the rapid acquisition of X-ray diffraction datasets by combining a high frame rate pixel array detector with ultrasonic acoustic levitation of protein crystals in liquid droplets. The rapid spinning of the crystal within a levitating droplet ensured an efficient sampling of the reciprocal space. The datasets were processed with a program suite developed for serial femtosecond crystallography (SFX). The structure, which was solved by molecular replacement, was found to be identical to the structure obtained by the conventional oscillation method for up to a 1.8-Å resolution limit. In particular, the absence of protein crystal damage resulting from the acoustic levitation was carefully established. These results represent a key step towards a fully automated sample handling and measurement pipeline, which has promising prospects for a high acquisition rate and high sample efficiency for room temperature X-ray crystallography. PMID:27150272

  6. X-ray Structure of Native Scorpion Toxin BmBKTx1 by Racemic Protein Crystallography Using Direct Methods

    SciTech Connect

    Mandal, Kalyaneswar; Pentelute, Brad L.; Tereshko, Valentina; Kossiakoff, Anthony A.; Kent, Stephen B.H.

    2009-04-08

    Racemic protein crystallography, enabled by total chemical synthesis, has allowed us to determine the X-ray structure of native scorpion toxin BmBKTx1; direct methods were used for phase determination. This is the first example of a protein racemate that crystallized in space group I41/a.

  7. pH Dependence of the Photoactive Yellow Protein Photocycle Investigated by Time-Resolved Crystallography

    SciTech Connect

    Tripathi, Shailesh; Šrajer, Vukica; Purwar, Namrta; Henning, Robert; Schmidt, Marius

    2012-05-24

    Visualizing the three-dimensional structures of a protein during its biological activity is key to understanding its mechanism. In general, protein structure and function are pH-dependent. Changing the pH provides new insights into the mechanisms that are involved in protein activity. Photoactive yellow protein (PYP) is a signaling protein that serves as an ideal model for time-dependent studies on light-activated proteins. Its photocycle is studied extensively under different pH conditions. However, the structures of the intermediates remain unknown until time-resolved crystallography is employed. With the newest beamline developments, a comprehensive time series of Laue data can now be collected from a single protein crystal. This allows us to vary the pH. Here we present the first structure, to our knowledge, of a short-lived protein-inhibitor complex formed in the pB state of the PYP photocycle at pH 4. A water molecule that is transiently stabilized in the chromophore active site prevents the relaxation of the chromophore back to the trans configuration. As a result, the dark-state recovery is slowed down dramatically. At pH 9, PYP stops cycling through the pB state altogether. The electrostatic environment in the chromophore-binding site is the likely reason for this altered kinetics at different pH values.

  8. pH Dependence of the Photoactive Yellow Protein Photocycle Investigated by Time-Resolved Crystallography

    PubMed Central

    Tripathi, Shailesh; Šrajer, Vukica; Purwar, Namrta; Henning, Robert; Schmidt, Marius

    2012-01-01

    Visualizing the three-dimensional structures of a protein during its biological activity is key to understanding its mechanism. In general, protein structure and function are pH-dependent. Changing the pH provides new insights into the mechanisms that are involved in protein activity. Photoactive yellow protein (PYP) is a signaling protein that serves as an ideal model for time-dependent studies on light-activated proteins. Its photocycle is studied extensively under different pH conditions. However, the structures of the intermediates remain unknown until time-resolved crystallography is employed. With the newest beamline developments, a comprehensive time series of Laue data can now be collected from a single protein crystal. This allows us to vary the pH. Here we present the first structure, to our knowledge, of a short-lived protein-inhibitor complex formed in the pB state of the PYP photocycle at pH 4. A water molecule that is transiently stabilized in the chromophore active site prevents the relaxation of the chromophore back to the trans configuration. As a result, the dark-state recovery is slowed down dramatically. At pH 9, PYP stops cycling through the pB state altogether. The electrostatic environment in the chromophore-binding site is the likely reason for this altered kinetics at different pH values. PMID:22339869

  9. Two-Dimensional Crystallization of Integral Membrane Proteins for Electron Crystallography

    PubMed Central

    Stokes, David L.; Rice, William J.; Hu, Minghui; Kim, Changki; Ubarretxena, Iban

    2011-01-01

    Although membrane proteins make up 30% of the proteome and are a common target for therapeutic drugs, determination of their atomic structure remains a technical challenge. Electron crystallography represents an alternative to the conventional methods of X-ray diffraction and NMR and relies on the formation of two-dimensional crystals. These crystals are produced by reconstituting purified, detergent-solubilized membrane proteins back into the native environment of a lipid bilayer. This chapter reviews methods for producing two-dimensional crystals and for screening them by negative stain electron microscopy. In addition, we show examples of the different morphologies that are commonly obtained and describe basic image analysis procedures that can be used to evaluate their promise for structure determination by cryoelectron microsopy. PMID:20665267

  10. Electron crystallography of PhoE porin, an outer membrane, channel- forming protein from E. coli

    SciTech Connect

    Walian, P.J.

    1989-11-01

    One approach to studying the structure of membrane proteins is the use of electron crystallography. Dr. Bing Jap has crystallized PhoE pore-forming protein (porin) from the outer membrane of escherichia coli (E. coli) into monolayer crystals. The findings of this research and those of Jap (1988, 1989) have determined these crystals to be highly ordered, yielding structural information to a resolution of better than 2.8 angstroms. The task of this thesis has been to collect and process the electron diffraction patterns necessary to generate a complete three-dimensional set of high resolution structure factor amplitudes of PhoE porin. Fourier processing of these amplitudes when combined with the corresponding phase data is expected to yield the three-dimensional structure of PhoE porin at better than 3.5 angstroms resolution. 92 refs., 33 figs., 3 tabs. (CBS)

  11. A split-beam probe-pump-probe scheme for femtosecond time resolved protein X-ray crystallography.

    PubMed

    van Thor, Jasper J; Madsen, Anders

    2015-01-01

    In order to exploit the femtosecond pulse duration of X-ray Free-Electron Lasers (XFEL) operating in the hard X-ray regime for ultrafast time-resolved protein crystallography experiments, critical parameters that determine the crystallographic signal-to-noise (I/σI) must be addressed. For single-crystal studies under low absorbed dose conditions, it has been shown that the intrinsic pulse intensity stability as well as mode structure and jitter of this structure, significantly affect the crystallographic signal-to-noise. Here, geometrical parameters are theoretically explored for a three-beam scheme: X-ray probe, optical pump, X-ray probe (or "probe-pump-probe") which will allow experimental determination of the photo-induced structure factor amplitude differences, ΔF, in a ratiometric manner, thereby internally referencing the intensity noise of the XFEL source. In addition to a non-collinear split-beam geometry which separates un-pumped and pumped diffraction patterns on an area detector, applying an additional convergence angle to both beams by focusing leads to integration over mosaic blocks in the case of well-ordered stationary protein crystals. Ray-tracing X-ray diffraction simulations are performed for an example using photoactive yellow protein crystals in order to explore the geometrical design parameters which would be needed. The specifications for an X-ray split and delay instrument that implements both an offset angle and focused beams are discussed, for implementation of a probe-pump-probe scheme at the European XFEL. We discuss possible extension of single crystal studies to serial femtosecond crystallography, particularly in view of the expected X-ray damage and ablation due to the first probe pulse. PMID:26798786

  12. A split-beam probe-pump-probe scheme for femtosecond time resolved protein X-ray crystallography

    DOE PAGESBeta

    van Thor, Jasper J.; Madsen, Anders

    2015-01-01

    In order to exploit the femtosecond pulse duration of X-ray Free-Electron Lasers (XFEL) operating in the hard X-ray regime for ultrafast time-resolved protein crystallography experiments, critical parameters that determine the crystallographic signal-to-noise (I/σI) must be addressed. For single-crystal studies under low absorbed dose conditions, it has been shown that the intrinsic pulse intensity stability as well as mode structure and jitter of this structure, significantly affect the crystallographic signal-to-noise. Here, geometrical parameters are theoretically explored for a three-beam scheme: X-ray probe, optical pump, X-ray probe (or “probe-pump-probe”) which will allow experimental determination of the photo-induced structure factor amplitude differences, ΔF,more » in a ratiometric manner, thereby internally referencing the intensity noise of the XFEL source. In addition to a non-collinear split-beam geometry which separates un-pumped and pumped diffraction patterns on an area detector, applying an additional convergence angle to both beams by focusing leads to integration over mosaic blocks in the case of well-ordered stationary protein crystals. Ray-tracing X-ray diffraction simulations are performed for an example using photoactive yellow protein crystals in order to explore the geometrical design parameters which would be needed. The specifications for an X-ray split and delay instrument that implements both an offset angle and focused beams are discussed, for implementation of a probe-pump-probe scheme at the European XFEL. We discuss possible extension of single crystal studies to serial femtosecond crystallography, particularly in view of the expected X-ray damage and ablation due to the first probe pulse.« less

  13. A split-beam probe-pump-probe scheme for femtosecond time resolved protein X-ray crystallography

    SciTech Connect

    van Thor, Jasper J.; Madsen, Anders

    2015-01-01

    In order to exploit the femtosecond pulse duration of X-ray Free-Electron Lasers (XFEL) operating in the hard X-ray regime for ultrafast time-resolved protein crystallography experiments, critical parameters that determine the crystallographic signal-to-noise (I/σI) must be addressed. For single-crystal studies under low absorbed dose conditions, it has been shown that the intrinsic pulse intensity stability as well as mode structure and jitter of this structure, significantly affect the crystallographic signal-to-noise. Here, geometrical parameters are theoretically explored for a three-beam scheme: X-ray probe, optical pump, X-ray probe (or “probe-pump-probe”) which will allow experimental determination of the photo-induced structure factor amplitude differences, ΔF, in a ratiometric manner, thereby internally referencing the intensity noise of the XFEL source. In addition to a non-collinear split-beam geometry which separates un-pumped and pumped diffraction patterns on an area detector, applying an additional convergence angle to both beams by focusing leads to integration over mosaic blocks in the case of well-ordered stationary protein crystals. Ray-tracing X-ray diffraction simulations are performed for an example using photoactive yellow protein crystals in order to explore the geometrical design parameters which would be needed. The specifications for an X-ray split and delay instrument that implements both an offset angle and focused beams are discussed, for implementation of a probe-pump-probe scheme at the European XFEL. We discuss possible extension of single crystal studies to serial femtosecond crystallography, particularly in view of the expected X-ray damage and ablation due to the first probe pulse.

  14. A split-beam probe-pump-probe scheme for femtosecond time resolved protein X-ray crystallography

    PubMed Central

    van Thor, Jasper J.; Madsen, Anders

    2015-01-01

    In order to exploit the femtosecond pulse duration of X-ray Free-Electron Lasers (XFEL) operating in the hard X-ray regime for ultrafast time-resolved protein crystallography experiments, critical parameters that determine the crystallographic signal-to-noise (I/σI) must be addressed. For single-crystal studies under low absorbed dose conditions, it has been shown that the intrinsic pulse intensity stability as well as mode structure and jitter of this structure, significantly affect the crystallographic signal-to-noise. Here, geometrical parameters are theoretically explored for a three-beam scheme: X-ray probe, optical pump, X-ray probe (or “probe-pump-probe”) which will allow experimental determination of the photo-induced structure factor amplitude differences, ΔF, in a ratiometric manner, thereby internally referencing the intensity noise of the XFEL source. In addition to a non-collinear split-beam geometry which separates un-pumped and pumped diffraction patterns on an area detector, applying an additional convergence angle to both beams by focusing leads to integration over mosaic blocks in the case of well-ordered stationary protein crystals. Ray-tracing X-ray diffraction simulations are performed for an example using photoactive yellow protein crystals in order to explore the geometrical design parameters which would be needed. The specifications for an X-ray split and delay instrument that implements both an offset angle and focused beams are discussed, for implementation of a probe-pump-probe scheme at the European XFEL. We discuss possible extension of single crystal studies to serial femtosecond crystallography, particularly in view of the expected X-ray damage and ablation due to the first probe pulse. PMID:26798786

  15. The use of polyoxometalates in protein crystallography – An attempt to widen a well-known bottleneck

    PubMed Central

    Bijelic, Aleksandar; Rompel, Annette

    2015-01-01

    Polyoxometalates (POMs) are discrete polynuclear metal-oxo anions with a fascinating variety of structures and unique chemical and physical properties. Their application in various fields is well covered in the literature, however little information about their usage in protein crystallization is available. This review summarizes the impact of the vast class of POMs on the formation of protein crystals, a well-known (frustrating) bottleneck in macromolecular crystallography, with the associated structure elucidation and a particular emphasis focused on POM's potential as a powerful crystallization additive for future research. The Protein Data Bank (PDB) was scanned for protein structures with incorporated POMs which were assigned a PDB ligand ID resulting in 30 PDB entries. These structures have been analyzed with regard to (i) the structure of POM itself in the immediate protein environment, (ii) the kind of interaction and position of the POM within the protein structure and (iii) the beneficial effects of POM on protein crystallography apparent so far. PMID:26339074

  16. Integrated description of protein dynamics from room-temperature X-ray crystallography and NMR

    PubMed Central

    Fenwick, R. Bryn; van den Bedem, Henry; Fraser, James S.; Wright, Peter E.

    2014-01-01

    Detailed descriptions of atomic coordinates and motions are required for an understanding of protein dynamics and their relation to molecular recognition, catalytic function, and allostery. Historically, NMR relaxation measurements have played a dominant role in the determination of the amplitudes and timescales (picosecond–nanosecond) of bond vector fluctuations, whereas high-resolution X-ray diffraction experiments can reveal the presence of and provide atomic coordinates for multiple, weakly populated substates in the protein conformational ensemble. Here we report a hybrid NMR and X-ray crystallography analysis that provides a more complete dynamic picture and a more quantitative description of the timescale and amplitude of fluctuations in atomic coordinates than is obtainable from the individual methods alone. Order parameters (S2) were calculated from single-conformer and multiconformer models fitted to room temperature and cryogenic X-ray diffraction data for dihydrofolate reductase. Backbone and side-chain order parameters derived from NMR relaxation experiments are in excellent agreement with those calculated from the room-temperature single-conformer and multiconformer models, showing that the picosecond timescale motions observed in solution occur also in the crystalline state. These motions are quenched in the crystal at cryogenic temperatures. The combination of NMR and X-ray crystallography in iterative refinement promises to provide an atomic resolution description of the alternate conformational substates that are sampled through picosecond to nanosecond timescale fluctuations of the protein structure. The method also provides insights into the structural heterogeneity of nonmethyl side chains, aromatic residues, and ligands, which are less commonly analyzed by NMR relaxation measurements. PMID:24474795

  17. Protein crystallography: From X-ray diffraction spots to a three dimensional image

    SciTech Connect

    Terwilliger, T.C.; Berendzen, J.

    1998-02-25

    Proteins are remarkable molecular machines that are essential for life. They can do many things ranging from the precise control of blood clotting to synthesizing complex organic compounds. Pictures of protein molecules are in high demand in biotechnology because they are important for applications such as drug discovery and for engineering enzymes for commercial use. X-ray crystallography is the most common method for determining the three-dimensional structures of protein molecules. When a crystal of a protein is placed in an X-ray beam, scattering of X-rays off the ordered molecules produces a diffraction pattern that can be measured on a position-sensitive CCD or image-plate detector. Protein crystals typically contain thousands of atoms and the diffraction data are generally measured to relatively low resolution. Consequently the direct methods approaches generally cannot be applied. Instead, if the crystal is modified by adding metal atoms at specific sites or by tuning the wavelength of the X-rays to cross an absorption edge of a metal atom in the crystal, then the information from these additional measurements is sufficient to first identify the /locations of the metal atoms. This information is then used along with the diffraction data to make a three-dimensional picture of electron densities. This picture can be used to determine the position of most or all of the atoms in the protein.

  18. Precise Manipulation and Patterning of Protein Crystals for Macromolecular Crystallography using Surface Acoustic Waves

    PubMed Central

    Guo, Feng; Zhou, Weijie; Li, Peng; Mao, Zhangming; Yennawar, Neela; French, Jarrod B.; Jun Huang, Tony

    2015-01-01

    Advances in modern X-ray sources and detector technology have made it possible for crystallographers to collect usable data on crystals of only a few micrometers or less in size. Despite these developments, sample handling techniques have significantly lagged behind and often prevent the full realization of current beamline capabilities. In order to address this shortcoming we have developed a surface acoustic wave-based method for manipulating and patterning crystals. This method, which does not damage the fragile protein crystals, can precisely manipulate and pattern micrometer and sub-micrometer sized crystals for data collection and screening. The technique is robust, inexpensive, and easy to implement. This method not only promises to significantly increase efficiency and throughput of both conventional and serial crystallography experiments, but also will make it possible to collect data on samples that were previously intractable. PMID:25641793

  19. Watching proteins function with 150-ps time-resolved X-ray crystallography

    NASA Astrophysics Data System (ADS)

    Anfinrud, Philip

    2007-03-01

    We have used time-resolved Laue crystallography to characterize ligand migration pathways and dynamics in wild-type and several mutant forms of myoglobin (Mb), a ligand-binding heme protein found in muscle tissue. In these pump-probe experiments, which were conducted on the ID09B time-resolved beamline at the European Synchrotron and Radiation Facility, a laser pulse photodissociates CO from an MbCO crystal and a suitably delayed X-ray pulse probes its structure via Laue diffraction. Single-site mutations in the vicinity of the heme pocket docking site were found to have a dramatic effect on ligand migration. To visualize this process, time-resolved electron density maps were stitched together into movies that unveil with <2-å spatial resolution and 150-ps time-resolution the correlated protein motions that accompany and/or mediate ligand migration. These studies help to illustrate at an atomic level relationships between protein structure, dynamics, and function.

  20. Nanoflow electrospinning serial femtosecond crystallography.

    PubMed

    Sierra, Raymond G; Laksmono, Hartawan; Kern, Jan; Tran, Rosalie; Hattne, Johan; Alonso-Mori, Roberto; Lassalle-Kaiser, Benedikt; Glöckner, Carina; Hellmich, Julia; Schafer, Donald W; Echols, Nathaniel; Gildea, Richard J; Grosse-Kunstleve, Ralf W; Sellberg, Jonas; McQueen, Trevor A; Fry, Alan R; Messerschmidt, Marc M; Miahnahri, Alan; Seibert, M Marvin; Hampton, Christina Y; Starodub, Dmitri; Loh, N Duane; Sokaras, Dimosthenis; Weng, Tsu-Chien; Zwart, Petrus H; Glatzel, Pieter; Milathianaki, Despina; White, William E; Adams, Paul D; Williams, Garth J; Boutet, Sébastien; Zouni, Athina; Messinger, Johannes; Sauter, Nicholas K; Bergmann, Uwe; Yano, Junko; Yachandra, Vittal K; Bogan, Michael J

    2012-11-01

    An electrospun liquid microjet has been developed that delivers protein microcrystal suspensions at flow rates of 0.14-3.1 µl min(-1) to perform serial femtosecond crystallography (SFX) studies with X-ray lasers. Thermolysin microcrystals flowed at 0.17 µl min(-1) and diffracted to beyond 4 Å resolution, producing 14,000 indexable diffraction patterns, or four per second, from 140 µg of protein. Nanoflow electrospinning extends SFX to biological samples that necessitate minimal sample consumption. PMID:23090408

  1. Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography

    SciTech Connect

    Schotte, Friedrich; Cho, Hyun Sun; Kaila, Ville R.I.; Kamikubo, Hironari; Dashdorj, Naranbaatar; Henry, Eric R.; Graber, Timothy J.; Henning, Robert; Wulff, Michael; Hummer, Gerhard; Kataoka, Mikio; Anfinrud, Philip A.

    2012-11-06

    To understand how signaling proteins function, it is necessary to know the time-ordered sequence of events that lead to the signaling state. We recently developed on the BioCARS 14-IDB beamline at the Advanced Photon Source the infrastructure required to characterize structural changes in protein crystals with near-atomic spatial resolution and 150-ps time resolution, and have used this capability to track the reversible photocycle of photoactive yellow protein (PYP) following trans-to-cis photoisomerization of its p-coumaric acid (pCA) chromophore over 10 decades of time. The first of four major intermediates characterized in this study is highly contorted, with the pCA carbonyl rotated nearly 90° out of the plane of the phenolate. A hydrogen bond between the pCA carbonyl and the Cys69 backbone constrains the chromophore in this unusual twisted conformation. Density functional theory calculations confirm that this structure is chemically plausible and corresponds to a strained cis intermediate. This unique structure is short-lived (~600 ps), has not been observed in prior cryocrystallography experiments, and is the progenitor of intermediates characterized in previous nanosecond time-resolved Laue crystallography studies. The structural transitions unveiled during the PYP photocycle include trans/cis isomerization, the breaking and making of hydrogen bonds, formation/relaxation of strain, and gated water penetration into the interior of the protein. This mechanistically detailed, near-atomic resolution description of the complete PYP photocycle provides a framework for understanding signal transduction in proteins, and for assessing and validating theoretical/computational approaches in protein biophysics.

  2. Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography

    PubMed Central

    Schotte, Friedrich; Cho, Hyun Sun; Kaila, Ville R. I.; Kamikubo, Hironari; Dashdorj, Naranbaatar; Henry, Eric R.; Graber, Timothy J.; Henning, Robert; Wulff, Michael; Hummer, Gerhard; Kataoka, Mikio; Anfinrud, Philip A.

    2012-01-01

    To understand how signaling proteins function, it is crucial to know the time-ordered sequence of events that lead to the signaling state. We recently developed on the BioCARS 14-IDB beamline at the Advanced Photon Source the infrastructure required to characterize structural changes in protein crystals with near-atomic spatial resolution and 150-ps time resolution, and have used this capability to track the reversible photocycle of photoactive yellow protein (PYP) following trans-to-cis photoisomerization of its p-coumaric acid (pCA) chromophore over 10 decades of time. The first of four major intermediates characterized in this study is highly contorted, with the pCA carbonyl rotated nearly 90° out of the plane of the phenolate. A hydrogen bond between the pCA carbonyl and the Cys69 backbone constrains the chromophore in this unusual twisted conformation. Density functional theory calculations confirm that this structure is chemically plausible and corresponds to a strained cis intermediate. This unique structure is short-lived (∼600 ps), has not been observed in prior cryocrystallography experiments, and is the progenitor of intermediates characterized in previous nanosecond time-resolved Laue crystallography studies. The structural transitions unveiled during the PYP photocycle include trans/cis isomerization, the breaking and making of hydrogen bonds, formation/relaxation of strain, and gated water penetration into the interior of the protein. This mechanistically detailed, near-atomic resolution description of the complete PYP photocycle provides a framework for understanding signal transduction in proteins, and for assessing and validating theoretical/computational approaches in protein biophysics. PMID:23132943

  3. Protein kinase A catalytic subunit primed for action: Time-lapse crystallography of Michaelis complex formation

    SciTech Connect

    Das, Amit; Gerlits, Oksana O.; Parks, Jerry M.; Langan, Paul; Kovalevskyi, Andrey Y.; Heller, William T.

    2015-11-12

    The catalytic subunit of the cyclic AMP-dependent protein kinase A (PKAc) catalyzes the transfer of the γ-phosphate of bound Mg2ATP to a serine or threonine residue of a protein substrate. Here, time-lapse X-ray crystallography was used to capture a series of complexes of PKAc with an oligopeptide substrate and unreacted Mg2ATP, including the Michaelis complex, that reveal important geometric rearrangements in and near the active site preceding the phosphoryl transfer reaction. Contrary to the prevailing view, Mg2+ binds first to the M1 site as a complex with ATP and is followed by Mg2+ binding to the M2 site. Furthermore, the target serine hydroxyl of the peptide substrate rotates away from the active site toward the bulk solvent, which breaks the hydrogen bond with D166. In conclusion, the serine hydroxyl of the substrate rotates back toward D166 to form the Michaelis complex with the active site primed for phosphoryl transfer.

  4. Electron crystallography of the scrapie prion protein complexed with heavy metals

    PubMed Central

    Wille, Holger; Govaerts, Cédric; Borovinskiy, Alexander; Latawiec, Diane; Downing, Kenneth H.; Cohen, Fred E.; Prusiner, Stanley B.

    2012-01-01

    The insolubility of the disease-causing isoform of the prion protein (PrPSc) has prevented studies of its three-dimensional structure at atomic resolution. Electron crystallography of two-dimensional crystals of N-terminally truncated PrPSc (PrP 27–30) and a miniprion (PrPSc106) provided the first insights at intermediate resolution on the molecular architecture of the prion. Here, we report on the structure of PrP 27–30 and PrPSc106 negatively stained with heavy metals. The interactions of the heavy metals with the crystal lattice were governed by tertiary and quaternary structural elements of the protein as well as the charge and size of the heavy metal salts. Staining with molybdate anions revealed three prominent densities near the center of the trimer that forms the unit cell, coinciding with the location of the β-helix that was proposed for the structure of PrPSc. Differential staining also confirmed the location of the internal deletion of PrPSc106 at or near these densities. PMID:17935686

  5. Protein kinase A catalytic subunit primed for action: Time-lapse crystallography of Michaelis complex formation

    DOE PAGESBeta

    Das, Amit; Gerlits, Oksana O.; Parks, Jerry M.; Langan, Paul; Kovalevskyi, Andrey Y.; Heller, William T.

    2015-11-12

    The catalytic subunit of the cyclic AMP-dependent protein kinase A (PKAc) catalyzes the transfer of the γ-phosphate of bound Mg2ATP to a serine or threonine residue of a protein substrate. Here, time-lapse X-ray crystallography was used to capture a series of complexes of PKAc with an oligopeptide substrate and unreacted Mg2ATP, including the Michaelis complex, that reveal important geometric rearrangements in and near the active site preceding the phosphoryl transfer reaction. Contrary to the prevailing view, Mg2+ binds first to the M1 site as a complex with ATP and is followed by Mg2+ binding to the M2 site. Furthermore, themore » target serine hydroxyl of the peptide substrate rotates away from the active site toward the bulk solvent, which breaks the hydrogen bond with D166. In conclusion, the serine hydroxyl of the substrate rotates back toward D166 to form the Michaelis complex with the active site primed for phosphoryl transfer.« less

  6. Protein Kinase A Catalytic Subunit Primed for Action: Time-Lapse Crystallography of Michaelis Complex Formation.

    PubMed

    Das, Amit; Gerlits, Oksana; Parks, Jerry M; Langan, Paul; Kovalevsky, Andrey; Heller, William T

    2015-12-01

    The catalytic subunit of the cyclic AMP-dependent protein kinase A (PKAc) catalyzes the transfer of the γ-phosphate of bound Mg2ATP to a serine or threonine residue of a protein substrate. Here, time-lapse X-ray crystallography was used to capture a series of complexes of PKAc with an oligopeptide substrate and unreacted Mg2ATP, including the Michaelis complex, that reveal important geometric rearrangements in and near the active site preceding the phosphoryl transfer reaction. Contrary to the prevailing view, Mg(2+) binds first to the M1 site as a complex with ATP and is followed by Mg(2+) binding to the M2 site. Concurrently, the target serine hydroxyl of the peptide substrate rotates away from the active site toward the bulk solvent, which breaks the hydrogen bond with D166. Lastly, the serine hydroxyl of the substrate rotates back toward D166 to form the Michaelis complex with the active site primed for phosphoryl transfer. PMID:26585512

  7. IMAGINE: first neutron protein structure and new capabilities for neutron macromolecular crystallography

    SciTech Connect

    Munshi, Parthapratim; Myles, Dean A A; Robertson, Lee; Stoica, Alexandru Dan; Crow, Lowell; Kovalevskyi, Andrii Y; Koritsanszky, Tibor S; Chakoumakos, Bryan C; Blessing, Robert; Meilleur, Flora

    2013-01-01

    We report the first high resolution neutron protein structure of perdeuterated rubredoxin from Pyrococcus furiosus (PfRd) determined using the new IMAGINE macromolecular neutron crystallography instrument at the Oak Ridge National Laboratory. Neutron diffraction data extending to 1.65 resolution were collected from a relatively small 0.7 mm3 PfRd crystal using 2.5 days (60 h) of beam time. The refined structure contains 371 out of 391, or 95%, of the deuterium atoms of the protein, and 58 solvent molecules. The IMAGINE instrument is designed to provide neutron data at or near atomic resolutions (1.5 ) from crystals with volume < 1.0 mm3 and with unit cell edges < 100 . Beam line features include elliptical focusing mirrors that deliver 3x107 n s-1 cm-2 into a 3.5 x 2.0 mm2 focal spot at the sample position, and variable short and long wavelength cutoff optics that provide automated exchange between multiple wavelength configurations ( min=2.0 , 2.8 , 3.3 - max =3.0 , 4.0 , 4.5 , ~20 ). Notably, the crystal used to collect this PfRd data is 5-10 times smaller than has been previously reported.

  8. Protein crystallography beamline (PX-BL21) at Indus-2 synchrotron.

    PubMed

    Kumar, Ashwani; Ghosh, Biplab; Poswal, H K; Pandey, K K; Hosur, M V; Dwivedi, Abhilash; Makde, Ravindra D; Sharma, Surinder M

    2016-03-01

    The protein crystallography beamline (PX-BL21), installed at the 1.5 T bending-magnet port at the Indian synchrotron (Indus-2), is now available to users. The beamline can be used for X-ray diffraction measurements on a single crystal of macromolecules such as proteins, nucleic acids and their complexes. PX-BL21 has a working energy range of 5-20 keV for accessing the absorption edges of heavy elements commonly used for phasing. A double-crystal monochromator [Si(111) and Si(220)] and a pair of rhodium-coated X-ray mirrors are used for beam monochromatization and manipulation, respectively. This beamline is equipped with a single-axis goniometer, Rayonix MX225 CCD detector, fluorescence detector, cryogenic sample cooler and automated sample changer. Additional user facilities include a workstation for on-site data processing and a biochemistry laboratory for sample preparation. In this article the beamline, other facilities and some recent scientific results are briefly described. PMID:26917153

  9. Nanoflow electrospinning serial femtosecond crystallography

    SciTech Connect

    Sierra, Raymond G.; Laksmono, Hartawan; Kern, Jan; Tran, Rosalie; Hattne, Johan; Alonso-Mori, Roberto; Lassalle-Kaiser, Benedikt; Glöckner, Carina; Hellmich, Julia; Schafer, Donald W.; Echols, Nathaniel; Gildea, Richard J.; Grosse-Kunstleve, Ralf W.; Sellberg, Jonas; McQueen, Trevor A.; Fry, Alan R.; Messerschmidt, Marc M.; Miahnahri, Alan; Seibert, M. Marvin; Hampton, Christina Y.; Starodub, Dmitri; Loh, N. Duane; Sokaras, Dimosthenis; Weng, Tsu-Chien; Zwart, Petrus H.; Glatzel, Pieter; Milathianaki, Despina; White, William E.; Adams, Paul D.; Williams, Garth J.; Boutet, Sébastien; Zouni, Athina; Messinger, Johannes; Sauter, Nicholas K.; Bergmann, Uwe; Yano, Junko; Yachandra, Vittal K.; Bogan, Michael J.

    2012-11-01

    A low flow rate liquid microjet method for delivery of hydrated protein crystals to X-ray lasers is presented. Linac Coherent Light Source data demonstrates serial femtosecond protein crystallography with micrograms, a reduction of sample consumption by orders of magnitude. An electrospun liquid microjet has been developed that delivers protein microcrystal suspensions at flow rates of 0.14–3.1 µl min{sup −1} to perform serial femtosecond crystallography (SFX) studies with X-ray lasers. Thermolysin microcrystals flowed at 0.17 µl min{sup −1} and diffracted to beyond 4 Å resolution, producing 14 000 indexable diffraction patterns, or four per second, from 140 µg of protein. Nanoflow electrospinning extends SFX to biological samples that necessitate minimal sample consumption.

  10. Hydrogens detected by subatomic resolution protein crystallography in a [NiFe] hydrogenase.

    PubMed

    Ogata, Hideaki; Nishikawa, Koji; Lubitz, Wolfgang

    2015-04-23

    The enzyme hydrogenase reversibly converts dihydrogen to protons and electrons at a metal catalyst. The location of the abundant hydrogens is of key importance for understanding structure and function of the protein. However, in protein X-ray crystallography the detection of hydrogen atoms is one of the major problems, since they display only weak contributions to diffraction and the quality of the single crystals is often insufficient to obtain sub-ångström resolution. Here we report the crystal structure of a standard [NiFe] hydrogenase (∼91.3 kDa molecular mass) at 0.89 Å resolution. The strictly anoxically isolated hydrogenase has been obtained in a specific spectroscopic state, the active reduced Ni-R (subform Ni-R1) state. The high resolution, proper refinement strategy and careful modelling allow the positioning of a large part of the hydrogen atoms in the structure. This has led to the direct detection of the products of the heterolytic splitting of dihydrogen into a hydride (H(-)) bridging the Ni and Fe and a proton (H(+)) attached to the sulphur of a cysteine ligand. The Ni-H(-) and Fe-H(-) bond lengths are 1.58 Å and 1.78Å, respectively. Furthermore, we can assign the Fe-CO and Fe-CN(-) ligands at the active site, and can obtain the hydrogen-bond networks and the preferred proton transfer pathway in the hydrogenase. Our results demonstrate the precise comprehensive information available from ultra-high-resolution structures of proteins as an alternative to neutron diffraction and other methods such as NMR structural analysis. PMID:25624102

  11. Time-resolved crystallography and protein design: signalling photoreceptors and optogenetics.

    PubMed

    Moffat, Keith

    2014-07-17

    Time-resolved X-ray crystallography and solution scattering have been successfully conducted on proteins on time-scales down to around 100 ps, set by the duration of the hard X-ray pulses emitted by synchrotron sources. The advent of hard X-ray free-electron lasers (FELs), which emit extremely intense, very brief, coherent X-ray pulses, opens the exciting possibility of time-resolved experiments with femtosecond time resolution on macromolecular structure, in both single crystals and solution. The X-ray pulses emitted by an FEL differ greatly in many properties from those emitted by a synchrotron, in ways that at first glance make time-resolved measurements of X-ray scattering with the required accuracy extremely challenging. This opens up several questions which I consider in this brief overview. Are there likely to be chemically and biologically interesting structural changes to be revealed on the femtosecond time-scale? How shall time-resolved experiments best be designed and conducted to exploit the properties of FELs and overcome challenges that they pose? To date, fast time-resolved reactions have been initiated by a brief laser pulse, which obviously requires that the system under study be light-sensitive. Although this is true for proteins of the visual system and for signalling photoreceptors, it is not naturally the case for most interesting biological systems. To generate more biological targets for time-resolved study, can this limitation be overcome by optogenetic, chemical or other means? PMID:24914168

  12. Time-resolved crystallography and protein design: signalling photoreceptors and optogenetics

    PubMed Central

    Moffat, Keith

    2014-01-01

    Time-resolved X-ray crystallography and solution scattering have been successfully conducted on proteins on time-scales down to around 100 ps, set by the duration of the hard X-ray pulses emitted by synchrotron sources. The advent of hard X-ray free-electron lasers (FELs), which emit extremely intense, very brief, coherent X-ray pulses, opens the exciting possibility of time-resolved experiments with femtosecond time resolution on macromolecular structure, in both single crystals and solution. The X-ray pulses emitted by an FEL differ greatly in many properties from those emitted by a synchrotron, in ways that at first glance make time-resolved measurements of X-ray scattering with the required accuracy extremely challenging. This opens up several questions which I consider in this brief overview. Are there likely to be chemically and biologically interesting structural changes to be revealed on the femtosecond time-scale? How shall time-resolved experiments best be designed and conducted to exploit the properties of FELs and overcome challenges that they pose? To date, fast time-resolved reactions have been initiated by a brief laser pulse, which obviously requires that the system under study be light-sensitive. Although this is true for proteins of the visual system and for signalling photoreceptors, it is not naturally the case for most interesting biological systems. To generate more biological targets for time-resolved study, can this limitation be overcome by optogenetic, chemical or other means? PMID:24914168

  13. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins.

    PubMed

    Bokhove, Marcel; Sadat Al Hosseini, Hamed; Saito, Takako; Dioguardi, Elisa; Gegenschatz-Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; de Sanctis, Daniele; Han, Ling; Jovine, Luca

    2016-04-01

    We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. PMID:26850170

  14. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins

    PubMed Central

    Bokhove, Marcel; Sadat Al Hosseini, Hamed; Saito, Takako; Dioguardi, Elisa; Gegenschatz-Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; de Sanctis, Daniele; Han, Ling; Jovine, Luca

    2016-01-01

    We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. PMID:26850170

  15. A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography

    SciTech Connect

    Bunker, Richard D.; Mandal, Kalyaneswar; Bashiri, Ghader; Chaston, Jessica J.; Pentelute, Bradley L.; Lott, J. Shaun; Kent, Stephen B. H.; Baker, Edward N.

    2015-04-07

    Racemic protein crystallography was used to determine the X-ray structure of the predicted Mycobacterium tuberculosis protein Rv1738, which had been completely recalcitrant to crystallization in its natural L-form. Native chemical ligation was used to synthesize both L-protein and D-protein enantiomers of Rv1738. Crystallization of the racemic {D-protein + L-protein} mixture was immediately successful. The resulting crystals diffracted to high resolution and also enabled facile structure determination because of the quantized phases of the data from centrosymmetric crystals. The X-ray structure of Rv1738 revealed striking similarity with bacterial hibernation factors, despite minimal sequence similarity. As a result, we predict that Rv1738, which is highly up-regulated in conditions that mimic the onset of persistence, helps trigger dormancy by association with the bacterial ribosome.

  16. Time-of-flight neutron diffraction study of bovine [gamma]-chymotrypsin at the Protein Crystallography Station

    SciTech Connect

    Lazar, Louis M.; Fisher, S. Zoe; Moulin, Aaron G.; Kovalevsky, Andrey; Novak, Walter R.P.; Langan, Paul; Petsko, Gregory A.; Ringe, Dagmar

    2012-02-06

    The overarching goal of this research project is to determine, for a subset of proteins, exact hydrogen positions using neutron diffraction, thereby improving H-atom placement in proteins so that they may be better used in various computational methods that are critically dependent upon said placement. In order to be considered applicable for neutron diffraction studies, the protein of choice must be amenable to ultrahigh-resolution X-ray crystallography, be able to form large crystals (1 mm{sup 3} or greater) and have a modestly sized unit cell (no dimension longer than 100 {angstrom}). As such, {gamma}-chymotrypsin is a perfect candidate for neutron diffraction. To understand and probe the role of specific active-site residues and hydrogen-bonding patterns in {gamma}-chymotrypsin, neutron diffraction studies were initiated at the Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center (LANSCE). A large single crystal was subjected to H/D exchange prior to data collection. Time-of-flight neutron diffraction data were collected to 2.0 {angstrom} resolution at the PCS with 85% completeness. Here, the first time-of-flight neutron data collection from {gamma}-chymotrypsin is reported.

  17. Operational experience of a large area x-ray camera for protein crystallography.

    SciTech Connect

    Joachimiak, A.; Jorden, A. R.; Loeffen, P. W.; Naday, I.; Sanishvili, R.; Westbrook, E. M.

    1999-07-13

    After 3 years experience of operating very large area (210mm x 210mm) CCD-based detectors at the Advanced Photon Source, operational experience is reported. Four such detectors have been built, two for Structural Biology Center (APS-1 and SBC-2), one for Basic Energy Sciences Synchrotrons Radiation Center (Gold-2) at Argonne National Laboratory's Advanced Photon Source and one for Osaka University by Oxford Instruments, for use at Spring 8 (PX-21O). The detector is specifically designed as a high resolution and fast readout camera for macromolecular crystallography. Design trade-offs for speed and size are reviewed in light of operational experience and future requirements are considered. Operational data and examples of crystallography data are presented, together with plans for more development.

  18. Nanoflow electrospinning serial femtosecond crystallography

    PubMed Central

    Sierra, Raymond G.; Laksmono, Hartawan; Kern, Jan; Tran, Rosalie; Hattne, Johan; Alonso-Mori, Roberto; Lassalle-Kaiser, Benedikt; Glöckner, Carina; Hellmich, Julia; Schafer, Donald W.; Echols, Nathaniel; Gildea, Richard J.; Grosse-Kunstleve, Ralf W.; Sellberg, Jonas; McQueen, Trevor A.; Fry, Alan R.; Messerschmidt, Marc M.; Miahnahri, Alan; Seibert, M. Marvin; Hampton, Christina Y.; Starodub, Dmitri; Loh, N. Duane; Sokaras, Dimosthenis; Weng, Tsu-Chien; Zwart, Petrus H.; Glatzel, Pieter; Milathianaki, Despina; White, William E.; Adams, Paul D.; Williams, Garth J.; Boutet, Sébastien; Zouni, Athina; Messinger, Johannes; Sauter, Nicholas K.; Bergmann, Uwe; Yano, Junko; Yachandra, Vittal K.; Bogan, Michael J.

    2012-01-01

    An electrospun liquid microjet has been developed that delivers protein microcrystal suspensions at flow rates of 0.14–3.1 µl min−1 to perform serial femtosecond crystallography (SFX) studies with X-ray lasers. Thermolysin microcrystals flowed at 0.17 µl min−1 and diffracted to beyond 4 Å resolution, producing 14 000 indexable diffraction patterns, or four per second, from 140 µg of protein. Nanoflow electrospinning extends SFX to biological samples that necessitate minimal sample consumption. PMID:23090408

  19. Strategies in RNA crystallography.

    PubMed

    Reyes, Francis E; Garst, Andrew D; Batey, Robert T

    2009-01-01

    A number of RNAs ranging from small helices to large megadalton ribonucleoprotein complexes have been solved to atomic resolution using X-ray crystallography. As with proteins, RNA crystallography involves a number of screening trials in which the concentration of macromolecule, precipitant, salt, and temperature are varied, an approach known as searching "condition space." In contrast to proteins, the nature of base pairing in nucleic acids creates predictable secondary structure that facilitates the rational design of RNA variants, allowing "sequence space" to be screened in parallel. This chapter reviews RNA-specific techniques and considerations for RNA crystallography and presents a complete workflow used by our laboratory for solving RNA structures starting with initial library construction, methods to investigate and improve RNA crystal quality, and finally phase determination and structure solution. PMID:20946787

  20. Radiation damage in protein serial femtosecond crystallography using an x-ray free-electron laser

    PubMed Central

    Lomb, Lukas; Barends, Thomas R. M.; Kassemeyer, Stephan; Aquila, Andrew; Epp, Sascha W.; Erk, Benjamin; Foucar, Lutz; Hartmann, Robert; Rudek, Benedikt; Rolles, Daniel; Rudenko, Artem; Shoeman, Robert L.; Andreasson, Jakob; Bajt, Sasa; Barthelmess, Miriam; Barty, Anton; Bogan, Michael J.; Bostedt, Christoph; Bozek, John D.; Caleman, Carl; Coffee, Ryan; Coppola, Nicola; DePonte, Daniel P.; Doak, R. Bruce; Ekeberg, Tomas; Fleckenstein, Holger; Fromme, Petra; Gebhardt, Maike; Graafsma, Heinz; Gumprecht, Lars; Hampton, Christina Y.; Hartmann, Andreas; Hauser, Günter; Hirsemann, Helmut; Holl, Peter; Holton, James M.; Hunter, Mark S.; Kabsch, Wolfgang; Kimmel, Nils; Kirian, Richard A.; Liang, Mengning; Maia, Filipe R. N. C.; Meinhart, Anton; Marchesini, Stefano; Martin, Andrew V.; Nass, Karol; Reich, Christian; Schulz, Joachim; Seibert, M. Marvin; Sierra, Raymond; Soltau, Heike; Spence, John C. H.; Steinbrener, Jan; Stellato, Francesco; Stern, Stephan; Timneanu, Nicusor; Wang, Xiaoyu; Weidenspointner, Georg; Weierstall, Uwe; White, Thomas A.; Wunderer, Cornelia; Chapman, Henry N.; Ullrich, Joachim; Strüder, Lothar; Schlichting, Ilme

    2013-01-01

    X-ray free-electron lasers deliver intense femtosecond pulses that promise to yield high resolution diffraction data of nanocrystals before the destruction of the sample by radiation damage. Diffraction intensities of lysozyme nanocrystals collected at the Linac Coherent Light Source using 2 keV photons were used for structure determination by molecular replacement and analyzed for radiation damage as a function of pulse length and fluence. Signatures of radiation damage are observed for pulses as short as 70 fs. Parametric scaling used in conventional crystallography does not account for the observed effects. PMID:24089594

  1. X-ray Crystallography Facility

    NASA Technical Reports Server (NTRS)

    1999-01-01

    University of Alabama engineer Stacey Giles briefs NASA astronaut Dr. Bornie Dunbar about the design and capabilities of the X-ray Crystallography Facility under development at the Center for Macromolecular Crystallography of the University of Alabama at Birmingham, AL, April 21, 1999. The X-ray Crystallography Facility is designed to speed the collection of protein structure information from crystals grown aboard the International Space Station. By measuring and mapping the protein crystal structure in space, researchers will avoid exposing the delicate crystals to the rigors of space travel and make important research data available to scientists much faster. The X-ray Crystallography facility is being designed and developed by the Center for Macromolecular Crystallography of the University of Alabama at Birmingham, a NASA Commercial Space Center.

  2. X-ray Crystallography Facility

    NASA Technical Reports Server (NTRS)

    1999-01-01

    University of Alabama engineer Lance Weiss briefs NASA astronaut Dr. Bornie Dunbar about the design and capabilities of the X-ray Crystallography Facility under development at the Center for Macromolecular Crystallography of the University of Alabama at Birmingham, AL, April 21, 1999. The X-ray Crystallography Facility is designed to speed the collection of protein structure information from crystals grown aboard the International Space Station. By measuring and mapping the protein crystal structure in space, researchers will avoid exposing the delicate crystals to the rigors of space travel and make important research data available to scientists much faster. The X-ray Crystallography facility is being designed and developed by the Center for Macromolecular Crystallography of the University of Alabama at Birmingham, a NASA Commercial Space Center.

  3. Diverse application platform for hard X-ray diffraction in SACLA (DAPHNIS): application to serial protein crystallography using an X-ray free-electron laser

    PubMed Central

    Tono, Kensuke; Nango, Eriko; Sugahara, Michihiro; Song, Changyong; Park, Jaehyun; Tanaka, Tomoyuki; Tanaka, Rie; Joti, Yasumasa; Kameshima, Takashi; Ono, Shun; Hatsui, Takaki; Mizohata, Eiichi; Suzuki, Mamoru; Shimamura, Tatsuro; Tanaka, Yoshiki; Iwata, So; Yabashi, Makina

    2015-01-01

    An experimental system for serial femtosecond crystallography using an X-ray free-electron laser (XFEL) has been developed. It basically consists of a sample chamber, fluid injectors and a two-dimensional detector. The chamber and the injectors are operated under helium atmosphere at 1 atm. The ambient pressure operation facilitates applications to fluid samples. Three kinds of injectors are employed to feed randomly oriented crystals in aqueous solution or highly viscous fluid. Experiments on lysozyme crystals were performed by using the 10 keV XFEL of the SPring-8 Angstrom Compact free-electron LAser (SACLA). The structure of model protein lysozyme from 1 µm crystals at a resolution of 2.4 Å was obtained. PMID:25931065

  4. Diverse application platform for hard X-ray diffraction in SACLA (DAPHNIS): application to serial protein crystallography using an X-ray free-electron laser.

    PubMed

    Tono, Kensuke; Nango, Eriko; Sugahara, Michihiro; Song, Changyong; Park, Jaehyun; Tanaka, Tomoyuki; Tanaka, Rie; Joti, Yasumasa; Kameshima, Takashi; Ono, Shun; Hatsui, Takaki; Mizohata, Eiichi; Suzuki, Mamoru; Shimamura, Tatsuro; Tanaka, Yoshiki; Iwata, So; Yabashi, Makina

    2015-05-01

    An experimental system for serial femtosecond crystallography using an X-ray free-electron laser (XFEL) has been developed. It basically consists of a sample chamber, fluid injectors and a two-dimensional detector. The chamber and the injectors are operated under helium atmosphere at 1 atm. The ambient pressure operation facilitates applications to fluid samples. Three kinds of injectors are employed to feed randomly oriented crystals in aqueous solution or highly viscous fluid. Experiments on lysozyme crystals were performed by using the 10 keV XFEL of the SPring-8 Angstrom Compact free-electron LAser (SACLA). The structure of model protein lysozyme from 1 µm crystals at a resolution of 2.4 Å was obtained. PMID:25931065

  5. Neutron Laue macromolecular crystallography

    SciTech Connect

    Meilleur, Flora; Myles, Dean A A; Blakeley, M. P.

    2006-01-01

    Recent progress in neutron protein crystallography such as the use of the Laue technique and improved neutron optics and detector technologies have dramatically improved the speed and precision with which neutron protein structures can now be determined. These studies are providing unique and complementary insights on hydrogen and hydration in protein crystal structures that are not available from X-ray structures alone. Parallel improvements in modern molecular biology now allow fully (per)deuterated protein samples to be produced for neutron scattering that essentially eradicate the large--and ultimately limiting--hydrogen incoherent scattering background that has hampered such studies in the past. High quality neutron data can now be collected to near atomic resolution ({approx}2.0 Angstroms) for proteins of up to {approx}50 kDa molecular weight using crystals of volume {approx}0.1 mm3 on the Laue diffractometer at ILL. The ability to flash-cool and collect high resolution neutron data from protein crystals at cryogenic temperature (15 K) has opened the way for kinetic crystallography on freeze trapped systems. Current instrument developments now promise to reduce crystal volume requirements by a further order of magnitude, making neutron protein crystallography a more accessible and routine technique.

  6. Racemic crystallography of synthetic protein enantiomers used to determine the X-ray structure of plectasin by direct methods

    PubMed Central

    Mandal, Kalyaneswar; Pentelute, Brad L; Tereshko, Valentina; Thammavongsa, Vilasak; Schneewind, Olaf; Kossiakoff, Anthony A; Kent, Stephen B H

    2009-01-01

    We describe the use of racemic crystallography to determine the X-ray structure of the natural product plectasin, a potent antimicrobial protein recently isolated from fungus. The protein enantiomers l-plectasin and d-plectasin were prepared by total chemical synthesis; interestingly, l-plectasin showed the expected antimicrobial activity, while d-plectasin was devoid of such activity. The mirror image proteins were then used for racemic crystallization. Synchrotron X-ray diffraction data were collected to atomic resolution from a racemic plectasin crystal; the racemate crystallized in the achiral centrosymmetric space group with one l-plectasin molecule and one d-plectasin molecule forming the unit cell. Dimer-like intermolecular interactions between the protein enantiomers were observed, which may account for the observed extremely low solvent content (13%–15%) and more highly ordered nature of the racemic crystals. The structure of the plectasin molecule was well defined for all 40 amino acids and was generally similar to the previously determined NMR structure, suggesting minimal impact of the crystal packing on the plectasin conformation. PMID:19472324

  7. Racemic crystallography of synthetic protein enantiomers used to determine the X-ray structure of plectasin by direct methods

    SciTech Connect

    Mandal, Kalyaneswar; Pentelute, Brad L.; Tereshko, Valentina; Thammavongsa, Vilasak; Schneewind, Olaf; Kossiakoff, Anthony A.; Kent, Stephen B.H.

    2009-06-30

    We describe the use of racemic crystallography to determine the X-ray structure of the natural product plectasin, a potent antimicrobial protein recently isolated from fungus. The protein enantiomers L-plectasin and D-plectasin were prepared by total chemical synthesis; interestingly, L-plectasin showed the expected antimicrobial activity, while D-plectasin was devoid of such activity. The mirror image proteins were then used for racemic crystallization. Synchrotron X-ray diffraction data were collected to atomic resolution from a racemic plectasin crystal; the racemate crystallized in the achiral centrosymmetric space group P1 with one L-plectasin molecule and one D-plectasin molecule forming the unit cell. Dimer-like intermolecular interactions between the protein enantiomers were observed, which may account for the observed extremely low solvent content (13%-15%) and more highly ordered nature of the racemic crystals. The structure of the plectasin molecule was well defined for all 40 amino acids and was generally similar to the previously determined NMR structure, suggesting minimal impact of the crystal packing on the plectasin conformation.

  8. Design, Synthesis, and Protein Crystallography of Biaryltriazoles as Potent Tautomerase Inhibitors of Macrophage Migration Inhibitory Factor

    PubMed Central

    Dziedzic, Pawel; Cisneros, José A.; Robertson, Michael J.; Hare, Alissa A.; Danford, Nadia E.; Baxter, Richard H. G.; Jorgensen, William L.

    2015-01-01

    Optimization is reported for biaryltriazoles as inhibitors of the tautomerase activity of human macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with numerous inflammatory diseases and cancer. A combined approach was taken featuring organic synthesis, enzymatic assaying, crystallography, and modeling including free-energy perturbation (FEP) calculations. X-ray crystal structures for 3a and 3b bound to MIF are reported and provided a basis for the modeling efforts. The accommodation of the inhibitors in the binding site is striking with multiple hydrogen bonds and aryl–aryl interactions. Additional modeling encouraged pursuit of 5-phenoxyquinolinyl analogues, which led to the very potent compound 3s. Activity was further enhanced by addition of a fluorine atom adjacent to the phenolic hydroxyl group as in 3w, 3z, 3aa, and 3bb to strengthen a key hydrogen bond. It is also shown that physical properties of the compounds can be modulated by variation of solvent-exposed substituents. Several of the compounds are likely the most potent known MIF tautomerase inhibitors; the most active ones are more than 1000-fold more active than the well-studied (R)-ISO-1 and more than 200-fold more active than the chromen-4-one Orita-13. PMID:25697265

  9. Volume-conserving trans-cis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallography

    NASA Astrophysics Data System (ADS)

    Jung, Yang Ouk; Lee, Jae Hyuk; Kim, Joonghan; Schmidt, Marius; Moffat, Keith; Šrajer, Vukica; Ihee, Hyotcherl

    2013-03-01

    Trans-to-cis isomerization, the key reaction in photoactive proteins, usually cannot occur through the standard one-bond-flip mechanism. Owing to spatial constraints imposed by a protein environment, isomerization probably proceeds through a volume-conserving mechanism in which highly choreographed atomic motions are expected, the details of which have not yet been observed directly. Here we employ time-resolved X-ray crystallography to visualize structurally the isomerization of the p-coumaric acid chromophore in photoactive yellow protein with a time resolution of 100 ps and a spatial resolution of 1.6 Å. The structure of the earliest intermediate (IT) resembles a highly strained transition state in which the torsion angle is located halfway between the trans- and cis-isomers. The reaction trajectory of IT bifurcates into two structurally distinct cis intermediates via hula-twist and bicycle-pedal pathways. The bifurcating reaction pathways can be controlled by weakening the hydrogen bond between the chromophore and an adjacent residue through E46Q mutation, which switches off the bicycle-pedal pathway.

  10. Elucidation of Lipid Binding Sites on Lung Surfactant Protein A Using X-ray Crystallography, Mutagenesis, and Molecular Dynamics Simulations.

    PubMed

    Goh, Boon Chong; Wu, Huixing; Rynkiewicz, Michael J; Schulten, Klaus; Seaton, Barbara A; McCormack, Francis X

    2016-07-01

    Surfactant protein A (SP-A) is a collagenous C-type lectin (collectin) that is critical for pulmonary defense against inhaled microorganisms. Bifunctional avidity of SP-A for pathogen-associated molecular patterns (PAMPs) such as lipid A and for dipalmitoylphosphatidylcholine (DPPC), the major component of surfactant membranes lining the air-liquid interface of the lung, ensures that the protein is poised for first-line interactions with inhaled pathogens. To improve our understanding of the motifs that are required for interactions with microbes and surfactant structures, we explored the role of the tyrosine-rich binding surface on the carbohydrate recognition domain of SP-A in the interaction with DPPC and lipid A using crystallography, site-directed mutagenesis, and molecular dynamics simulations. Critical binding features for DPPC binding include a three-walled tyrosine cage that binds the choline headgroup through cation-π interactions and a positively charged cluster that binds the phosphoryl group. This basic cluster is also critical for binding of lipid A, a bacterial PAMP and target for SP-A. Molecular dynamics simulations further predict that SP-A binds lipid A more tightly than DPPC. These results suggest that the differential binding properties of SP-A favor transfer of the protein from surfactant DPPC to pathogen membranes containing appropriate lipid PAMPs to effect key host defense functions. PMID:27324153

  11. Volume-conserving trans-cis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallography

    SciTech Connect

    Jung, Yang Ouk; Lee, Jae Hyuk; Kim, Joonghan; Schmidt, Marius; Moffat, Keith; Šrajer, Vukica; Ihee, Hyotcherl

    2013-02-03

    Trans-to-cis isomerization, the key reaction in photoactive proteins, usually cannot occur through the standard one-bond-flip mechanism. Owing to spatial constraints imposed by a protein environment, isomerization probably proceeds through a volume-conserving mechanism in which highly choreographed atomic motions are expected, the details of which have not yet been observed directly. Here we employ time-resolved X-ray crystallography to visualize structurally the isomerization of the p-coumaric acid chromophore in photoactive yellow protein with a time resolution of 100 ps and a spatial resolution of 1.6 Å. The structure of the earliest intermediate (IT) resembles a highly strained transition state in which the torsion angle is located halfway between the trans- and cis-isomers. The reaction trajectory of IT bifurcates into two structurally distinct cis intermediates via hula-twist and bicycle-pedal pathways. The bifurcating reaction pathways can be controlled by weakening the hydrogen bond between the chromophore and an adjacent residue through E46Q mutation, which switches off the bicycle-pedal pathway.

  12. Ultrahigh resolution protein crystallography: Concanavalin A to 0.94 A and beyond

    SciTech Connect

    Deacon, A.M.; Gleichmann, T.; Harrop, S.J.; Helliwell, J.R.; Kalb Gilboa, A.J.; Yariv, J.

    1996-09-01

    Many years ago the idea of collecting voluminous quantities of weak reflection intensities from a protein crystal, at high resolution, was a particular challenge [J.R. Helliwell (1979) Daresbury Study Weekend DL/SCI R13, pp. 1{endash}6]. The combination of insertion devices with very high x-ray fluxes at short x-ray wavelengths, sensitive CCD detectors, and freezing of crystals have provided the means to certainly match those best hopes. So much so that the data can best be described as ultrahigh resolution, at least as evidenced in our studies of the 25000 molecular weight plant protein concanavalin A. (The intrinsic property of this protein is to bind sugar molecules; it is implicated in cell-to-cell recognition processes and is widely used as a laboratory diagnostic tool.) At CHESS we have used a 0.9 A wavelength beam on station A1, fed by a 24 pole multipole wiggler. Both an imaging plate system and the Princeton 1k CCD detector [M. Tate {ital et} {ital al}., J. Appl. Cryst. {bold 28}, 196 (1995)] have been used on this experimental setup to collect diffraction data sets from frozen concanavalin A crystals (saccharide-free crystal form). The rapid readout of the CCD was most convenient compared with the image plate and its associated scanning and erasing. Moreover the data processing results towards the edges of the detectors, 0.98 A, show that the CCD is much better than the image plate at recording these weaker data (Rmerge(I) 13{percent} versus 44{percent}, respectively). The poor performance of the image plate with weak signals has of course been documented by the Daresbury detector group [R. Lewis, J. Synchrotron Radiation {bold 1}, 43 (1994)]. However, the aperture of the CCD used was limiting here. Very recently, in another run at CHESS with the CCD on A1, we have been able to record diffraction data to 0.94 A by further offsetting the detector. We again found that the reflections are still strong at the edge. (Abstract Truncated)

  13. Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography

    SciTech Connect

    Mandal, Kalyaneswar; Uppalapati, Maruti; Ault-Riché, Dana; Kenney, John; Lowitz, Joshua; Sidhu, Sachdev S.; Kent, Stephen B.H.

    2012-10-23

    Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF{sub 165} to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {l_brace}D-protein antagonist + L-protein form of VEGF-A{r_brace}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 {angstrom}. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 {angstrom}{sup 2} in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.

  14. Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography.

    PubMed

    Mandal, Kalyaneswar; Uppalapati, Maruti; Ault-Riché, Dana; Kenney, John; Lowitz, Joshua; Sidhu, Sachdev S; Kent, Stephen B H

    2012-09-11

    Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF(165) to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {D-protein antagonist + L-protein form of VEGF-A}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 Å. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 Å(2) in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2. PMID:22927390

  15. Protein crystallography for non-crystallographers, or how to get the best (but not more) from published macromolecular structures

    PubMed Central

    Wlodawer, Alexander; Minor, Wladek; Dauter, Zbigniew; Jaskolski, Mariusz

    2015-01-01

    The number of macromolecular structures deposited in the Protein Data Bank now exceeds 45 000, with the vast majority determined using crystallographic methods. Thousands of studies describing such structures have been published in the scientific literature, and 14 Nobel prizes in chemistry or medicine have been awarded to protein crystallographers. As important as these structures are for understanding the processes that take place in living organisms and also for practical applications such as drug design, many non-crystallographers still have problems with critical evaluation of the structural literature data. This review attempts to provide a brief outline of technical aspects of crystallography and to explain the meaning of some parameters that should be evaluated by users of macromolecular structures in order to interpret, but not over-interpret, the information present in the coordinate files and in their description. A discussion of the extent of the information that can be gleaned from the coordinates of structures solved at different resolution, as well as problems and pitfalls encountered in structure determination and interpretation are also covered. PMID:18034855

  16. Structural Properties of Human CaMKII Ca2+ /Calmodulin-Dependent Protein Kinase II using X-ray Crystallography

    NASA Astrophysics Data System (ADS)

    Cao, Yumeng Melody; McSpadden, Ethan; Kuriyan, John; Department of Molecular; Cell Biology; Department of Chemistry Team

    To this day, human memory storage remains a mystery as we can at most describe the process vaguely on a cellular level. Switch-like properties of Calcium/Calmodulin-Dependent Protein Kinase II make it a leading candidate in understanding the molecular basis of human memory. The protein crystal was placed in the beam of a synchrotron source and the x-ray crystallography data was collected as reflections on a diffraction pattern that undergo Fourier transform to obtain the electron density. We observed two drastic differences from our solved structure at 2.75Å to a similar construct of the mouse CaMKII association domain. Firstly, our structure is a 6-fold symmetric dodecamer, whereas the previously published construct was a 7-fold symmetric tetradecamer. This suggests the association domain of human CaMKII is a dynamic structure that is triggered subunit exchange process. Secondly, in our structure the N-terminal tag is docked as an additional beta-strand on an uncapped beta-sheet present in each association domain protomer. This is concrete evidence of the involvement of the polypeptide docking site in the molecular mechanism underlining subunit exchange. In the future, we would like to selectively inhibit the exchange process while not disrupting the other functionalities of CaMKII.

  17. Milestones in Electron Crystallography

    PubMed Central

    Renault, Ludovic; Chou, Hui-Ting; Chiu, Po-Lin; Hill, Rena M.; Zeng, Xiangyan; Gipson, Bryant; Zhang, Zi Yan; Cheng, Anchi; Unger, Vinzenz; Stahlberg, Henning

    2007-01-01

    Summary Electron crystallography determines the structure of membrane embedded proteins in the two-dimensionally crystallized state by cryo-transmission electron microscopy imaging and computer structure reconstruction. Milestones on the path to the structure are high-level expression, purification of functional protein, reconstitution into two-dimensional lipid membrane crystals, high-resolution imaging, and structure determination by computer image processing. Here we review the current state of these methods. We also created an Internet information exchange platform for electron crystallography, where guidelines for the imaging and data processing method are maintained. The server (http://2dx.org) provides the electron crystallography community with a central information exchange platform, which is structured in blog and Wiki form, allowing visitors to add comments or discussions. It currently offers a detailed step-by-step introduction to image processing with the MRC software program. The server is also a repository for the 2dx software package, a user-friendly image processing system for 2D membrane protein crystals. PMID:17103018

  18. Neutron Nucleic Acid Crystallography.

    PubMed

    Chatake, Toshiyuki

    2016-01-01

    The hydration shells surrounding nucleic acids and hydrogen-bonding networks involving water molecules and nucleic acids are essential interactions for the structural stability and function of nucleic acids. Water molecules in the hydration shells influence various conformations of DNA and RNA by specific hydrogen-bonding networks, which often contribute to the chemical reactivity and molecular recognition of nucleic acids. However, X-ray crystallography could not provide a complete description of structural information with respect to hydrogen bonds. Indeed, X-ray crystallography is a powerful tool for determining the locations of water molecules, i.e., the location of the oxygen atom of H2O; however, it is very difficult to determine the orientation of the water molecules, i.e., the orientation of the two hydrogen atoms of H2O, because X-ray scattering from the hydrogen atom is very small.Neutron crystallography is a specialized tool for determining the positions of hydrogen atoms. Neutrons are not diffracted by electrons, but are diffracted by atomic nuclei; accordingly, neutron scattering lengths of hydrogen and its isotopes are comparable to those of non-hydrogen atoms. Therefore, neutron crystallography can determine both of the locations and orientations of water molecules. This chapter describes the current status of neutron nucleic acid crystallographic research as well as the basic principles of neutron diffraction experiments performed on nucleic acid crystals: materials, crystallization, diffraction experiments, and structure determination. PMID:26227050

  19. Five-dimensional crystallography

    PubMed Central

    Schmidt, Marius; Graber, Tim; Henning, Robert; Srajer, Vukica

    2010-01-01

    A method for determining a comprehensive chemical kinetic mechanism in macromolecular reactions is presented. The method is based on five-dimensional crystallography, where, in addition to space and time, temperature is also taken into consideration and an analysis based on singular value decomposition is applied. First results of such a time-resolved crystallographic study are presented. Temperature-dependent time-resolved X-ray diffraction measurements were conducted on the newly upgraded BioCARS 14-ID-B beamline at the Advanced Photon Source and aimed at elucidating a comprehensive kinetic mechanism of the photoactive yellow protein photocycle. Extensive time series of crystallographic data were collected at two temperatures, 293 K and 303 K. Relaxation times of the reaction extracted from these time series exhibit measurable differences for the two temperatures, hence demonstrating that five-dimensional crystallography is feasible. PMID:20164643

  20. The Role of Protein Crystallography in Defining the Mechanisms of Biogenesis and Catalysis in Copper Amine Oxidase

    PubMed Central

    Klema, Valerie J.; Wilmot, Carrie M.

    2012-01-01

    Copper amine oxidases (CAOs) are a ubiquitous group of enzymes that catalyze the conversion of primary amines to aldehydes coupled to the reduction of O2 to H2O2. These enzymes utilize a wide range of substrates from methylamine to polypeptides. Changes in CAO activity are correlated with a variety of human diseases, including diabetes mellitus, Alzheimer’s disease, and inflammatory disorders. CAOs contain a cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), that is required for catalytic activity and synthesized through the post-translational modification of a tyrosine residue within the CAO polypeptide. TPQ generation is a self-processing event only requiring the addition of oxygen and Cu(II) to the apoCAO. Thus, the CAO active site supports two very different reactions: TPQ synthesis, and the two electron oxidation of primary amines. Crystal structures are available from bacterial through to human sources, and have given insight into substrate preference, stereospecificity, and structural changes during biogenesis and catalysis. In particular both these processes have been studied in crystallo through the addition of native substrates. These latter studies enable intermediates during physiological turnover to be directly visualized, and demonstrate the power of this relatively recent development in protein crystallography. PMID:22754303

  1. The role of protein crystallography in defining the mechanisms of biogenesis and catalysis in copper amine oxidase.

    PubMed

    Klema, Valerie J; Wilmot, Carrie M

    2012-01-01

    Copper amine oxidases (CAOs) are a ubiquitous group of enzymes that catalyze the conversion of primary amines to aldehydes coupled to the reduction of O(2) to H(2)O(2). These enzymes utilize a wide range of substrates from methylamine to polypeptides. Changes in CAO activity are correlated with a variety of human diseases, including diabetes mellitus, Alzheimer's disease, and inflammatory disorders. CAOs contain a cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), that is required for catalytic activity and synthesized through the post-translational modification of a tyrosine residue within the CAO polypeptide. TPQ generation is a self-processing event only requiring the addition of oxygen and Cu(II) to the apoCAO. Thus, the CAO active site supports two very different reactions: TPQ synthesis, and the two electron oxidation of primary amines. Crystal structures are available from bacterial through to human sources, and have given insight into substrate preference, stereospecificity, and structural changes during biogenesis and catalysis. In particular both these processes have been studied in crystallo through the addition of native substrates. These latter studies enable intermediates during physiological turnover to be directly visualized, and demonstrate the power of this relatively recent development in protein crystallography. PMID:22754303

  2. Mechanism of the nucleotidyl-transfer reaction in DNA polymerase revealed by time-resolved protein crystallography

    PubMed Central

    Nakamura, Teruya; Zhao, Ye; Yamagata, Yuriko; Hua, Yue-jin; Yang, Wei

    2013-01-01

    Nucleotidyl-transfer reaction catalyzed by DNA polymerase is a fundamental enzymatic reaction for DNA synthesis. Until now, a number of structural and kinetic studies on DNA polymerases have proposed a two-metalion mechanism of the nucleotidyl-transfer reaction. However, the actual reaction process has never been visualized. Recently, we have followed the nucleotidyl-transfer reaction process by human DNA polymerase η using time-resolved protein crystallography. In sequence, two Mg2+ ions bind to the active site, the nucleophile 3′-OH is deprotonated, the deoxyribose at the primer end converts from C2′-endo to C3′-endo, and the nucleophile and the α-phosphate of the substrate dATP approach each other to form the new bond. In this process, we observed transient elements, which are a water molecule to deprotonate the 3′-OH and an additional Mg2+ ion to stabilize the intermediate state. Particularly, the third Mg2+ ion observed in this study may be a general feature of the two-metalion mechanism.

  3. X-ray crystallography and computational docking for the detection and development of protein-ligand interactions.

    PubMed

    Kershaw, N M; Wright, G S A; Sharma, R; Antonyuk, S V; Strange, R W; Berry, N G; O'Neill, P M; Hasnain, S S

    2013-01-01

    Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterised by the selective dysfunction and death of the upper and lower motor neurons. Median survival rates are between 3 and 5 years after diagnosis. Mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) have been linked to a subset of familial forms of ALS (fALS). Herein, we describe a fragment- based drug discovery (FBDD) approach for the investigation of small molecule binding sites in SOD1. X-ray crystallography has been used as the primary screening method and has been shown to directly detect protein-ligand interactions which cannot be unambiguously identified using other biophysical methods. The structural requirements for effective binding at Trp32 are detailed for a series of quinazoline-containing compounds. The investigation of an additional site that binds a range of catecholamines and the use of computational modelling to assist fragment evolution is discussed. This study also highlights the importance of ligand solubility for successful Xray crystallographic campaigns in lead compound design. PMID:23278398

  4. Structural rigidity of a large cavity-containing protein revealed by high-pressure crystallography

    PubMed Central

    Collins, Marcus D.; Quillin, Michael L.; Hummer, Gerhard; Matthews, Brian W.; Gruner, Sol M.

    2007-01-01

    Steric constraints, charged interactions and many other forces important to protein structure and function can be explored by mutagenic experiments. Research of this kind has led to a wealth of knowledge about what stabilizes proteins in their folded states. To gain a more complete picture requires that we perturb these structures in a continuous manner, something mutagenesis cannot achieve. With high pressure crystallographic methods it is now possible to explore the detailed properties of proteins while continuously varying thermodynamic parameters. In this paper, we detail the structural response of the cavity-containing mutant L99A of T4 lysozyme, as well as its pseudo wild-type (WT*) counterpart, to hydrostatic pressure. Surprisingly, the cavity has almost no effect on the pressure response: virtually the same changes are observed in WT* as in L99A under pressure. The cavity is most rigid, while other regions deform substantially. This implies that while some residues may increase the thermodynamic stability of a protein, they may also be structurally irrelevant. As recently shown, the cavity fills with water at pressures above 100 MPa while retaining its overall size. The resultant picture of the protein is one in which conformationally fluctuating side groups provide a liquid-like environment, but which also contribute to the rigidity of the peptide backbone. PMID:17292912

  5. Direct detection of x-rays for protein crystallography employing a thick, large area CCD

    DOEpatents

    Atac, Muzaffer; McKay, Timothy

    1999-01-01

    An apparatus and method for directly determining the crystalline structure of a protein crystal. The crystal is irradiated by a finely collimated x-ray beam. The interaction of the x-ray beam with the crystal produces scattered x-rays. These scattered x-rays are detected by means of a large area, thick CCD which is capable of measuring a significant number of scattered x-rays which impact its surface. The CCD is capable of detecting the position of impact of the scattered x-ray on the surface of the CCD and the quantity of scattered x-rays which impact the same cell or pixel. This data is then processed in real-time and the processed data is outputted to produce a image of the structure of the crystal. If this crystal is a protein the molecular structure of the protein can be determined from the data received.

  6. A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography.

    PubMed

    Bunker, Richard D; Mandal, Kalyaneswar; Bashiri, Ghader; Chaston, Jessica J; Pentelute, Bradley L; Lott, J Shaun; Kent, Stephen B H; Baker, Edward N

    2015-04-01

    Protein 3D structure can be a powerful predictor of function, but it often faces a critical roadblock at the crystallization step. Rv1738, a protein from Mycobacterium tuberculosis that is strongly implicated in the onset of nonreplicating persistence, and thereby latent tuberculosis, resisted extensive attempts at crystallization. Chemical synthesis of the L- and D-enantiomeric forms of Rv1738 enabled facile crystallization of the D/L-racemic mixture. The structure was solved by an ab initio approach that took advantage of the quantized phases characteristic of diffraction by centrosymmetric crystals. The structure, containing L- and D-dimers in a centrosymmetric space group, revealed unexpected homology with bacterial hibernation-promoting factors that bind to ribosomes and suppress translation. This suggests that the functional role of Rv1738 is to contribute to the shutdown of ribosomal protein synthesis during the onset of nonreplicating persistence of M. tuberculosis. PMID:25831534

  7. A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography

    PubMed Central

    Bunker, Richard D.; Mandal, Kalyaneswar; Bashiri, Ghader; Chaston, Jessica J.; Pentelute, Bradley L.; Lott, J. Shaun; Kent, Stephen B. H.; Baker, Edward N.

    2015-01-01

    Protein 3D structure can be a powerful predictor of function, but it often faces a critical roadblock at the crystallization step. Rv1738, a protein from Mycobacterium tuberculosis that is strongly implicated in the onset of nonreplicating persistence, and thereby latent tuberculosis, resisted extensive attempts at crystallization. Chemical synthesis of the l- and d-enantiomeric forms of Rv1738 enabled facile crystallization of the d/l-racemic mixture. The structure was solved by an ab initio approach that took advantage of the quantized phases characteristic of diffraction by centrosymmetric crystals. The structure, containing l- and d-dimers in a centrosymmetric space group, revealed unexpected homology with bacterial hibernation-promoting factors that bind to ribosomes and suppress translation. This suggests that the functional role of Rv1738 is to contribute to the shutdown of ribosomal protein synthesis during the onset of nonreplicating persistence of M. tuberculosis. PMID:25831534

  8. Watching a protein as it functions with picosecond X-ray crystallography and femtosecond IR spectroscopy

    NASA Astrophysics Data System (ADS)

    Anfinrud, Philip

    2003-03-01

    Proteins are engaged in a myriad of tasks that are essential to life. To understand in mechanistic detail how proteins function, it is crucial to know the time ordering of events that give rise to their designed (or modified) function. Myoglobin (Mb), a ligand-binding heme protein, has long served as a model system for investigating ligand transport and binding in proteins. Using femtosecond time-resolved polarized IR spectroscopy, the dynamics of ligand motion after photodetachment of CO from MbCO have been probed. These studies reveal the time-dependent orientation of CO, the existence of a docking site that mediates the transport of ligands to and from the active binding site, as well as the dynamics of ligand binding and escape. To probe the structural evolution of a protein as it executes its function, a multinational collaboration has been established to acquire time-resolved X-ray crystal structures on the ID09B beam line at the European Synchrotron and Radiation Facility. Recent improvements in the experimental methodology have extended the time resolution down to 150 picoseconds and improved the spatial resolution of the electron density maps to below 2 Å. Using this technique, we have acquired time-resolved high resolution structures of L29F-MbCO, a mutant of myoglobin that exhibits unusually rapid ligand migration dynamics. The frame-by-frame structural evolution allows us to literally "watch" this protein as it functions. Conformational changes far more dramatic than the structural differences between the carboxy and deoxy states are observed. The correlated motion of CO and several side chains provides a structural explanation for the rapid expulsion of toxic CO from the nearby ligand docking site.

  9. Advances in membrane protein crystallography: in situ and in meso data collection

    SciTech Connect

    Weyand, Simone; Tate, Christopher G.

    2015-05-23

    Membrane protein structural biology has made tremendous advances over the last decade but there are still many challenges associated with crystallization, data collection and structure determination. Two independent groups, Axford et al. [(2015), Acta Cryst. D71, 1228–1237] and Huang et al. [(2015), Acta Cryst. D71, 1238–1256], have published methods that make a major contribution to addressing these challenges.

  10. Membrane Protein Structure Determination Using Crystallography and Lipidic Mesophases - Recent Advances and Successes

    PubMed Central

    Caffrey, Martin; Li, Dianfan; Dukkipati, Abhiram

    2012-01-01

    The crystal structure of the β2-adrenergic receptor in complex with an agonist and its cognate G protein has just recently been solved. It is now possible to explore in molecular detail the means by which this paradigmatic transmembrane receptor binds agonist, communicates the impulse or signalling event across the membrane and sets in motion a series of G protein-directed intracellular responses. The structure was determined using crystals of the ternary complex grown in a rationally designed lipidic mesophase by the so-called in meso method. The method is proving to be particularly useful in the G protein-coupled receptor field where the structures of thirteen distinct receptor types have been solved in the past five years. In addition to receptors, the method has proven useful with a wide variety of integral membrane protein classes that include bacterial and eukaryotic rhodopsins, a light harvesting complex II (LHII), photosynthetic reaction centers, cytochrome oxidases, β-barrels, an exchanger, and an integral membrane peptide. This attests to the versatility and range of the method and supports the view that the in meso method should be included in the arsenal of the serious membrane structural biologist. For this to happen however, the reluctance in adopting it attributable, in part, to the anticipated difficulties associated with handling the sticky, viscous cubic mesophase in which crystals grow must be overcome. Harvesting and collecting diffraction data with the mesophase-grown crystals is also viewed with some trepidation. It is acknowledged that there are challenges associated with the method. Over the years, we have endeavored to establish how the method works at a molecular level and to make it user-friendly. To these ends, tools for handling the mesophase in the pico- to nano-liter volume range have been developed for highly efficient crystallization screening in manual and robotic modes. Methods have been implemented for evaluating the functional

  11. Structural investigation of ribonuclease A conformational preferences using high pressure protein crystallography

    NASA Astrophysics Data System (ADS)

    Kurpiewska, Katarzyna; Dziubek, Kamil; Katrusiak, Andrzej; Font, Josep; Ribò, Marc; Vilanova, Maria; Lewiński, Krzysztof

    2016-04-01

    Hydrostatic pressure in range 0.1-1.5 GPa is used to modify biological system behaviour mostly in biophysical studies of proteins in solution. Due to specific influence on the system equilibrium high pressure can act as a filter that enables to identify and investigate higher energy protein conformers. The idea of the presented experiments is to examine the behaviour of RNase A molecule under high pressure before and after introduction of destabilizing mutation. For the first time crystal structures of wild-type bovine pancreatic ribonuclease A and its markedly less stable variant modified at position Ile106 were determined at different pressures. X-ray diffraction experiments at high pressure showed that the secondary structure of RNase A is well preserved even beyond 0.67 GPa at room temperature. Detailed structural analysis of ribonuclease A conformation observed under high pressure revealed that pressure influences hydrogen bonds pattern, cavity size and packing of molecule.

  12. DARPin-Based Crystallization Chaperones Exploit Molecular Geometry as a Screening Dimension in Protein Crystallography.

    PubMed

    Batyuk, Alexander; Wu, Yufan; Honegger, Annemarie; Heberling, Matthew M; Plückthun, Andreas

    2016-04-24

    DARPin libraries, based on a Designed Ankyrin Repeat Protein consensus framework, are a rich source of binding partners for a wide variety of proteins. Their modular structure, stability, ease of in vitro selection and high production yields make DARPins an ideal starting point for further engineering. The X-ray structures of around 30 different DARPin complexes demonstrate their ability to facilitate crystallization of their target proteins by restricting flexibility and preventing undesired interactions of the target molecule. However, their small size (18 kDa), very hydrophilic surface and repetitive structure can limit the DARPins' ability to provide essential crystal contacts and their usefulness as a search model for addressing the crystallographic phase problem in molecular replacement. To optimize DARPins for their application as crystallization chaperones, rigid domain-domain fusions of the DARPins to larger proteins, proven to yield high-resolution crystal structures, were generated. These fusions were designed in such a way that they affect only one of the terminal capping repeats of the DARPin and do not interfere with residues involved in target binding, allowing to exchange at will the binding specificities of the DARPin in the fusion construct. As a proof of principle, we designed rigid fusions of a stabilized version of Escherichia coli TEM-1 β-lactamase to the C-terminal capping repeat of various DARPins in six different relative domain orientations. Five crystal structures representing four different fusion constructs, alone or in complex with the cognate target, show the predicted relative domain orientations and prove the validity of the concept. PMID:26975886

  13. The Significance of G Protein-Coupled Receptor Crystallography for Drug Discovery

    PubMed Central

    Salon, John A.; Lodowski, David T.

    2011-01-01

    Crucial as molecular sensors for many vital physiological processes, seven-transmembrane domain G protein-coupled receptors (GPCRs) comprise the largest family of proteins targeted by drug discovery. Together with structures of the prototypical GPCR rhodopsin, solved structures of other liganded GPCRs promise to provide insights into the structural basis of the superfamily's biochemical functions and assist in the development of new therapeutic modalities and drugs. One of the greatest technical and theoretical challenges to elucidating and exploiting structure-function relationships in these systems is the emerging concept of GPCR conformational flexibility and its cause-effect relationship for receptor-receptor and receptor-effector interactions. Such conformational changes can be subtle and triggered by relatively small binding energy effects, leading to full or partial efficacy in the activation or inactivation of the receptor system at large. Pharmacological dogma generally dictates that these changes manifest themselves through kinetic modulation of the receptor's G protein partners. Atomic resolution information derived from increasingly available receptor structures provides an entrée to the understanding of these events and practically applying it to drug design. Supported by structure-activity relationship information arising from empirical screening, a unified structural model of GPCR activation/inactivation promises to both accelerate drug discovery in this field and improve our fundamental understanding of structure-based drug design in general. This review discusses fundamental problems that persist in drug design and GPCR structural determination. PMID:21969326

  14. Towards a rational approach for heavy-atom derivative screening in protein crystallography

    SciTech Connect

    Agniswamy, Johnson; Joyce, M. Gordon; Hammer, Carl H.; Sun, Peter D.

    2008-04-01

    Heavy-atom derivatization is routinely used in protein structure determination and is thus of critical importance in structural biology. In order to replace the current trial-and-error heavy-atom derivative screening with a knowledge-based rational derivative-selection method, the reactivity of more than 40 heavy-atom compounds over a wide range of buffer and pH values was systematically examined using peptides which contained a single reactive amino-acid residue. Heavy-atom derivatization is routinely used in protein structure determination and is thus of critical importance in structural biology. In order to replace the current trial-and-error heavy-atom derivative screening with a knowledge-based rational derivative-selection method, the reactivity of more than 40 heavy-atom compounds over a wide range of buffer and pH values was systematically examined using peptides which contained a single reactive amino-acid residue. Met-, Cys- and His-containing peptides were derivatized against Hg, Au and Pt compounds, while Tyr-, Glu-, Asp-, Asn- and Gln-containing peptides were assessed against Pb compounds. A total of 1668 reactive conditions were examined using mass spectrometry and were compiled into heavy-atom reactivity tables. The results showed that heavy-atom derivatization reactions are highly linked to buffer and pH, with the most accommodating buffer being MES at pH 6. A group of 21 compounds were identified as most successful irrespective of ligand or buffer/pH conditions. To assess the applicability of the peptide heavy-atom reactivity to proteins, lysozyme crystals were derivatized with a list of peptide-reactive compounds that included both known and new compounds for lysozyme derivatization. The results showed highly consistent heavy-atom reactivities between the peptides and lysozyme.

  15. A history of neutrons in biology: the development of neutron protein crystallography at BNL and LANL.

    PubMed

    Schoenborn, Benno P

    2010-11-01

    The first neutron diffraction data were collected from crystals of myoglobin almost 42 years ago using a step-scan diffractometer with a single detector. Since then, major advances have been made in neutron sources, instrumentation and data collection and analysis, and in biochemistry. Fundamental discoveries about enzyme mechanisms, biological complex structures, protein hydration and H-atom positions have been and continue to be made using neutron diffraction. The promise of neutrons has not changed since the first crystal diffraction data were collected. Today, with the developments of beamlines at spallation neutron sources and the use of the Laue method for data collection, the field of neutrons in structural biology has renewed vitality. PMID:21041948

  16. Protein, DNA, and Virus Crystallography with a Focused Imaging Proportional Counter

    NASA Astrophysics Data System (ADS)

    Durbin, R. M.; Burns, R.; Moulai, J.; Metcalf, P.; Freymann, D.; Blum, M.; Anderson, J. E.; Harrison, S. C.; Wiley, D. C.

    1986-05-01

    A set of programs has been developed for rapid collection of x-ray intensity data from protein and virus crystals with a commercially available two-dimensional focused geometry electronic detector. The detector is compact and portable, with unusually high spatial resolution comparable to that used in oscillation photography. It has allowed x-ray data collection on weakly diffracting crystals with large unit cells, as well as more conventional ``diffractometer-quality'' crystals. The quality of the data is compared with that from oscillation photography and automated diffractometry in the range of unit cells from 96.3 to 383.2 angstroms. Isomorphous and anomalous difference Pattersons, based on detector data, are shown for a variable surface glycoprotein mercury derivative and for a repressor-DNA bromine derivative, which has been solved at 7 angstroms with detector data only.

  17. Measuring and modeling diffuse scattering in protein X-ray crystallography

    DOE PAGESBeta

    Van Benschoten, Andrew H.; Liu, Lin; Gonzalez, Ana; Brewster, Aaron S.; Sauter, Nicholas K.; Fraser, James S.; Wall, Michael E.

    2016-03-28

    X-ray diffraction has the potential to provide rich information about the structural dynamics of macromolecules. To realize this potential, both Bragg scattering, which is currently used to derive macromolecular structures, and diffuse scattering, which reports on correlations in charge density variations, must be measured. Until now, measurement of diffuse scattering from protein crystals has been scarce because of the extra effort of collecting diffuse data. Here, we present 3D measurements of diffuse intensity collected from crystals of the enzymes cyclophilin A and trypsin. The measurements were obtained from the same X-ray diffraction images as the Bragg data, using best practicesmore » for standard data collection. To model the underlying dynamics in a practical way that could be used during structure refinement, we tested translation–libration–screw (TLS), liquid-like motions (LLM), and coarse-grained normal-modes (NM) models of protein motions. The LLM model provides a global picture of motions and was refined against the diffuse data, whereas the TLS and NM models provide more detailed and distinct descriptions of atom displacements, and only used information from the Bragg data. Whereas different TLS groupings yielded similar Bragg intensities, they yielded different diffuse intensities, none of which agreed well with the data. In contrast, both the LLM and NM models agreed substantially with the diffuse data. In conclusion, these results demonstrate a realistic path to increase the number of diffuse datasets available to the wider biosciences community and indicate that dynamics-inspired NM structural models can simultaneously agree with both Bragg and diffuse scattering.« less

  18. Measuring and modeling diffuse scattering in protein X-ray crystallography.

    PubMed

    Van Benschoten, Andrew H; Liu, Lin; Gonzalez, Ana; Brewster, Aaron S; Sauter, Nicholas K; Fraser, James S; Wall, Michael E

    2016-04-12

    X-ray diffraction has the potential to provide rich information about the structural dynamics of macromolecules. To realize this potential, both Bragg scattering, which is currently used to derive macromolecular structures, and diffuse scattering, which reports on correlations in charge density variations, must be measured. Until now, measurement of diffuse scattering from protein crystals has been scarce because of the extra effort of collecting diffuse data. Here, we present 3D measurements of diffuse intensity collected from crystals of the enzymes cyclophilin A and trypsin. The measurements were obtained from the same X-ray diffraction images as the Bragg data, using best practices for standard data collection. To model the underlying dynamics in a practical way that could be used during structure refinement, we tested translation-libration-screw (TLS), liquid-like motions (LLM), and coarse-grained normal-modes (NM) models of protein motions. The LLM model provides a global picture of motions and was refined against the diffuse data, whereas the TLS and NM models provide more detailed and distinct descriptions of atom displacements, and only used information from the Bragg data. Whereas different TLS groupings yielded similar Bragg intensities, they yielded different diffuse intensities, none of which agreed well with the data. In contrast, both the LLM and NM models agreed substantially with the diffuse data. These results demonstrate a realistic path to increase the number of diffuse datasets available to the wider biosciences community and indicate that dynamics-inspired NM structural models can simultaneously agree with both Bragg and diffuse scattering. PMID:27035972

  19. Measuring and modeling diffuse scattering in protein X-ray crystallography

    PubMed Central

    Van Benschoten, Andrew H.; Liu, Lin; Gonzalez, Ana; Brewster, Aaron S.; Sauter, Nicholas K.; Wall, Michael E.

    2016-01-01

    X-ray diffraction has the potential to provide rich information about the structural dynamics of macromolecules. To realize this potential, both Bragg scattering, which is currently used to derive macromolecular structures, and diffuse scattering, which reports on correlations in charge density variations, must be measured. Until now, measurement of diffuse scattering from protein crystals has been scarce because of the extra effort of collecting diffuse data. Here, we present 3D measurements of diffuse intensity collected from crystals of the enzymes cyclophilin A and trypsin. The measurements were obtained from the same X-ray diffraction images as the Bragg data, using best practices for standard data collection. To model the underlying dynamics in a practical way that could be used during structure refinement, we tested translation–libration–screw (TLS), liquid-like motions (LLM), and coarse-grained normal-modes (NM) models of protein motions. The LLM model provides a global picture of motions and was refined against the diffuse data, whereas the TLS and NM models provide more detailed and distinct descriptions of atom displacements, and only used information from the Bragg data. Whereas different TLS groupings yielded similar Bragg intensities, they yielded different diffuse intensities, none of which agreed well with the data. In contrast, both the LLM and NM models agreed substantially with the diffuse data. These results demonstrate a realistic path to increase the number of diffuse datasets available to the wider biosciences community and indicate that dynamics-inspired NM structural models can simultaneously agree with both Bragg and diffuse scattering. PMID:27035972

  20. Protonation states of histidine and other key residues in deoxy normal human adult hemoglobin by neutron protein crystallography

    SciTech Connect

    Kovalevsky, Andrey; Chatake, Toshiyuki; Shibayama, Naoya; Park, Sam-Yong; Ishikawa, Takuya; Mustyakimov, Marat; Fisher, S. Zoe; Langan, Paul; Morimoto, Yukio

    2010-11-01

    Using neutron diffraction analysis, the protonation states of 35 of 38 histidine residues were determined for the deoxy form of normal human adult hemoglobin. Distal and buried histidines may contribute to the increased affinity of the deoxy state for hydrogen ions and its decreased affinity for oxygen compared with the oxygenated form. The protonation states of the histidine residues key to the function of deoxy (T-state) human hemoglobin have been investigated using neutron protein crystallography. These residues can reversibly bind protons, thereby regulating the oxygen affinity of hemoglobin. By examining the OMIT F{sub o} − F{sub c} and 2F{sub o} − F{sub c} neutron scattering maps, the protonation states of 35 of the 38 His residues were directly determined. The remaining three residues were found to be disordered. Surprisingly, seven pairs of His residues from equivalent α or β chains, αHis20, αHis50, αHis58, αHis89, βHis63, βHis143 and βHis146, have different protonation states. The protonation of distal His residues in the α{sub 1}β{sub 1} heterodimer and the protonation of αHis103 in both subunits demonstrates that these residues may participate in buffering hydrogen ions and may influence the oxygen binding. The observed protonation states of His residues are compared with their ΔpK{sub a} between the deoxy and oxy states. Examination of inter-subunit interfaces provided evidence for interactions that are essential for the stability of the deoxy tertiary structure.

  1. Simulations of radiation damage as a function of the temporal pulse profile in femtosecond X-ray protein crystallography.

    PubMed

    Jönsson, H Olof; Tîmneanu, Nicuşor; Östlin, Christofer; Scott, Howard A; Caleman, Carl

    2015-03-01

    Serial femtosecond X-ray crystallography of protein nanocrystals using ultrashort and intense pulses from an X-ray free-electron laser has proved to be a successful method for structural determination. However, due to significant variations in diffraction pattern quality from pulse to pulse only a fraction of the collected frames can be used. Experimentally, the X-ray temporal pulse profile is not known and can vary with every shot. This simulation study describes how the pulse shape affects the damage dynamics, which ultimately affects the biological interpretation of electron density. The instantaneously detected signal varies during the pulse exposure due to the pulse properties, as well as the structural and electronic changes in the sample. Here ionization and atomic motion are simulated using a radiation transfer plasma code. Pulses with parameters typical for X-ray free-electron lasers are considered: pulse energies ranging from 10(4) to 10(7) J cm(-2) with photon energies from 2 to 12 keV, up to 100 fs long. Radiation damage in the form of sample heating that will lead to a loss of crystalline periodicity and changes in scattering factor due to electronic reconfigurations of ionized atoms are considered here. The simulations show differences in the dynamics of the radiation damage processes for different temporal pulse profiles and intensities, where ionization or atomic motion could be predominant. The different dynamics influence the recorded diffracted signal in any given resolution and will affect the subsequent structure determination. PMID:25723927

  2. Developments in the Implementation of Acoustic Droplet Ejection for Protein Crystallography.

    PubMed

    Wu, Ping; Noland, Cameron; Ultsch, Mark; Edwards, Bonnie; Harris, David; Mayer, Robert; Harris, Seth F

    2016-02-01

    Acoustic droplet ejection (ADE) enables crystallization experiments at the low-nanoliter scale, resulting in rapid vapor diffusion equilibration dynamics and efficient reagent usage in the empirical discovery of structure-enabling protein crystallization conditions. We extend our validation of this technology applied to the diverse physicochemical property space of aqueous crystallization reagents where dynamic fluid analysis coupled to ADE aids in accurate and precise dispensations. Addition of crystallization seed stocks, chemical additives, or small-molecule ligands effectively modulates crystallization, and we here provide examples in optimization of crystal morphology and diffraction quality by the acoustic delivery of ultra-small volumes of these cofactors. Additional applications are discussed, including set up of in situ proteolysis and alternate geometries of crystallization that leverage the small scale afforded by acoustic delivery. Finally, we describe parameters of a system of automation in which the acoustic liquid handler is integrated with a robotic arm, plate centrifuge, peeler, sealer, and stacks, which allows unattended high-throughput crystallization experimentation. PMID:26275619

  3. Improving Protocols for Protein Mapping through Proper Comparison to Crystallography Data

    PubMed Central

    Lexa, Katrina W.; Carlson, Heather A.

    2013-01-01

    Computational approaches to fragment-based drug design (FBDD) can complement experiments and facilitate the identification of potential hot spots along the protein surface. However, the evaluation of computational methods for mapping binding sites frequently focuses upon the ability to reproduce crystallographic coordinates to within a low RMSD threshold. This dependency on the deposited coordinate data overlooks the original electron density from the experiment, thus techniques may be developed based upon subjective - or even erroneous - atomic coordinates. This can become a significant drawback in applications to systems where the location of hot spots is unknown. Based on comparison to crystallographic density, we previously showed that mixed-solvent molecular dynamics (MixMD) accurately identifies the active site for HEWL, with acetonitrile as an organic solvent. Here, we concentrated on the influence of protic solvent on simulation and refined the optimal MixMD approach for extrapolation of the method to systems without established sites. Our results establish an accurate approach for comparing simulations to experiment. We have outlined the most efficient strategy for MixMD, based on simulation length and number of runs. The development outlined here makes MixMD a robust method which should prove useful across a broad range of target structures. Lastly, our results with MixMD match experimental data so well that consistency between simulations and density may be a useful way to aid the identification of probes vs waters during the refinement of future MSCS crystallographic structures. PMID:23327200

  4. Improving protocols for protein mapping through proper comparison to crystallography data.

    PubMed

    Lexa, Katrina W; Carlson, Heather A

    2013-02-25

    Computational approaches to fragment-based drug design (FBDD) can complement experiments and facilitate the identification of potential hot spots along the protein surface. However, the evaluation of computational methods for mapping binding sites frequently focuses upon the ability to reproduce crystallographic coordinates to within a low RMSD threshold. This dependency on the deposited coordinate data overlooks the original electron density from the experiment, thus techniques may be developed based upon subjective-or even erroneous-atomic coordinates. This can become a significant drawback in applications to systems where the location of hot spots is unknown. On the basis of comparison to crystallographic density, we previously showed that mixed-solvent molecular dynamics (MixMD) accurately identifies the active site for HEWL, with acetonitrile as an organic solvent. Here, we concentrated on the influence of protic solvent on simulation and refined the optimal MixMD approach for extrapolation of the method to systems without established sites. Our results establish an accurate approach for comparing simulations to experiment. We have outlined the most efficient strategy for MixMD, based on simulation length and number of runs. The development outlined here makes MixMD a robust method which should prove useful across a broad range of target structures. Lastly, our results with MixMD match experimental data so well that consistency between simulations and density may be a useful way to aid the identification of probes vs waters during the refinement of future multiple solvent crystallographic structures. PMID:23327200

  5. Preliminary time-of-flight neutron diffraction studies of Escherichia coli ABC transport receptor phosphate-binding protein at the Protein Crystallography Station

    PubMed Central

    Sippel, K. H.; Bacik, J.; Quiocho, F. A.; Fisher, S. Z.

    2014-01-01

    Inorganic phosphate is an essential molecule for all known life. Organisms have developed many mechanisms to ensure an adequate supply, even in low-phosphate conditions. In prokaryotes phosphate transport is instigated by the phosphate-binding protein (PBP), the initial receptor for the ATP-binding cassette (ABC) phosphate transporter. In the crystal structure of the PBP–phosphate complex, the phosphate is completely desolvated and sequestered in a deep cleft and is bound by 13 hydrogen bonds: 12 to protein NH and OH donor groups and one to a carboxylate acceptor group. The carboxylate plays a key recognition role by accepting a phosphate hydrogen. PBP phosphate affinity is relatively consistent across a broad pH range, indicating the capacity to bind monobasic (H2PO4 −) and dibasic (HPO4 2−) phosphate; however, the mechanism by which it might accommodate the second hydrogen of monobasic phosphate is unclear. To answer this question, neutron diffraction studies were initiated. Large single crystals with a volume of 8 mm3 were grown and subjected to hydrogen/deuterium exchange. A 2.5 Å resolution data set was collected on the Protein Crystallography Station at the Los Alamos Neutron Science Center. Initial refinement of the neutron data shows significant nuclear density, and refinement is ongoing. This is the first report of a neutron study from this superfamily. PMID:24915101

  6. Radiation Damage and Racemic Protein Crystallography Reveal the Unique Structure of the GASA/Snakin Protein Superfamily.

    PubMed

    Yeung, Ho; Squire, Christopher J; Yosaatmadja, Yuliana; Panjikar, Santosh; López, Gemma; Molina, Antonio; Baker, Edward N; Harris, Paul W R; Brimble, Margaret A

    2016-07-01

    Proteins from the GASA/snakin superfamily are common in plant proteomes and have diverse functions, including hormonal crosstalk, development, and defense. One 63-residue member of this family, snakin-1, an antimicrobial protein from potatoes, has previously been chemically synthesized in a fully active form. Herein the 1.5 Å structure of snakin-1, determined by a novel combination of racemic protein crystallization and radiation-damage-induced phasing (RIP), is reported. Racemic crystals of snakin-1 and quasi-racemic crystals incorporating an unnatural 4-iodophenylalanine residue were prepared from chemically synthesized d- and l-proteins. Breakage of the C-I bonds in the quasi-racemic crystals facilitated structure determination by RIP. The crystal structure reveals a unique protein fold with six disulfide crosslinks, presenting a distinct electrostatic surface that may target the protein to microbial cell surfaces. PMID:27145301

  7. X-ray Crystallography Facility

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Edward Snell, a National Research Council research fellow at NASA's Marshall Space Flight Center (MSFC), prepares a protein crystal for analysis by x-ray crystallography as part of NASA's structural biology program. The small, individual crystals are bombarded with x-rays to produce diffraction patterns, a map of the intensity of the x-rays as they reflect through the crystal.

  8. The future of crystallography in drug discovery

    PubMed Central

    Zheng, Heping; Hou, Jing; Zimmerman, Matthew D; Wlodawer, Alexander; Minor, Wladek

    2014-01-01

    Introduction X-ray crystallography plays an important role in structure-based drug design (SBDD), and accurate analysis of crystal structures of target macromolecules and macromolecule–ligand complexes is critical at all stages. However, whereas there has been significant progress in improving methods of structural biology, particularly in X-ray crystallography, corresponding progress in the development of computational methods (such as in silico high-throughput screening) is still on the horizon. Crystal structures can be overinterpreted and thus bias hypotheses and follow-up experiments. As in any experimental science, the models of macromolecular structures derived from X-ray diffraction data have their limitations, which need to be critically evaluated and well understood for structure-based drug discovery. Areas covered This review describes how the validity, accuracy and precision of a protein or nucleic acid structure determined by X-ray crystallography can be evaluated from three different perspectives: i) the nature of the diffraction experiment; ii) the interpretation of an electron density map; and iii) the interpretation of the structural model in terms of function and mechanism. The strategies to optimally exploit a macromolecular structure are also discussed in the context of ‘Big Data’ analysis, biochemical experimental design and structure-based drug discovery. Expert opinion Although X-ray crystallography is one of the most detailed ‘microscopes’ available today for examining macromolecular structures, the authors would like to re-emphasize that such structures are only simplified models of the target macromolecules. The authors also wish to reinforce the idea that a structure should not be thought of as a set of precise coordinates but rather as a framework for generating hypotheses to be explored. Numerous biochemical and biophysical experiments, including new diffraction experiments, can and should be performed to verify or falsify

  9. From crystallography to life

    NASA Astrophysics Data System (ADS)

    Allen, Roland E.

    2014-06-01

    2014 is the International Year of Crystallography, an extremely broad field which has had enormous impact in biology and materials science. Both experimental facilities and methods for interpreting the data have become increasingly sophisticated during the past century, and many highly complex systems have now been characterized, including large proteins and other biological macromolecules. A very few representative examples are mentioned here, including crystallographic studies of proteins that regulate programmed cell death (apoptosis), and structure determinations of G-protein coupled receptors (GPCRs), respectively the subjects of the 2014 Aminoff Prize and the 2012 Nobel Prize in chemistry. Normal apoptosis is essential for human embryonic development, prevention of cancer, and other processes within multicellular organisms. GPCRs are the targets of about half of all modern medicinal drugs, since they are responsible for the majority of cellular responses to hormones and neurotransmitters, as well as the senses of sight, taste, and smell. In materials, the behavior of electrons (both ordinary and exotic) is largely determined by the arrangement of the atoms. As examples, we mention carbon-based materials (diamond, buckyballs, nanotubes, and graphene) and high-temperature superconductors (cuprate and iron-based).

  10. Status of the crystallography beamlines at SSRF

    NASA Astrophysics Data System (ADS)

    He, Jianhua; Gao, Xingyu

    2015-02-01

    The Shanghai Synchrotron Radiation Facility (SSRF), an advanced intermediate-energy third-generation light source in China, was completed with seven phase-I beamlines opening to users in May 2009. Among these beamlines, there are two dedicated crystallography beamlines, one for macromolecular crystallography and one for crystallography in materials science, condensed matter physics and other relevant fields. The macromolecular crystallography beamline BL17U1, based on an in-vacuum undulator, has achieved very high brightness at the sample position with its flux of 4.1 × 1012 photons/s at 12 keV and focused beam size of FWHM (H × V) 67 × 23 μm2 in a small beam divergence over an energy range of 5-18 keV. Nowadays, there are about 200 user groups at this beamline with more than 330 structures solved each year. In the past, lots of significant results have been obtained at this beamline, such as the structural determination of important membrane proteins and proteins of viruses. In addition, three new macromolecular crystallography beamlines of different features have just been constructed and will soon open to users. To meet the rapidly growing user demands and the important scientific challenges, a few more dedicated crystallography beamlines have been proposed in the Phase-II Beamlines Project.

  11. Lipidic cubic phase serial millisecond crystallography using synchrotron radiation

    PubMed Central

    Nogly, Przemyslaw; James, Daniel; Wang, Dingjie; White, Thomas A.; Zatsepin, Nadia; Shilova, Anastasya; Nelson, Garrett; Liu, Haiguang; Johansson, Linda; Heymann, Michael; Jaeger, Kathrin; Metz, Markus; Wickstrand, Cecilia; Wu, Wenting; Båth, Petra; Berntsen, Peter; Oberthuer, Dominik; Panneels, Valerie; Cherezov, Vadim; Chapman, Henry; Schertler, Gebhard; Neutze, Richard; Spence, John; Moraes, Isabel; Burghammer, Manfred; Standfuss, Joerg; Weierstall, Uwe

    2015-01-01

    Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven proton-pump bacteriorhodopsin (bR) at a resolution of 2.4 Å. The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway. PMID:25866654

  12. Developments in optics and performance at BL13-XALOC, the macromolecular crystallography beamline at the Alba Synchrotron

    PubMed Central

    Juanhuix, Jordi; Gil-Ortiz, Fernando; Cuní, Guifré; Colldelram, Carles; Nicolás, Josep; Lidón, Julio; Boter, Eva; Ruget, Claude; Ferrer, Salvador; Benach, Jordi

    2014-01-01

    BL13-XALOC is currently the only macromolecular crystallography beamline at the 3 GeV ALBA synchrotron near Barcelona, Spain. The optics design is based on an in-vacuum undulator, a Si(111) channel-cut crystal monochromator and a pair of KB mirrors. It allows three main operation modes: a focused configuration, where both mirrors can focus the beam at the sample position to 52 µm × 5.5 µm FWHM (H × V); a defocused configuration that can match the size of the beam to the dimensions of the crystals or to focus the beam at the detector; and an unfocused configuration, where one or both mirrors are removed from the photon beam path. To achieve a uniform defocused beam, the slope errors of the mirrors were reduced down to 55 nrad RMS by employing a novel method that has been developed at the ALBA high-accuracy metrology laboratory. Thorough commissioning with X-ray beam and user operation has demonstrated an excellent energy and spatial stability of the beamline. The end-station includes a high-accuracy single-axis diffractometer, a removable mini-kappa stage, an automated sample-mounting robot and a photon-counting detector that allows shutterless operation. The positioning tables of the diffractometer and the detector are based on a novel and highly stable design. This equipment, together with the operation flexibility of the beamline, allows a large variety of types of crystals to be tackled, from medium-sized crystals with large unit-cell parameters to microcrystals. Several examples of data collections measured during beamline commissioning are described. The beamline started user operation on 18 July 2012. PMID:24971961

  13. Warm dense crystallography

    NASA Astrophysics Data System (ADS)

    Valenza, Ryan A.; Seidler, Gerald T.

    2016-03-01

    The intense femtosecond-scale pulses from x-ray free electron lasers (XFELs) are able to create and interrogate interesting states of matter characterized by long-lived nonequilibrium semicore or core electron occupancies or by the heating of dense phases via the relaxation cascade initiated by the photoelectric effect. We address here the latter case of "warm dense matter" (WDM) and investigate the observable consequences of x-ray heating of the electronic degrees of freedom in crystalline systems. We report temperature-dependent density functional theory calculations for the x-ray diffraction from crystalline LiF, graphite, diamond, and Be. We find testable, strong signatures of condensed-phase effects that emphasize the importance of wide-angle scattering to study nonequilibrium states. These results also suggest that the reorganization of the valence electron density at eV-scale temperatures presents a confounding factor to achieving atomic resolution in macromolecular serial femtosecond crystallography (SFX) studies at XFELs, as performed under the "diffract before destroy" paradigm.

  14. Neutron proton crystallography station (PCS)

    SciTech Connect

    Fisher, Zoe; Kovalevsky, Andrey; Johnson, Hannah; Mustyakimov, Marat

    2009-01-01

    The PCS (Protein Crystallography Station) at Los Alamos Neutron Science Center (LANSCE) is a unique facility in the USA that is designed and optimized for detecting and collecting neutron diffraction data from macromolecular crystals. PCS utilizes the 20 Hz spallation neutron source at LANSCE to enable time-of-flight measurements using 0.6-7.0 {angstrom} neutrons. This increases the neutron flux on the sample by using a wavelength range that is optimal for studying macromolecular crystal structures. The diagram below show a schematic of PCS and photos of the detector and instrument cave.

  15. Structural analysis of flexible proteins in solution by SmallAngle X-ray Scattering combined with crystallography

    SciTech Connect

    Tsutakawa, Susan E.; Hura, Greg L.; Frankel, Ken A.; Cooper,Priscilla K.; Tainer, John A.

    2006-05-25

    In the last few years, SAXS of biological materials has been rapidly evolving and promises to move structural analysis to a new level. Recent innovations in SAXS data analysis allow ab initio shape predictions of proteins in solution. Furthermore, experimental scattering data can be compared to calculated scattering curves from the growing data base of solved structures and also identify aggregation and unfolded proteins. Combining SAXS results with atomic resolution structures enables detailed characterizations in solution of mass, radius, conformations, assembly, and shape changes associated with protein folding and functions. SAXS can efficiently reveal the spatial organization of protein domains, including domains missing from or disordered in known crystal structures, and establish cofactor or substrate-induced conformational changes. For flexible domains or unstructured regions that are not amenable for study by many other structural techniques, SAXS provides a unique technology. Here, we present SAXS shape predictions for PCNA that accurately predict a trimeric ring assembly and for a full-length DNA repair glycosylase with a large unstructured region. These new results in combination with illustrative published data show how SAXS combined with high resolution crystal structures efficiently establishes architectures, assemblies, conformations, and unstructured regions for proteins and protein complexes in solution.

  16. Performance of protein stability predictors.

    PubMed

    Khan, Sofia; Vihinen, Mauno

    2010-06-01

    Stability is a fundamental property affecting function, activity, and regulation of biomolecules. Stability changes are often found for mutated proteins involved in diseases. Stability predictors computationally predict protein-stability changes caused by mutations. We performed a systematic analysis of 11 online stability predictors' performances. These predictors are CUPSAT, Dmutant, FoldX, I-Mutant2.0, two versions of I-Mutant3.0 (sequence and structure versions), MultiMutate, MUpro, SCide, Scpred, and SRide. As input, 1,784 single mutations found in 80 proteins were used, and these mutations did not include those used for training. The programs' performances were also assessed according to where the mutations were found in the proteins, that is, in secondary structures and on the surface or in the core of a protein, and according to protein structure type. The extents to which the mutations altered the occupied volumes at the residue sites and the charge interactions were also characterized. The predictions of all programs were in line with the experimental data. I-Mutant3.0 (utilizing structural information), Dmutant, and FoldX were the most reliable predictors. The stability-center predictors performed with similar accuracy. However, at best, the predictions were only moderately accurate ( approximately 60%) and significantly better tools would be needed for routine analysis of mutation effects. PMID:20232415

  17. Using macromolecular-crystallography beamline and microfluidic platform for small-angle diffraction studies of lipidic matrices for membrane-protein crystallization

    NASA Astrophysics Data System (ADS)

    Kondrashkina, E.; Khvostichenko, D. S.; Perry, S. L.; Von Osinski, J.; Kenis, P. J. A.; Brister, K.

    2013-03-01

    Macromolecular-crystallography (MX) beamlines routinely provide a possibility to change X-ray beam energy, focus the beam to a size of tens of microns, align a sample on a microdiffractometer using on-axis video microscope, and collect data with an area-detector positioned in three dimensions. These capabilities allow for running complementary measurements of small-angle X-ray scattering and diffraction (SAXS) at the same beamline with such additions to the standard MX setup as a vacuum path between the sample and the detector, a modified beam stop, and a custom sample cell. On the 21-ID-D MX beamline at the Advanced Photon Source we attach a vacuum flight tube to the area detector support and use the support motion for aligning a beam stop built into the rear end of the flight tube. At 8 KeV energy and 1 m sample-to-detector distance we can achieve a small-angle resolution of 0.01A-1 in the reciprocal space. Measuring SAXS with this setup, we have studied phase diagrams of lipidic mesophases used as matrices for membrane-protein crystallization. The outcome of crystallization trials is significantly affected by the structure of the lipidic mesophases, which is determined by the composition of the crystallization mixture. We use a microfluidic chip for the mesophase formulation and in situ SAXS data collection. Using the MX beamline and the microfluidic platform we have demonstrated the viability of the high-throughput SAXS studies facilitating screening of lipidic matrices for membrane-protein crystallization.

  18. Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source

    PubMed Central

    Classen, Scott; Hura, Greg L.; Holton, James M.; Rambo, Robert P.; Rodic, Ivan; McGuire, Patrick J.; Dyer, Kevin; Hammel, Michal; Meigs, George; Frankel, Kenneth A.; Tainer, John A.

    2013-01-01

    The SIBYLS beamline (12.3.1) of the Advanced Light Source at Lawrence Berkeley National Laboratory, supported by the US Department of Energy and the National Institutes of Health, is optimized for both small-angle X-ray scattering (SAXS) and macromolecular crystallography (MX), making it unique among the world’s mostly SAXS or MX dedicated beamlines. Since SIBYLS was commissioned, assessments of the limitations and advantages of a combined SAXS and MX beamline have suggested new strategies for integration and optimal data collection methods and have led to additional hardware and software enhancements. Features described include a dual mode monochromator [containing both Si(111) crystals and Mo/B4C multilayer elements], rapid beamline optics conversion between SAXS and MX modes, active beam stabilization, sample-loading robotics, and mail-in and remote data collection. These features allow users to gain valuable insights from both dynamic solution scattering and high-resolution atomic diffraction experiments performed at a single synchrotron beamline. Key practical issues considered for data collection and analysis include radiation damage, structural ensembles, alternative conformers and flexibility. SIBYLS develops and applies efficient combined MX and SAXS methods that deliver high-impact results by providing robust cost-effective routes to connect structures to biology and by performing experiments that aid beamline designs for next generation light sources. PMID:23396808

  19. Crystallography without Crystals: An Overview

    NASA Astrophysics Data System (ADS)

    Ourmazd, Abbas

    2007-03-01

    Protein X-ray crystallography, an ``outgrowth of physics,'' is now the mainstay of biology, biochemistry, and the pharmaceutical industry. However, roughly 40% of biological molecules do not crystallize. And although more than half a million proteins have been sequenced, the structure of less than 40,000 has been determined. By obviating the need for purification and crystallization, the ability to determine the structure of individual biological molecules would constitute a fundamental breakthrough. The confluence of four developments has generated intense interest in achieving this by short-pulse X-ray scattering: *The advent of algorithms capable of ``solving the phase problem'' with practical demonstrations in astronomy, high-energy electron diffraction, and protein crystallography [1,2,3]. *Development of sophisticated techniques for determining the relative orientation of electron microscope images of biological entities such as cells and large macromolecules [4]. *Development of techniques for producing beams of hydrated proteins [3,5]. *The promise of ultra-bright, short pulses of X-rays from X-ray Free Electron Lasers (XFELs) under construction in the US, Europe, and Japan. I will describe how these and other key developments have brought the prospect of single-molecule structure determination ``tantalizingly close,'' perhaps even closer than generally realized in the literature. [1] J. R. Fienup, Appl. Opt. 21, 2758 (1982). [2] J. Miao et al. PNAS 98, 6641 (2001). [3] J.C.H. Spence et al. Acta Cryst. A61, 237 (2005) [4] J. Frank, Three-Dimensional Electron Microscopy of Macromolecular Assemblies (OUP Press, 2006) [5] J.B. Fenn, J. Biomolecular Techniques 13, 101 (2002).

  20. High-resolution neutron protein crystallography with radically small crystal volumes: application of perdeuteration to human aldose reductase.

    PubMed

    Hazemann, I; Dauvergne, M T; Blakeley, M P; Meilleur, F; Haertlein, M; Van Dorsselaer, A; Mitschler, A; Myles, D A A; Podjarny, A

    2005-10-01

    Neutron diffraction data have been collected to 2.2 Angstrom resolution from a small (0.15 mm(3)) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase [h-AR(D)], subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm(3) are reported. Neutron data were recorded to 2 Angstrom resolution, with subsequent data analysis using data to 2.2 Angstrom. This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples. PMID:16204895

  1. High-resolution neutron protein crystallography with radically small crystal volumes: Application of perdeuteration to human aldose reductase

    SciTech Connect

    Hazemann, I.; Dauvergne, M. T.; Blakeley, M. P.; Meilleur, Flora; Haertlein, M.; Van Dorsselaer, A.; Mitschler, A.; Myles, Dean A A; Podjarny, A.

    2005-08-01

    Neutron diffraction data have been collected to 2.2 {angstrom} resolution from a small (0.15 mm{sup 3}) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase [h-AR(D)], subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm{sup 3} are reported. Neutron data were recorded to 2 {angstrom} resolution, with subsequent data analysis using data to 2.2 {angstrom}. This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples.

  2. X-ray crystallography facility for the international space station

    SciTech Connect

    McdDonald, William T.; Lewis, Johanna L.; Smith, Craig D.; DeLucas, Lawrence J.

    1997-01-10

    Directed by NASA's Office of Space Access and Technology (OSAT), the University of Alabama at Birmingham (UAB) Center for Macromolecular Crystallography (CMC) recently completed a Design Feasibility Study for the X-ray Crystallography Facility (XCF) for the International Space Station (ISS). The XCF is a facility for growing macromolecular protein crystals; harvesting, selecting, and mounting sample crystals, and snap-freezing the samples, if necessary; performing x-ray diffraction; and downlinking the diffraction data to the ground. Knowledge of the structure of protein molecules is essential for the development of pharmaceuticals by structure-based drug design techniques. Currently, x-ray diffraction of high quality protein crystals is the only method of determining the structure of these macromolecules. High quality protein crystals have been grown in microgravity onboard the Space Shuttle Orbiter for more than 10 years, but these crystals always have been returned to Earth for x-ray diffraction. The XCF will allow crystal growth, harvesting, mounting, and x-ray diffraction onboard the ISS, maximizing diffraction data quality and timeliness. This paper presents the XCF design concept, describing key feasibility issues for the ISS application and advanced technologies and operational features which resolve those issues. The conclusion is that the XCF design is feasible and can be operational onboard the ISS by early in 2002.

  3. Serial femtosecond crystallography: the first five years

    PubMed Central

    Schlichting, Ilme

    2015-01-01

    Protein crystallography using synchrotron radiation sources has had a tremendous impact on biology, having yielded the structures of thousands of proteins and given detailed insight into their mechanisms. However, the technique is limited by the requirement for macroscopic crystals, which can be difficult to obtain, as well as by the often severe radiation damage caused in diffraction experiments, in particular when using tiny crystals. To slow radiation damage, data collection is typically performed at cryogenic temperatures. With the advent of free-electron lasers (FELs) capable of delivering extremely intense femtosecond X-ray pulses, this situation appears to be remedied, allowing the structure determination of undamaged macromolecules using either macroscopic or microscopic crystals. The latter are exposed to the FEL beam in random orientations and their diffraction data are collected at cryogenic or room temperature in a serial fashion, since each crystal is destroyed upon a single exposure. The new approaches required for crystal growth and delivery, and for diffraction data analysis, including de novo phasing, are reviewed. The opportunities and challenges of SFX are described, including applications such as time-resolved measurements and the analysis of radiation damage-prone systems. PMID:25866661

  4. Automated High Throughput Drug Target Crystallography

    SciTech Connect

    Rupp, B

    2005-02-18

    The molecular structures of drug target proteins and receptors form the basis for 'rational' or structure guided drug design. The majority of target structures are experimentally determined by protein X-ray crystallography, which as evolved into a highly automated, high throughput drug discovery and screening tool. Process automation has accelerated tasks from parallel protein expression, fully automated crystallization, and rapid data collection to highly efficient structure determination methods. A thoroughly designed automation technology platform supported by a powerful informatics infrastructure forms the basis for optimal workflow implementation and the data mining and analysis tools to generate new leads from experimental protein drug target structures.

  5. High-Throughput Methods for Electron Crystallography

    PubMed Central

    Stokes, David L.; Ubarretxena-Belandia, Iban; Gonen, Tamir; Engel, Andreas

    2013-01-01

    Membrane proteins play a tremendously important role in cell physiology and serve as a target for an increasing number of drugs. Structural information is key to understanding their function and for developing new strategies for combating disease. However, the complex physical chemistry associated with membrane proteins has made them more difficult to study than their soluble cousins. Electron crystallography has historically been a successful method for solving membrane protein structures and has the advantage of providing the natural environment of a lipid membrane. Specifically, when membrane proteins form two-dimensional arrays within a lipid bilayer, images and diffraction can be recorded by electron microscopy. The corresponding data can be combined to produce a three-dimensional reconstruction which, under favorable conditions, can extend to atomic resolution. Like X-ray crystallography, the quality of the structures are very much dependent on the order and size of the crystals. However, unlike X-ray crystallography, high-throughput methods for screening crystallization trials for electron crystallography are not in general use. In this chapter, we describe two alternative and potentially complementary methods for high-throughput screening of membrane protein crystallization within the lipid bilayer. The first method relies on the conventional use of dialysis for removing detergent and thus reconstituting the bilayer; an array of dialysis wells in the standard 96-well format allows the use of a liquid-handling robot and greatly increases throughput. The second method relies on detergent complexation by cyclodextrin; a specialized pipetting robot has been designed not only to titrate cyclodextrin, but to use light scattering to monitor the reconstitution process. In addition, the use of liquid-handling robots for making negatively stained grids and methods for automatically imaging samples in the electron microscope are described. PMID:23132066

  6. Crystallography of icosahedral crystals

    NASA Astrophysics Data System (ADS)

    Bak, P.

    The crystallography of icosahedral crystals is constructed. The actual three-dimensional crystal is represented by a three-dimensional cut in a regular six-dimensional periodic crystal with symmetry described by a six-dimensional space group, and the positions of atoms correspond to an arrangement of hypersurface segments. The resulting crystal cannot in general be viewed as a space-filling arrangemment of a small number of different Penrose tiles. The intensities of Bragg spots are given directly as the intensities of Bragg spots of the six-dimensional crystal.

  7. Missed opportunities in crystallography.

    PubMed

    Dauter, Zbigniew; Jaskolski, Mariusz

    2014-09-01

    Scrutinized from the perspective of time, the giants in the history of crystallography more than once missed a nearly obvious chance to make another great discovery, or went in the wrong direction. This review analyzes such missed opportunities focusing on macromolecular crystallographers (using Perutz, Pauling, Franklin as examples), although cases of particular historical (Kepler), methodological (Laue, Patterson) or structural (Pauling, Ramachandran) relevance are also described. Linus Pauling, in particular, is presented several times in different circumstances, as a man of vision, oversight, or even blindness. His example underscores the simple truth that also in science incessant creativity is inevitably connected with some probability of fault. PMID:24814223

  8. Neutron Laue diffraction in macromolecular crystallography

    NASA Astrophysics Data System (ADS)

    Myles, D. A. A.; Bon, C.; Langan, P.; Cipriani, F.; Castagna, J. C.; Lehmann, M. S.; Wilkinson, C.

    The time scales required for conventional neutron diffraction analysis of biological single crystals at, or near, atomic resolution are prohibitive - such studies are rarely performed. Laue (white beam) diffraction can provide a more rapid and efficient survey of reciprocal space, maximising the flux at the sample and stimulating large numbers of reflections simultaneously. A LAue DIffractometer (LADI), designed specifically for macromolecular crystallography, has been installed on a cold neutron guide at ILL. The detector comprises a large Gd 2O 3-doped neutron-sensitive image plate (400 × 800 mm) mounted on a cylindrical camera (318 mm diameter) that is read in phonographic mode after exposure. Detector response has been evaluated and performance indicators are given. Narrow (Quasi-Laue) band-passes (d/ gl/ λ = 8-20%) are often required for large unit-cell biological crystals in order to reduce reflection overlap and incoherent background. Laue and Quasi-Laue data have now been collected for a number of proteins and other biological crystals. Recent results are presented and future prospects reviewed.

  9. Crystallography: past and present

    NASA Astrophysics Data System (ADS)

    Hodeau, J.-L.; Guinebretiere, R.

    2007-12-01

    In the 19th century, crystallography referred to the study of crystal shapes. Such studies by Haüy and Bravais allowed the establishment of important hypotheses such as (i) “les molécules intégrantes qui sont censées être les plus petits solides que l’on puisse extraire d’un minéral” [1], (ii) the definition of the crystal lattice and (iii) “le cristal est clivable parallèlement à deux ou trois formes cristallines” [2]. This morphological crystallography defined a crystal like “a chemically homogeneous solid, wholly or partly bounded by natural planes that intersect at predetermined angles” [3]. It described the main symmetry elements and operations, nomenclatures of different crystal forms and also the theory of twinning. A breakthrough appeared in 1912 with the use of X-rays by M. von Laue and W.H. and W.L. Bragg. This experimental development allowed the determination of the atomic content of each unit cell constituting the crystal and defined a crystal as “any solid in which an atomic pattern is repeated periodically in three dimensions, that is, any solid that “diffracts” an incident X-ray beam” [3]. Mathematical tools like the Patterson methods, the direct methods, were developed. The way for solving crystalline structure was opened first for simple compounds and at that time crystallography was associated mainly with perfect crystals. In the fifties, crystallographers already had most apparatus and fundamental methods at their disposal; however, we had to wait for the development of computers to see the full use of these tools. Furthermore the development of new sources of neutrons, electrons and synchrotron X-rays allowed the studies of complex compounds like large macromolecules in biology. Nowadays, one of the new frontiers for crystallographers is to relate the crystal structure to its physical-chemical-biological properties, this means that an accurate structural determination is needed to focus on a selective part of the

  10. Site-specific Incorporation of 3-Iodo-L-tyrosine into Proteins and Single-wavelength Anomalous Dispersion Phasing with Soft X-ray in Protein Crystallography

    NASA Astrophysics Data System (ADS)

    Murayama, Kazutaka; Sakamoto, Kensaku

    Iodine is a good anomalous scatter for radiations from in-house X-ray generators (Cu/CrKα). Non-natural amino acid, 3-iodo-L-tyrosine, is able to be site-specifically incorporated into proteins with amber suppresser tRNA and mutated TyrRS from M. jannaschii in the E. coli expression system. To determine the crystal structure of acetyl transferase from T. thermophilus, iodotyrosine-containing proteins were prepared and crystallized. Structure determination was successfully conducted with the protein variant with iodotyrosine at position 111. Anomalous signals from iodotyrosine with Cu/CrKα radiations were both sufficient to calculate clear electron density map. In the crystal structure, iodotyrosine did not significantly disturb the native structure.

  11. Serial crystallography captures enzyme catalysis in copper nitrite reductase at atomic resolution from one crystal.

    PubMed

    Horrell, Sam; Antonyuk, Svetlana V; Eady, Robert R; Hasnain, S Samar; Hough, Michael A; Strange, Richard W

    2016-07-01

    Relating individual protein crystal structures to an enzyme mechanism remains a major and challenging goal for structural biology. Serial crystallography using multiple crystals has recently been reported in both synchrotron-radiation and X-ray free-electron laser experiments. In this work, serial crystallography was used to obtain multiple structures serially from one crystal (MSOX) to study in crystallo enzyme catalysis. Rapid, shutterless X-ray detector technology on a synchrotron MX beamline was exploited to perform low-dose serial crystallography on a single copper nitrite reductase crystal, which survived long enough for 45 consecutive 100 K X-ray structures to be collected at 1.07-1.62 Å resolution, all sampled from the same crystal volume. This serial crystallography approach revealed the gradual conversion of the substrate bound at the catalytic type 2 Cu centre from nitrite to nitric oxide, following reduction of the type 1 Cu electron-transfer centre by X-ray-generated solvated electrons. Significant, well defined structural rearrangements in the active site are evident in the series as the enzyme moves through its catalytic cycle, namely nitrite reduction, which is a vital step in the global denitrification process. It is proposed that such a serial crystallography approach is widely applicable for studying any redox or electron-driven enzyme reactions from a single protein crystal. It can provide a 'catalytic reaction movie' highlighting the structural changes that occur during enzyme catalysis. The anticipated developments in the automation of data analysis and modelling are likely to allow seamless and near-real-time analysis of such data on-site at some of the powerful synchrotron crystallographic beamlines. PMID:27437114

  12. Serial crystallography captures enzyme catalysis in copper nitrite reductase at atomic resolution from one crystal

    PubMed Central

    Horrell, Sam; Antonyuk, Svetlana V.; Eady, Robert R.; Hasnain, S. Samar; Hough, Michael A.; Strange, Richard W.

    2016-01-01

    Relating individual protein crystal structures to an enzyme mechanism remains a major and challenging goal for structural biology. Serial crystallography using multiple crystals has recently been reported in both synchrotron-radiation and X-ray free-electron laser experiments. In this work, serial crystallography was used to obtain multiple structures serially from one crystal (MSOX) to study in crystallo enzyme catalysis. Rapid, shutterless X-ray detector technology on a synchrotron MX beamline was exploited to perform low-dose serial crystallography on a single copper nitrite reductase crystal, which survived long enough for 45 consecutive 100 K X-ray structures to be collected at 1.07–1.62 Å resolution, all sampled from the same crystal volume. This serial crystallography approach revealed the gradual conversion of the substrate bound at the catalytic type 2 Cu centre from nitrite to nitric oxide, following reduction of the type 1 Cu electron-transfer centre by X-ray-generated solvated electrons. Significant, well defined structural rearrangements in the active site are evident in the series as the enzyme moves through its catalytic cycle, namely nitrite reduction, which is a vital step in the global denitrification process. It is proposed that such a serial crystallography approach is widely applicable for studying any redox or electron-driven enzyme reactions from a single protein crystal. It can provide a ‘catalytic reaction movie’ highlighting the structural changes that occur during enzyme catalysis. The anticipated developments in the automation of data analysis and modelling are likely to allow seamless and near-real-time analysis of such data on-site at some of the powerful synchrotron crystallographic beamlines. PMID:27437114

  13. In situ macromolecular crystallography using microbeams

    PubMed Central

    Axford, Danny; Owen, Robin L.; Aishima, Jun; Foadi, James; Morgan, Ann W.; Robinson, James I.; Nettleship, Joanne E.; Owens, Raymond J.; Moraes, Isabel; Fry, Elizabeth E.; Grimes, Jonathan M.; Harlos, Karl; Kotecha, Abhay; Ren, Jingshan; Sutton, Geoff; Walter, Thomas S.; Stuart, David I.; Evans, Gwyndaf

    2012-01-01

    Despite significant progress in high-throughput methods in macromolecular crystallography, the production of diffraction-quality crystals remains a major bottleneck. By recording diffraction in situ from crystals in their crystallization plates at room temperature, a number of problems associated with crystal handling and cryoprotection can be side-stepped. Using a dedicated goniometer installed on the microfocus macromolecular crystallography beamline I24 at Diamond Light Source, crystals have been studied in situ with an intense and flexible microfocus beam, allowing weakly diffracting samples to be assessed without a manual crystal-handling step but with good signal to noise, despite the background scatter from the plate. A number of case studies are reported: the structure solution of bovine enterovirus 2, crystallization screening of membrane proteins and complexes, and structure solution from crystallization hits produced via a high-throughput pipeline. These demonstrate the potential for in situ data collection and structure solution with microbeams. PMID:22525757

  14. Advanced beamline automation for biological crystallography experiments.

    PubMed

    Cork, Carl; O'Neill, James; Taylor, John; Earnest, Thomas

    2006-08-01

    An automated crystal-mounting/alignment system has been developed at Lawrence Berkeley National Laboratory and has been installed on three of the protein-crystallography experimental stations at the Advanced Light Source (ALS); it is currently being implemented at synchrotron crystallography beamlines at CHESS, NSLS and the APS. The benefits to using an automounter system include (i) optimization of the use of synchrotron beam time, (ii) facilitation of advanced data-collection techniques, (iii) collection of higher quality data, (iv) reduction of the risk to crystals and (v) exploration of systematic studies of experimental protocols. Developments on the next-generation automounter with improvements in robustness, automated alignment and sample tracking are under way, with an end-to-end data-flow process being developed to allow remote data collection and monitoring. PMID:16855300

  15. Resolution of structural heterogeneity in dynamic crystallography

    PubMed Central

    Ren, Zhong; Chan, Peter W. Y.; Moffat, Keith; Pai, Emil F.; Royer, William E.; Šrajer, Vukica; Yang, Xiaojing

    2013-01-01

    Dynamic behavior of proteins is critical to their function. X-­ray crystallography, a powerful yet mostly static technique, faces inherent challenges in acquiring dynamic information despite decades of effort. Dynamic ‘structural changes’ are often indirectly inferred from ‘structural differences’ by comparing related static structures. In contrast, the direct observation of dynamic structural changes requires the initiation of a biochemical reaction or process in a crystal. Both the direct and the indirect approaches share a common challenge in analysis: how to interpret the structural heterogeneity intrinsic to all dynamic processes. This paper presents a real-space approach to this challenge, in which a suite of analytical methods and tools to identify and refine the mixed structural species present in multiple crystallographic data sets have been developed. These methods have been applied to representative scenarios in dynamic crystallography, and reveal structural information that is otherwise difficult to interpret or inaccessible using conventional methods. PMID:23695239

  16. Fixed target combined with spectral mapping: approaching 100% hit rates for serial crystallography.

    PubMed

    Oghbaey, Saeed; Sarracini, Antoine; Ginn, Helen M; Pare-Labrosse, Olivier; Kuo, Anling; Marx, Alexander; Epp, Sascha W; Sherrell, Darren A; Eger, Bryan T; Zhong, Yinpeng; Loch, Rolf; Mariani, Valerio; Alonso-Mori, Roberto; Nelson, Silke; Lemke, Henrik T; Owen, Robin L; Pearson, Arwen R; Stuart, David I; Ernst, Oliver P; Mueller-Werkmeister, Henrike M; Miller, R J Dwayne

    2016-08-01

    The advent of ultrafast highly brilliant coherent X-ray free-electron laser sources has driven the development of novel structure-determination approaches for proteins, and promises visualization of protein dynamics on sub-picosecond timescales with full atomic resolution. Significant efforts are being applied to the development of sample-delivery systems that allow these unique sources to be most efficiently exploited for high-throughput serial femtosecond crystallography. Here, the next iteration of a fixed-target crystallography chip designed for rapid and reliable delivery of up to 11 259 protein crystals with high spatial precision is presented. An experimental scheme for predetermining the positions of crystals in the chip by means of in situ spectroscopy using a fiducial system for rapid, precise alignment and registration of the crystal positions is presented. This delivers unprecedented performance in serial crystallography experiments at room temperature under atmospheric pressure, giving a raw hit rate approaching 100% with an effective indexing rate of approximately 50%, increasing the efficiency of beam usage and allowing the method to be applied to systems where the number of crystals is limited. PMID:27487825

  17. High-pressure crystallography of periodic and aperiodic crystals.

    PubMed

    Hejny, Clivia; Minkov, Vasily S

    2015-03-01

    More than five decades have passed since the first single-crystal X-ray diffraction experiments at high pressure were performed. These studies were applied historically to geochemical processes occurring in the Earth and other planets, but high-pressure crystallography has spread across different fields of science including chemistry, physics, biology, materials science and pharmacy. With each passing year, high-pressure studies have become more precise and comprehensive because of the development of instrumentation and software, and the systems investigated have also become more complicated. Starting with crystals of simple minerals and inorganic compounds, the interests of researchers have shifted to complicated metal-organic frameworks, aperiodic crystals and quasicrystals, molecular crystals, and even proteins and viruses. Inspired by contributions to the microsymposium 'High-Pressure Crystallography of Periodic and Aperiodic Crystals' presented at the 23rd IUCr Congress and General Assembly, the authors have tried to summarize certain recent results of single-crystal studies of molecular and aperiodic structures under high pressure. While the selected contributions do not cover the whole spectrum of high-pressure research, they demonstrate the broad diversity of novel and fascinating results and may awaken the reader's interest in this topic. PMID:25866659

  18. High-pressure crystallography of periodic and aperiodic crystals

    PubMed Central

    Hejny, Clivia; Minkov, Vasily S.

    2015-01-01

    More than five decades have passed since the first single-crystal X-ray diffraction experiments at high pressure were performed. These studies were applied historically to geochemical processes occurring in the Earth and other planets, but high-pressure crystallography has spread across different fields of science including chemistry, physics, biology, materials science and pharmacy. With each passing year, high-pressure studies have become more precise and comprehensive because of the development of instrumentation and software, and the systems investigated have also become more complicated. Starting with crystals of simple minerals and inorganic compounds, the interests of researchers have shifted to complicated metal–organic frameworks, aperiodic crystals and quasicrystals, molecular crystals, and even proteins and viruses. Inspired by contributions to the microsymposium ‘High-Pressure Crystallography of Periodic and Aperiodic Crystals’ presented at the 23rd IUCr Congress and General Assembly, the authors have tried to summarize certain recent results of single-crystal studies of molecular and aperiodic structures under high pressure. While the selected contributions do not cover the whole spectrum of high-pressure research, they demonstrate the broad diversity of novel and fascinating results and may awaken the reader’s interest in this topic. PMID:25866659

  19. Structural physiology based on electron crystallography

    PubMed Central

    Fujiyoshi, Yoshinori

    2011-01-01

    There are many questions in brain science, which are extremely interesting but very difficult to answer. For example, how do education and other experiences during human development influence the ability and personality of the adult? The molecular mechanisms underlying such phenomena are still totally unclear. However, technological and instrumental advancements of electron microscopy have facilitated comprehension of the structures of biological components, cells, and organelles. Electron crystallography is especially good for studying the structure and function of membrane proteins, which are key molecules of signal transduction in neural and other cells. Electron crystallography is now an established technique to analyze the structures of membrane proteins in lipid bilayers, which are close to their natural biological environment. By utilizing cryo-electron microscopes with helium cooled specimen stages, which were developed through a personal motivation to understand functions of neural systems from a structural point of view, structures of membrane proteins were analyzed at a resolution higher than 3 Å. This review has four objectives. First, it is intended to introduce the new research field of structural physiology. Second, it introduces some of the personal struggles, which were involved in developing the cryo-electron microscope. Third, it discusses some of the technology for the structural analysis of membrane proteins based on cryo-electron microscopy. Finally, it reviews structural and functional analyses of membrane proteins. PMID:21416541

  20. Metalloprotein Crystallography: More than a Structure.

    PubMed

    Bowman, Sarah E J; Bridwell-Rabb, Jennifer; Drennan, Catherine L

    2016-04-19

    Metal ions and metallocofactors play important roles in a broad range of biochemical reactions. Accordingly, it has been estimated that as much as 25-50% of the proteome uses transition metal ions to carry out a variety of essential functions. The metal ions incorporated within metalloproteins fulfill functional roles based on chemical properties, the diversity of which arises as transition metals can adopt different redox states and geometries, dictated by the identity of the metal and the protein environment. The coupling of a metal ion with an organic framework in metallocofactors, such as heme and cobalamin, further expands the chemical functionality of metals in biology. The three-dimensional visualization of metal ions and complex metallocofactors within a protein scaffold is often a starting point for enzymology, highlighting the importance of structural characterization of metalloproteins. Metalloprotein crystallography, however, presents a number of implicit challenges including correctly incorporating the relevant metal or metallocofactor, maintaining the proper environment for the protein to be purified and crystallized (including providing anaerobic, cold, or aphotic environments), and being mindful of the possibility of X-ray induced damage to the proteins or incorporated metal ions. Nevertheless, the incorporated metals or metallocofactors also present unique advantages in metalloprotein crystallography. The significant resonance that metals undergo with X-ray photons at wavelengths used for protein crystallography and the rich electronic properties of metals, which provide intense and spectroscopically unique signatures, allow a metalloprotein crystallographer to use anomalous dispersion to determine phases for structure solution and to use simultaneous or parallel spectroscopic techniques on single crystals. These properties, coupled with the improved brightness of beamlines, the ability to tune the wavelength of the X-ray beam, the availability of

  1. Metalloprotein Crystallography: More than a Structure

    PubMed Central

    2016-01-01

    Conspectus Metal ions and metallocofactors play important roles in a broad range of biochemical reactions. Accordingly, it has been estimated that as much as 25–50% of the proteome uses transition metal ions to carry out a variety of essential functions. The metal ions incorporated within metalloproteins fulfill functional roles based on chemical properties, the diversity of which arises as transition metals can adopt different redox states and geometries, dictated by the identity of the metal and the protein environment. The coupling of a metal ion with an organic framework in metallocofactors, such as heme and cobalamin, further expands the chemical functionality of metals in biology. The three-dimensional visualization of metal ions and complex metallocofactors within a protein scaffold is often a starting point for enzymology, highlighting the importance of structural characterization of metalloproteins. Metalloprotein crystallography, however, presents a number of implicit challenges including correctly incorporating the relevant metal or metallocofactor, maintaining the proper environment for the protein to be purified and crystallized (including providing anaerobic, cold, or aphotic environments), and being mindful of the possibility of X-ray induced damage to the proteins or incorporated metal ions. Nevertheless, the incorporated metals or metallocofactors also present unique advantages in metalloprotein crystallography. The significant resonance that metals undergo with X-ray photons at wavelengths used for protein crystallography and the rich electronic properties of metals, which provide intense and spectroscopically unique signatures, allow a metalloprotein crystallographer to use anomalous dispersion to determine phases for structure solution and to use simultaneous or parallel spectroscopic techniques on single crystals. These properties, coupled with the improved brightness of beamlines, the ability to tune the wavelength of the X-ray beam, the

  2. Automated sample mounting and alignment system for biological crystallography at a synchrotron source.

    PubMed

    Snell, Gyorgy; Cork, Carl; Nordmeyer, Robert; Cornell, Earl; Meigs, George; Yegian, Derek; Jaklevic, Joseph; Jin, Jian; Stevens, Raymond C; Earnest, Thomas

    2004-04-01

    High-throughput data collection for macromolecular crystallography requires an automated sample mounting and alignment system for cryo-protected crystals that functions reliably when integrated into protein-crystallography beamlines at synchrotrons. Rapid mounting and dismounting of the samples increases the efficiency of the crystal screening and data collection processes, where many crystals can be tested for the quality of diffraction. The sample-mounting subsystem has random access to 112 samples, stored under liquid nitrogen. Results of extensive tests regarding the performance and reliability of the system are presented. To further increase throughput, we have also developed a sample transport/storage system based on "puck-shaped" cassettes, which can hold sixteen samples each. Seven cassettes fit into a standard dry shipping Dewar. The capabilities of a robotic crystal mounting and alignment system with instrumentation control software and a relational database allows for automated screening and data collection to be developed. PMID:15062077

  3. Automated sample mounting and technical advance alignment system for biological crystallography at a synchrotron source

    SciTech Connect

    Snell, Gyorgy; Cork, Carl; Nordmeyer, Robert; Cornell, Earl; Meigs, George; Yegian, Derek; Jaklevic, Joseph; Jin, Jian; Stevens, Raymond C.; Earnest, Thomas

    2004-01-07

    High-throughput data collection for macromolecular crystallography requires an automated sample mounting system for cryo-protected crystals that functions reliably when integrated into protein-crystallography beamlines at synchrotrons. Rapid mounting and dismounting of the samples increases the efficiency of the crystal screening and data collection processes, where many crystals can be tested for the quality of diffraction. The sample-mounting subsystem has random access to 112 samples, stored under liquid nitrogen. Results of extensive tests regarding the performance and reliability of the system are presented. To further increase throughput, we have also developed a sample transport/storage system based on ''puck-shaped'' cassettes, which can hold sixteen samples each. Seven cassettes fit into a standard dry shipping Dewar. The capabilities of a robotic crystal mounting and alignment system with instrumentation control software and a relational database allows for automated screening and data collection to be developed.

  4. Native sulfur/chlorine SAD phasing for serial femtosecond crystallography

    SciTech Connect

    Nakane, Takanori; Song, Changyong; Suzuki, Mamoru; Nango, Eriko; Kobayashi, Jun; Masuda, Tetsuya; Inoue, Shigeyuki; Mizohata, Eiichi; Nakatsu, Toru; Tanaka, Tomoyuki; Tanaka, Rie; Shimamura, Tatsuro; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Yabashi, Makina; Nureki, Osamu; Iwata, So; Sugahara, Michihiro

    2015-11-27

    Sulfur SAD phasing facilitates the structure determination of diverse native proteins using femtosecond X-rays from free-electron lasers via serial femtosecond crystallography. Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 Å is successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures.

  5. A technique for determining the deuterium/hydrogen contrast map in neutron macromolecular crystallography.

    PubMed

    Chatake, Toshiyuki; Fujiwara, Satoru

    2016-01-01

    A difference in the neutron scattering length between hydrogen and deuterium leads to a high density contrast in neutron Fourier maps. In this study, a technique for determining the deuterium/hydrogen (D/H) contrast map in neutron macromolecular crystallography is developed and evaluated using ribonuclease A. The contrast map between the D2O-solvent and H2O-solvent crystals is calculated in real space, rather than in reciprocal space as performed in previous neutron D/H contrast crystallography. The present technique can thus utilize all of the amplitudes of the neutron structure factors for both D2O-solvent and H2O-solvent crystals. The neutron D/H contrast maps clearly demonstrate the powerful detectability of H/D exchange in proteins. In fact, alternative protonation states and alternative conformations of hydroxyl groups are observed at medium resolution (1.8 Å). Moreover, water molecules can be categorized into three types according to their tendency towards rotational disorder. These results directly indicate improvement in the neutron crystal structure analysis. This technique is suitable for incorporation into the standard structure-determination process used in neutron protein crystallography; consequently, more precise and efficient determination of the D-atom positions is possible using a combination of this D/H contrast technique and standard neutron structure-determination protocols. PMID:26894536

  6. Oil-free hyaluronic acid matrix for serial femtosecond crystallography

    PubMed Central

    Sugahara, Michihiro; Song, Changyong; Suzuki, Mamoru; Masuda, Tetsuya; Inoue, Shigeyuki; Nakane, Takanori; Yumoto, Fumiaki; Nango, Eriko; Tanaka, Rie; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Yabashi, Makina; Nureki, Osamu; Numata, Keiji; Iwata, So

    2016-01-01

    The grease matrix was originally introduced as a microcrystal-carrier for serial femtosecond crystallography and has been expanded to applications for various types of proteins, including membrane proteins. However, the grease-based matrix has limited application for oil-sensitive proteins. Here we introduce a grease-free, water-based hyaluronic acid matrix. Applications for proteinase K and lysozyme proteins were able to produce electron density maps at 2.3-Å resolution. PMID:27087008

  7. Oil-free hyaluronic acid matrix for serial femtosecond crystallography.

    PubMed

    Sugahara, Michihiro; Song, Changyong; Suzuki, Mamoru; Masuda, Tetsuya; Inoue, Shigeyuki; Nakane, Takanori; Yumoto, Fumiaki; Nango, Eriko; Tanaka, Rie; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Yabashi, Makina; Nureki, Osamu; Numata, Keiji; Iwata, So

    2016-01-01

    The grease matrix was originally introduced as a microcrystal-carrier for serial femtosecond crystallography and has been expanded to applications for various types of proteins, including membrane proteins. However, the grease-based matrix has limited application for oil-sensitive proteins. Here we introduce a grease-free, water-based hyaluronic acid matrix. Applications for proteinase K and lysozyme proteins were able to produce electron density maps at 2.3-Å resolution. PMID:27087008

  8. Oil-free hyaluronic acid matrix for serial femtosecond crystallography

    NASA Astrophysics Data System (ADS)

    Sugahara, Michihiro; Song, Changyong; Suzuki, Mamoru; Masuda, Tetsuya; Inoue, Shigeyuki; Nakane, Takanori; Yumoto, Fumiaki; Nango, Eriko; Tanaka, Rie; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Yabashi, Makina; Nureki, Osamu; Numata, Keiji; Iwata, So

    2016-04-01

    The grease matrix was originally introduced as a microcrystal-carrier for serial femtosecond crystallography and has been expanded to applications for various types of proteins, including membrane proteins. However, the grease-based matrix has limited application for oil-sensitive proteins. Here we introduce a grease-free, water-based hyaluronic acid matrix. Applications for proteinase K and lysozyme proteins were able to produce electron density maps at 2.3-Å resolution.

  9. Introduction to a general crystallography.

    PubMed

    Janner, A

    2001-07-01

    The definition of an extended crystallographic group is given, based on an n-dimensional Euclidean space, carrier of a faithful integral representation of a permutation group of atomic positions. The Euclidean crystallography of normal crystals and the higher-dimensional one applied to incommensurately modulated crystals, intergrowth crystals and quasicrystals are special cases of a general crystallography. The same is true for the multimetrical crystallographic characterization of ice and of snow crystals. This approach can also be applied to single molecules, leading to what may be denoted as molecular crystallography. It possibly allows non-trivial structural relations between atomic positions belonging to the asymmetric unit of the molecular point group to be obtained. Two simple molecules, polycyclic aromatic hydrocarbons, are treated as illustrative examples. PMID:11418747

  10. X-ray crystallography

    NASA Technical Reports Server (NTRS)

    2001-01-01

    X-rays diffracted from a well-ordered protein crystal create sharp patterns of scattered light on film. A computer can use these patterns to generate a model of a protein molecule. To analyze the selected crystal, an X-ray crystallographer shines X-rays through the crystal. Unlike a single dental X-ray, which produces a shadow image of a tooth, these X-rays have to be taken many times from different angles to produce a pattern from the scattered light, a map of the intensity of the X-rays after they diffract through the crystal. The X-rays bounce off the electron clouds that form the outer structure of each atom. A flawed crystal will yield a blurry pattern; a well-ordered protein crystal yields a series of sharp diffraction patterns. From these patterns, researchers build an electron density map. With powerful computers and a lot of calculations, scientists can use the electron density patterns to determine the structure of the protein and make a computer-generated model of the structure. The models let researchers improve their understanding of how the protein functions. They also allow scientists to look for receptor sites and active areas that control a protein's function and role in the progress of diseases. From there, pharmaceutical researchers can design molecules that fit the active site, much like a key and lock, so that the protein is locked without affecting the rest of the body. This is called structure-based drug design.

  11. Graphene-based microfluidics for serial crystallography.

    PubMed

    Sui, Shuo; Wang, Yuxi; Kolewe, Kristopher W; Srajer, Vukica; Henning, Robert; Schiffman, Jessica D; Dimitrakopoulos, Christos; Perry, Sarah L

    2016-08-01

    Microfluidic strategies to enable the growth and subsequent serial crystallographic analysis of micro-crystals have the potential to facilitate both structural characterization and dynamic structural studies of protein targets that have been resistant to single-crystal strategies. However, adapting microfluidic crystallization platforms for micro-crystallography requires a dramatic decrease in the overall device thickness. We report a robust strategy for the straightforward incorporation of single-layer graphene into ultra-thin microfluidic devices. This architecture allows for a total material thickness of only ∼1 μm, facilitating on-chip X-ray diffraction analysis while creating a sample environment that is stable against significant water loss over several weeks. We demonstrate excellent signal-to-noise in our X-ray diffraction measurements using a 1.5 μs polychromatic X-ray exposure, and validate our approach via on-chip structure determination using hen egg white lysozyme (HEWL) as a model system. Although this work is focused on the use of graphene for protein crystallography, we anticipate that this technology should find utility in a wide range of both X-ray and other lab on a chip applications. PMID:27241728

  12. Electron Crystallography – The Waking Beauty of Structural Biology

    PubMed Central

    Pope, Christopher R; Unger, Vinzenz M

    2012-01-01

    Since its debut in the mid 70ties, electron crystallography has been a valuable alternative in the structure determination of biological macromolecules. Its reliance on single- or double-layered two-dimensionally ordered arrays and the ability to obtain structural information from small and disordered crystals make this approach particularly useful for the study of membrane proteins in a lipid bilayer environment. Despite its unique advantages, technological hurdles have kept electron crystallography from reaching its full potential. Addressing the issues, recent initiatives developed high-throughput pipelines for crystallization and screening. Adding progress in automating data collection, image analysis and phase extension methods, electron crystallography is poised to raise its profile and may lead the way in exploring the structural biology of macromolecular complexes. PMID:22525160

  13. Crystallography of ribosomal particles

    NASA Astrophysics Data System (ADS)

    Yonath, A.; Frolow, F.; Shoham, M.; Müssig, J.; Makowski, I.; Glotz, C.; Jahn, W.; Weinstein, S.; Wittmann, H. G.

    1988-07-01

    Several forms of three-dimensional crystals and two-dimensional sheets of intact ribosomes and their subunits have been obtained as a result of: (a) an extensive systematic investigation of the parameters involved in crystallization, (b) a development of an experimental procedure for controlling the volumes of the crystallization droplets, (c) a study of the nucleation process, and (d) introducing a delicate seeding procedure coupled with variations in the ratios of mono- and divalent ions in the crystallization medium. In all cases only biologically active particles could be crystallized, and the crystalline material retains its integrity and activity. Crystallographic data have been collected from crystals of 50S ribosomal subunits, using synchrotron radiation at temperatures between + 19 and - 180°C. Although at 4°C the higher resolution reflections decay within minutes in the synchrotron beam, at cryo-temperature there was hardly any radiation damage, and a complete set of data to about 6Åresolution could be collected from a single crystal. Heavy-atom clusters were used for soaking as well as for specific binding to the surface of the ribosomal subunits prior to crystallization. The 50S ribosomal subunits from a mutant of B. stearothermophilus which lacks the ribosomal protein BL11 crystallize isomorphously with in the native ones. Models, aimed to be used for low resolution phasing, have been reconstructed from two-dimensional sheets of 70S ribosomes and 50S subunits at 47 and 30Å, respectively. These models show the overall structure of these particles, the contact areas between the large and small subunits, the space where protein synthesis might take place and a tunnel which may provide the path for the nascent protein chain.

  14. Sub-atomic resolution X-ray crystallography and neutron crystallography: promise, challenges and potential.

    PubMed

    Blakeley, Matthew P; Hasnain, Samar S; Antonyuk, Svetlana V

    2015-07-01

    The International Year of Crystallography saw the number of macromolecular structures deposited in the Protein Data Bank cross the 100000 mark, with more than 90000 of these provided by X-ray crystallography. The number of X-ray structures determined to sub-atomic resolution (i.e. ≤1 Å) has passed 600 and this is likely to continue to grow rapidly with diffraction-limited synchrotron radiation sources such as MAX-IV (Sweden) and Sirius (Brazil) under construction. A dozen X-ray structures have been deposited to ultra-high resolution (i.e. ≤0.7 Å), for which precise electron density can be exploited to obtain charge density and provide information on the bonding character of catalytic or electron transfer sites. Although the development of neutron macromolecular crystallography over the years has been far less pronounced, and its application much less widespread, the availability of new and improved instrumentation, combined with dedicated deuteration facilities, are beginning to transform the field. Of the 83 macromolecular structures deposited with neutron diffraction data, more than half (49/83, 59%) were released since 2010. Sub-mm(3) crystals are now regularly being used for data collection, structures have been determined to atomic resolution for a few small proteins, and much larger unit-cell systems (cell edges >100 Å) are being successfully studied. While some details relating to H-atom positions are tractable with X-ray crystallography at sub-atomic resolution, the mobility of certain H atoms precludes them from being located. In addition, highly polarized H atoms and protons (H(+)) remain invisible with X-rays. Moreover, the majority of X-ray structures are determined from cryo-cooled crystals at 100 K, and, although radiation damage can be strongly controlled, especially since the advent of shutterless fast detectors, and by using limited doses and crystal translation at micro-focus beams, radiation damage can still take place. Neutron

  15. Sub-atomic resolution X-ray crystallography and neutron crystallography: promise, challenges and potential

    PubMed Central

    Blakeley, Matthew P.; Hasnain, Samar S.; Antonyuk, Svetlana V.

    2015-01-01

    The International Year of Crystallography saw the number of macromolecular structures deposited in the Protein Data Bank cross the 100000 mark, with more than 90000 of these provided by X-ray crystallography. The number of X-ray structures determined to sub-atomic resolution (i.e. ≤1 Å) has passed 600 and this is likely to continue to grow rapidly with diffraction-limited synchrotron radiation sources such as MAX-IV (Sweden) and Sirius (Brazil) under construction. A dozen X-ray structures have been deposited to ultra-high resolution (i.e. ≤0.7 Å), for which precise electron density can be exploited to obtain charge density and provide information on the bonding character of catalytic or electron transfer sites. Although the development of neutron macromolecular crystallography over the years has been far less pronounced, and its application much less widespread, the availability of new and improved instrumentation, combined with dedicated deuteration facilities, are beginning to transform the field. Of the 83 macromolecular structures deposited with neutron diffraction data, more than half (49/83, 59%) were released since 2010. Sub-mm3 crystals are now regularly being used for data collection, structures have been determined to atomic resolution for a few small proteins, and much larger unit-cell systems (cell edges >100 Å) are being successfully studied. While some details relating to H-atom positions are tractable with X-ray crystallography at sub-atomic resolution, the mobility of certain H atoms precludes them from being located. In addition, highly polarized H atoms and protons (H+) remain invisible with X-rays. Moreover, the majority of X-ray structures are determined from cryo-cooled crystals at 100 K, and, although radiation damage can be strongly controlled, especially since the advent of shutterless fast detectors, and by using limited doses and crystal translation at micro-focus beams, radiation damage can still take place. Neutron

  16. Crystal growth and crystallography

    NASA Technical Reports Server (NTRS)

    Chernov, A. A.

    1998-01-01

    Selected topics that may be of interest for both crystal-structure and crystal-growth communities are overviewed. The growth of protein crystals, along with that of some other compounds, is one of the topics, and recent insights into related phenomena are considered as examples of applications of general principles. The relationship between crystal growth shape and structure is reviewed and an attempt to introduce semiquantitative characterization of binding for proteins is made. The concept of kinks for complex structures is briefly discussed. Even at sufficiently low supersaturations, the fluctuation of steps may not be sufficient to implement the Gibbs-Thomson law if the kink density is low enough. Subsurface ordering of liquids and growth of rough interfaces from melts is discussed. Crystals growing in microgravity from solution should be more perfect if they preferentially trap stress-inducing impurities, thus creating an impurity-depleted zone around themselves. Evidently, such a zone is developed only around the crystals growing in the absence of convection. Under terrestrial conditions, the self-purified depleted zone is destroyed by convection, the crystal traps more impurity and grows stressed. The stress relief causes mosaicity. In systems containing stress-inducing but poorly trapped impurities, the crystals grown in the absence of convection should be worse than those of their terrestrial counterparts.

  17. Raman Crystallography and Other Biochemical Applications of Raman Microscopy

    NASA Astrophysics Data System (ADS)

    Carey, Paul R.

    2006-05-01

    Recent studies using a Raman microscope have shown that single protein crystals provide an ideal platform to undertake Raman difference spectroscopic analyses under nonresonance conditions. This approach, termed Raman crystallography, provides a means of characterizing chemical events within the crystal such as ligand binding and enzyme reactions. In many cases Raman crystallography goes hand in hand with X-ray crystallographic studies because the Raman results can inform the X-ray crystallographer about the status of chemical events in the crystal prior to flash freezing and X-ray analysis. In turn, the combined data from the Raman and X-ray analyses are highly synergistic and offer novel perspectives on structure and dynamics in enzyme active sites. In a related area, protein misfolding, Raman microscopy can provide detailed insights into the chemistry of the amyloid plaques associated with Alzheimer's disease and into the intermediates on the α-synuclein protein misfolding pathway implicated in Parkinson's disease.

  18. Combining biophysical screening and X-ray crystallography for fragment-based drug discovery.

    PubMed

    Hennig, Michael; Ruf, Armin; Huber, Walter

    2012-01-01

    Over the past decade, fragment-based drug discovery (FBDD) has gained importance for the generation of novel ideas to inspire synthetic chemistry. In order to identify small molecules that bind to a target protein, multiple approaches have been utilized by various groups in the pharmaceutical industry and by academic groups. The combination of fragment screening by biophysical methods and in particular with surface plasmon resonance technologies (SPR) together with the visualization of the binding properties by X-ray crystallography offers a number of benefits. Screening by SPR identifies ligands for a target protein as well as provides an assessment of the binding properties with respect to affinity, stoichiometry, and specificity of the interaction. Despite the huge technology advances of the past years, X-ray crystallography is still a resource-intensive technology, and SPR binding data provides excellent measures to prioritize X-ray experiments and consequently enable a better success rate in obtaining structural information. Information on the chemical structures of fragments binding to a protein can be used to perform similarity searches in compound libraries in order to establish structure-activity relationships as well as to explore particular scaffolds. At Roche we have applied this workflow for a number of targets and the experiences will be outlined in this review. PMID:21837555

  19. Micro-crystallography comes of age

    PubMed Central

    Smith, Janet L.; Fischetti, Robert F.; Yamamoto, Masaki

    2012-01-01

    The latest revolution in macromolecular crystallography was incited by the development of dedicated, user friendly, micro-crystallography beamlines. Brilliant X-ray beams of diameter 20 microns or less, now available at most synchrotron sources, enable structure determination from samples that previously were inaccessible. Relative to traditional crystallography, crystals with one or more small dimensions have diffraction patterns with vastly improved signal-to-noise when recorded with an appropriately matched beam size. Structures can be solved from isolated, well diffracting regions within inhomogeneous samples. This review summarizes the technological requirements and approaches to producing micro-beams and how they continue to change the practice of crystallography. PMID:23021872

  20. Modulation of kinase-inhibitor interactions by auxiliary protein binding: Crystallography studies on Aurora A interactions with VX-680 and with TPX2

    SciTech Connect

    Zhao, Baoguang; Smallwood, Angela; Yang, Jingsong; Koretke, Kristin; Nurse, Kelvin; Calamari, Amy; Kirkpatrick, Robert B.; Lai, Zhihong

    2008-10-24

    VX-680, also known as MK-0457, is an ATP-competitive small molecule inhibitor of the Aurora kinases that has entered phase II clinical trials for the treatment of cancer. We have solved the cocrystal structure of AurA/TPX2/VX-680 at 2.3 {angstrom} resolution. In the crystal structure, VX-680 binds to the active conformation of AurA. The glycine-rich loop in AurA adopts a unique bent conformation, forming a {pi}-{pi} interaction with the phenyl group of VX-680. In contrast, in the published AurA/VX-680 structure, VX-680 binds to AurA in the inactive conformation, interacting with a hydrophobic pocket only present in the inactive conformation. These data suggest that TPX2, a protein cofactor, can alter the binding mode of VX-680 with AurA. More generally, the presence of physiologically relevant cofactor proteins can alter the kinetics, binding interactions, and inhibition of enzymes, and studies with these multiprotein complexes may be beneficial to the discovery and optimization of enzyme inhibitors as therapeutic agents.

  1. Native sulfur/chlorine SAD phasing for serial femtosecond crystallography

    PubMed Central

    Nakane, Takanori; Song, Changyong; Suzuki, Mamoru; Nango, Eriko; Kobayashi, Jun; Masuda, Tetsuya; Inoue, Shigeyuki; Mizohata, Eiichi; Nakatsu, Toru; Tanaka, Tomoyuki; Tanaka, Rie; Shimamura, Tatsuro; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Yabashi, Makina; Nureki, Osamu; Iwata, So; Sugahara, Michihiro

    2015-01-01

    Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 Å is successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures. PMID:26627659

  2. Novel protein-inhibitor interactions in site 3 of Ca(2+)-bound S100B as discovered by X-ray crystallography.

    PubMed

    Cavalier, Michael C; Melville, Zephan; Aligholizadeh, Ehson; Raman, E Prabhu; Yu, Wenbo; Fang, Lei; Alasady, Milad; Pierce, Adam D; Wilder, Paul T; MacKerell, Alexander D; Weber, David J

    2016-06-01

    Structure-based drug discovery is under way to identify and develop small-molecule S100B inhibitors (SBiXs). Such inhibitors have therapeutic potential for treating malignant melanoma, since high levels of S100B downregulate wild-type p53 tumor suppressor function in this cancer. Computational and X-ray crystallographic studies of two S100B-SBiX complexes are described, and both compounds (apomorphine hydrochloride and ethidium bromide) occupy an area of the S100B hydrophobic cleft which is termed site 3. These data also reveal novel protein-inhibitor interactions which can be used in future drug-design studies to improve SBiX affinity and specificity. Of particular interest, apomorphine hydrochloride showed S100B-dependent killing in melanoma cell assays, although the efficacy exceeds its affinity for S100B and implicates possible off-target contributions. Because there are no structural data available for compounds occupying site 3 alone, these studies contribute towards the structure-based approach to targeting S100B by including interactions with residues in site 3 of S100B. PMID:27303795

  3. The success story of crystallography.

    PubMed

    Schwarzenbach, Dieter

    2012-01-01

    Diffractionists usually place the birth of crystallography in 1912 with the first X-ray diffraction experiment of Friedrich, Knipping and Laue. This discovery propelled the mathematical branch of mineralogy to global importance and enabled crystal structure determination. Knowledge of the geometrical structure of matter at atomic resolution had revolutionary consequences for all branches of the natural sciences: physics, chemistry, biology, earth sciences and material science. It is scarcely possible for a single person in a single article to trace and appropriately value all of these developments. This article presents the limited, subjective view of its author and a limited selection of references. The bulk of the article covers the history of X-ray structure determination from the NaCl structure to aperiodic structures and macromolecular structures. The theoretical foundations were available by 1920. The subsequent success of crystallography was then due to the development of diffraction equipment, the theory of the solution of the phase problem, symmetry theory and computers. The many structures becoming known called for the development of crystal chemistry and of data banks. Diffuse scattering from disordered structures without and with partial long-range order allows determination of short-range order. Neutron and electron scattering and diffraction are also mentioned. PMID:22186283

  4. A novel inert crystal delivery medium for serial femtosecond crystallography

    SciTech Connect

    Conrad, Chelsie E.; Basu, Shibom; James, Daniel; Wang, Dingjie; Schaffer, Alexander; Roy-Chowdhury, Shatabdi; Zatsepin, Nadia A.; Aquila, Andrew; Coe, Jesse; Gati, Cornelius; Hunter, Mark S.; Koglin, Jason E.; Kupitz, Christopher; Nelson, Garrett; Subramanian, Ganesh; White, Thomas A.; Zhao, Yun; Zook, James; Boutet, Sébastien; Cherezov, Vadim; Spence, John C. H.; Fromme, Raimund; Weierstall, Uwe; Fromme, Petra

    2015-06-30

    Serial femtosecond crystallography (SFX) has opened a new era in crystallography by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5Å resolution using 300µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.

  5. A novel inert crystal delivery medium for serial femtosecond crystallography

    DOE PAGESBeta

    Conrad, Chelsie E.; Basu, Shibom; James, Daniel; Wang, Dingjie; Schaffer, Alexander; Roy-Chowdhury, Shatabdi; Zatsepin, Nadia A.; Aquila, Andrew; Coe, Jesse; Gati, Cornelius; et al

    2015-06-30

    Serial femtosecond crystallography (SFX) has opened a new era in crystallography by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, themore » structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.« less

  6. A novel inert crystal delivery medium for serial femtosecond crystallography

    SciTech Connect

    Conrad, Chelsie E.; Basu, Shibom; James, Daniel; Wang, Dingjie; Schaffer, Alexander; Roy-Chowdhury, Shatabdi; Zatsepin, Nadia A.; Aquila, Andrew; Coe, Jesse; Gati, Cornelius; Hunter, Mark S.; Koglin, Jason E.; Kupitz, Christopher; Nelson, Garrett; Subramanian, Ganesh; White, Thomas A.; Zhao, Yun; Zook, James; Boutet, Sébastien; Cherezov, Vadim; Spence, John C. H.; Fromme, Raimund; Weierstall, Uwe; Fromme, Petra

    2015-06-30

    Serial femtosecond crystallography (SFX) has opened a new era in crystallography by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.

  7. A novel inert crystal delivery medium for serial femtosecond crystallography.

    PubMed

    Conrad, Chelsie E; Basu, Shibom; James, Daniel; Wang, Dingjie; Schaffer, Alexander; Roy-Chowdhury, Shatabdi; Zatsepin, Nadia A; Aquila, Andrew; Coe, Jesse; Gati, Cornelius; Hunter, Mark S; Koglin, Jason E; Kupitz, Christopher; Nelson, Garrett; Subramanian, Ganesh; White, Thomas A; Zhao, Yun; Zook, James; Boutet, Sébastien; Cherezov, Vadim; Spence, John C H; Fromme, Raimund; Weierstall, Uwe; Fromme, Petra

    2015-07-01

    Serial femtosecond crystallography (SFX) has opened a new era in crystallo-graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes. PMID:26177184

  8. Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography.

    PubMed

    Mueller, C; Marx, A; Epp, S W; Zhong, Y; Kuo, A; Balo, A R; Soman, J; Schotte, F; Lemke, H T; Owen, R L; Pai, E F; Pearson, A R; Olson, J S; Anfinrud, P A; Ernst, O P; Dwayne Miller, R J

    2015-09-01

    We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linac Coherent Light Source (LCLS, Menlo Park, California, USA). The chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs. PMID:26798825

  9. Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography

    PubMed Central

    Mueller, C.; Marx, A.; Epp, S. W.; Zhong, Y.; Kuo, A.; Balo, A. R.; Soman, J.; Schotte, F.; Lemke, H. T.; Owen, R. L.; Pai, E. F.; Pearson, A. R.; Olson, J. S.; Anfinrud, P. A.; Ernst, O. P.; Dwayne Miller, R. J.

    2015-01-01

    We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linac Coherent Light Source (LCLS, Menlo Park, California, USA). The chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs. PMID:26798825

  10. Implications of the focal beam profile in serial femtosecond crystallography

    SciTech Connect

    Galli, Lorenzo; Chapman, Henry N.; Metcalf, Peter

    2015-05-12

    The photon density profile of an X-ray free-electron laser (XFEL) beam at the focal position is a critical parameter for serial femtosecond crystallography (SFX), but is difficult to measure because of the destructive power of the beam. A novel high intensity radiation induced phasing method (HIRIP) has been proposed as a general experimental approach for protein structure determination, but has proved to be sensitive to variations of the X-ray intensity, with uniform incident fluence desired for best performance. Here we show that experimental SFX data collected at the nano-focus chamber of the Coherent X-ray Imaging end-station at the Linac Coherent Light Source using crystals with a limited size distribution suggests an average profile of the X-ray beam that has a large variation of intensity. We propose a new method to improve the quality of high fluence data for HI-RIP, by identifying and removing diffraction patterns from crystals exposed to the low intensity region of the beam. The method requires crystals of average size comparable to the width of the focal spot.

  11. Sample mounts for microcrystal crystallography

    NASA Technical Reports Server (NTRS)

    Thorne, Robert E. (Inventor); Stum, Zachary (Inventor); O'Neill, Kevin (Inventor); Kmetko, Jan (Inventor)

    2007-01-01

    Sample mounts (10) for mounting microcrystals of biological macromolecules for X-ray crystallography are prepared by using patterned thin polyimide films (12) that have curvature imparted thereto, for example, by being attached to a curved outer surface of a small metal rod (16). The patterned film (12) preferably includes a tapered tip end (24) for holding a crystal. Preferably, a small sample aperture is disposed in the film for reception of the crystal. A second, larger aperture can also be provided that is connected to the sample aperture by a drainage channel, allowing removal of excess liquid and easier manipulation in viscous solutions. The curvature imparted to the film (12) increases the film's rigidity and allows a convenient scoop-like action for retrieving crystals. The polyimide contributes minimally to background and absorption, and can be treated to obtain desired hydrophobicity or hydrophilicity.

  12. Sample mounts for microcrystal crystallography

    NASA Technical Reports Server (NTRS)

    Thorne, Robert E. (Inventor); Stum, Zachary (Inventor); O'Neill, Kevin (Inventor); Kmetko, Jan (Inventor)

    2009-01-01

    Sample mounts (10) for mounting microcrystals of biological macromolecules for X-ray crystallography are prepared by using patterned thin polyimide films (12) that have curvature imparted thereto, for example, by being attached to a curved outer surface of a small metal rod (16). The patterned film (12) preferably includes a tip end (24) for holding a crystal. Preferably, a small sample aperture is disposed in the film for reception of the crystal. A second, larger aperture can also be provided that is connected to the sample aperture by a drainage channel, allowing removal of excess liquid and easier manipulation in viscous solutions. The curvature imparted to the film (12) increases the film's rigidity and allows a convenient scoop-like action for retrieving crystals. The polyimide contributes minimally to background and absorption, and can be treated to obtain desired hydrophobicity or hydrophilicity.

  13. Liquid sample delivery techniques for serial femtosecond crystallography

    PubMed Central

    Weierstall, Uwe

    2014-01-01

    X-ray free-electron lasers overcome the problem of radiation damage in protein crystallography and allow structure determination from micro- and nanocrystals at room temperature. To ensure that consecutive X-ray pulses do not probe previously exposed crystals, the sample needs to be replaced with the X-ray repetition rate, which ranges from 120 Hz at warm linac-based free-electron lasers to 1 MHz at superconducting linacs. Liquid injectors are therefore an essential part of a serial femtosecond crystallography experiment at an X-ray free-electron laser. Here, we compare different techniques of injecting microcrystals in solution into the pulsed X-ray beam in vacuum. Sample waste due to mismatch of the liquid flow rate to the X-ray repetition rate can be addressed through various techniques. PMID:24914163

  14. Wrinkling crystallography on spherical surfaces

    PubMed Central

    Brojan, Miha; Terwagne, Denis; Lagrange, Romain; Reis, Pedro M.

    2015-01-01

    We present the results of an experimental investigation on the crystallography of the dimpled patterns obtained through wrinkling of a curved elastic system. Our macroscopic samples comprise a thin hemispherical shell bound to an equally curved compliant substrate. Under compression, a crystalline pattern of dimples self-organizes on the surface of the shell. Stresses are relaxed by both out-of-surface buckling and the emergence of defects in the quasi-hexagonal pattern. Three-dimensional scanning is used to digitize the topography. Regarding the dimples as point-like packing units produces spherical Voronoi tessellations with cells that are polydisperse and distorted, away from their regular shapes. We analyze the structure of crystalline defects, as a function of system size. Disclinations are observed and, above a threshold value, dislocations proliferate rapidly with system size. Our samples exhibit striking similarities with other curved crystals of charged particles and colloids. Differences are also found and attributed to the far-from-equilibrium nature of our patterns due to the random and initially frozen material imperfections which act as nucleation points, the presence of a physical boundary which represents an additional source of stress, and the inability of dimples to rearrange during crystallization. Even if we do not have access to the exact form of the interdimple interaction, our experiments suggest a broader generality of previous results of curved crystallography and their robustness on the details of the interaction potential. Furthermore, our findings open the door to future studies on curved crystals far from equilibrium. PMID:25535355

  15. Crystallography, Evolution, and the Structure of Viruses

    PubMed Central

    Rossmann, Michael G.

    2012-01-01

    My undergraduate education in mathematics and physics was a good grounding for graduate studies in crystallographic studies of small organic molecules. As a postdoctoral fellow in Minnesota, I learned how to program an early electronic computer for crystallographic calculations. I then joined Max Perutz, excited to use my skills in the determination of the first protein structures. The results were even more fascinating than the development of techniques and provided inspiration for starting my own laboratory at Purdue University. My first studies on dehydrogenases established the conservation of nucleotide-binding structures. Having thus established myself as an independent scientist, I could start on my most cherished ambition of studying the structure of viruses. About a decade later, my laboratory had produced the structure of a small RNA plant virus and then, in another six years, the first structure of a human common cold virus. Many more virus structures followed, but soon it became essential to supplement crystallography with electron microscopy to investigate viral assembly, viral infection of cells, and neutralization of viruses by antibodies. A major guide in all these studies was the discovery of evolution at the molecular level. The conservation of three-dimensional structure has been a recurring theme, from my experiences with Max Perutz in the study of hemoglobin to the recognition of the conserved nucleotide-binding fold and to the recognition of the jelly roll fold in the capsid protein of a large variety of viruses. PMID:22318719

  16. Fluorescent Approaches to High Throughput Crystallography

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Forsythe, Elizabeth; Achari, Amiruddha

    2005-01-01

    X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1 %, of a macromolecule solution with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "hits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low

  17. Fluorescent Approaches to High Throughput Crystallography

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Forsythe, Elizabeth

    2005-01-01

    X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, 51%, of a macromolecule solution with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear hits. Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics

  18. Fluorescent Approaches to High Throughput Crystallography

    NASA Technical Reports Server (NTRS)

    Minamitani, Elizabeth Forsythe; Pusey, Marc L.

    2004-01-01

    X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1%, of a macromolecule solution with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of a macromolecules purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals will show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "bits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment

  19. Fluorescent Approaches to High Throughput Crystallography

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Forsythe, Elizabeth

    2004-01-01

    X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically can not reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1%, of a macromolecule solution with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "hits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low

  20. First Results from a Microfocus X-Ray System for Macromolecular Crystallography

    NASA Technical Reports Server (NTRS)

    Gubarev, Mikhail; Ciszak, Ewa; Ponomarev, Igor; Gibson, Walter; Joy, Marshall

    1999-01-01

    The design and performance of a 40 Watt laboratory crystallography system optimized for the structure determination of small protein crystals are described. This system combines a microfocus x-ray generator (40 microns FWHM spot size at a power level of 40 Watts) and a short focal length (F = 2.6 mm) polycapillary collimating optic, and produces a small diameter quasi-parallel x-ray beam. Measurements of x-ray flux, divergence and spectral purity of the resulting x-ray beam are presented. The x-ray flux in a 250 microns diameter aperture produced by the microfocus system is 14.7 times higher .than that from a 3.15 kW rotating anode generator equipped with graphite monochromator. Crystallography data taken with the microfocus system are presented, and indicate that the divergence and spectral purity of the x-ray are sufficient to refine the diffraction data using a standard crystallographic software. Significant additional improvements in flux and beam divergence are possible, and plans for achieving these coals are discussed.

  1. An Optical Crystallography Instructional Package on Videocassettes.

    ERIC Educational Resources Information Center

    Birnie, Richard W.

    1980-01-01

    Describes a self-teaching instructional package on color videocassettes, supplemented with audio descriptions, prepared from original super-8mm cinephotomicrographs for use in optical crystallography courses. Production techniques are also reviewed. (Author/JN)

  2. X-ray crystallography at the heart of life science.

    PubMed

    Yonath, Ada

    2011-10-01

    X-ray crystallography is the fundamental research tool that shaped our notion on biological structure & function at the molecular level. It generates the information vital to understand life processes by providing the information required for creating accurate three-dimensional models (namely mapping the position of each and every atom that makes up the studied object). The use of this method begun in the middle of last century following Max von Laue discovery of the phenomenon of diffraction of X-rays by crystals, and the successful application of this discovery for the determination of the electronic distribution within simple inorganic molecules by Sir William Henry Bragg and his son, William Lawrence Bragg. The idea of extension of this method to biological molecules met initially with considerable skepticism. For over two decades many respected scientists doubted whether it could be done. Yet, despite its bottlenecks (some of which are described below), the superiority of X-ray crystallography over all other approaches for shedding light on functional aspects at the molecular level became evident once the first structure was determined. The power of this method inspired continuous efforts and spectacular innovations, which vastly accelerated its incredible expansion. Consequently, over the last six decades biological crystallography has produced a constantly growing number of structures, some of which were considered formidable. This remarkable advance yielded numerous new insights into intricate functional aspects. Owing to space limitation this article focuses on selected studies performed recently and highlights some recent exciting developments. PMID:21824762

  3. From crystal morphology to molecular and scale crystallography

    NASA Astrophysics Data System (ADS)

    Janner, A.; Janssen, T.

    2015-08-01

    A number of topics, ranging from morphology of aperiodic crystals to indexed enclosing forms of axial-symmetric proteins, nucleic acids and viruses, have been selected among those investigated by the authors in 50 years of research. The basic symmetries involved in fields like superspace, molecular and scale crystallography, are considered from a personal point of view in their time evolution. A number of specific subjects follow, chosen among a few highlights and presented according to the experience of the authors: snow crystals, calaverite {{AuTe}}2, the incommensurately modulated crystals {{Rb}}2{{ZnBr}}4, {[{N}{({{CH}}3)}4]}2{{ZnCl}}4 and the mitochondrial ferritin.

  4. A Compact X-Ray System for Macromolecular Crystallography

    NASA Technical Reports Server (NTRS)

    Gubarev, Mikhail; Ciszak, Ewa; Ponomarev, Igor; Gibson, Walter; Joy, Marshall

    2000-01-01

    We describe the design and performance of a high flux x-ray system for a macromolecular crystallography that combines a microfocus x-ray generator (40 micrometer full width at half maximum spot size at a power level of 46.5 W) and a collimating polycapillary optic. The Cu Ka lpha x-ray flux produced by this optimized system through a 500,um diam orifice is 7.0 times greater than the x-ray flux previously reported by Gubarev et al. [M. Gubarev et al., J. Appl. Crystallogr. 33, 882 (2000)]. The x-ray flux from the microfocus system is also 2.6 times higher than that produced by a rotating anode generator equipped with a graded multilayer monochromator (green optic, Osmic Inc. CMF24-48-Cu6) and 40% less than that produced by a rotating anode generator with the newest design of graded multilayer monochromator (blue optic, Osmic, Inc. CMF12-38-Cu6). Both rotating anode generators operate at a power level of 5000 W, dissipating more than 100 times the power of our microfocus x-ray system. Diffraction data collected from small test crystals are of high quality. For example, 42 540 reflections collected at ambient temperature from a lysozyme crystal yielded R(sub sym)=5.0% for data extending to 1.70 A, and 4.8% for the complete set of data to 1.85 A. The amplitudes of the observed reflections were used to calculate difference electron density maps that revealed positions of structurally important ions and water molecules in the crystal of lysozyme using the phases calculated from the protein model.

  5. A Compact X-Ray System for Macromolecular Crystallography. 5

    NASA Technical Reports Server (NTRS)

    Gubarev, Mikhail; Ciszak, Ewa; Ponomarev, Igor; Joy, Marshall

    2000-01-01

    We describe the design and performance of a high flux x-ray system for macromolecular crystallography that combines a microfocus x-ray generator (40 gm FWHM spot size at a power level of 46.5Watts) and a 5.5 mm focal distance polycapillary optic. The Cu K(sub alpha) X-ray flux produced by this optimized system is 7.0 times above the X-ray flux previously reported. The X-ray flux from the microfocus system is also 3.2 times higher than that produced by the rotating anode generator equipped with a long focal distance graded multilayer monochromator (Green optic; CMF24-48-Cu6) and 30% less than that produced by the rotating anode generator with the newest design of graded multilayer monochromator (Blue optic; CMF12-38-Cu6). Both rotating anode generators operate at a power level of 5000 Watts, dissipating more than 100 times the power of our microfocus x-ray system. Diffraction data collected from small test crystals are of high quality. For example, 42,540 reflections collected at ambient temperature from a lysozyme crystal yielded R(sub sym) 5.0% for the data extending to 1.7A, and 4.8% for the complete set of data to 1.85A. The amplitudes of the reflections were used to calculate difference electron density maps that revealed positions of structurally important ions and water molecules in the crystal of lysozyme using the phases calculated from the protein model.

  6. In vivo crystallography at X-ray free-electron lasers: the next generation of structural biology?

    PubMed

    Gallat, François-Xavier; Matsugaki, Naohiro; Coussens, Nathan P; Yagi, Koichiro J; Boudes, Marion; Higashi, Tetsuya; Tsuji, Daisuke; Tatano, Yutaka; Suzuki, Mamoru; Mizohata, Eiichi; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Park, Jaehyun; Song, Changyong; Hatsui, Takaki; Yabashi, Makina; Nango, Eriko; Itoh, Kohji; Coulibaly, Fasséli; Tobe, Stephen; Ramaswamy, S; Stay, Barbara; Iwata, So; Chavas, Leonard M G

    2014-07-17

    The serendipitous discovery of the spontaneous growth of protein crystals inside cells has opened the field of crystallography to chemically unmodified samples directly available from their natural environment. On the one hand, through in vivo crystallography, protocols for protein crystal preparation can be highly simplified, although the technique suffers from difficulties in sampling, particularly in the extraction of the crystals from the cells partly due to their small sizes. On the other hand, the extremely intense X-ray pulses emerging from X-ray free-electron laser (XFEL) sources, along with the appearance of serial femtosecond crystallography (SFX) is a milestone for radiation damage-free protein structural studies but requires micrometre-size crystals. The combination of SFX with in vivo crystallography has the potential to boost the applicability of these techniques, eventually bringing the field to the point where in vitro sample manipulations will no longer be required, and direct imaging of the crystals from within the cells will be achievable. To fully appreciate the diverse aspects of sample characterization, handling and analysis, SFX experiments at the Japanese SPring-8 angstrom compact free-electron laser were scheduled on various types of in vivo grown crystals. The first experiments have demonstrated the feasibility of the approach and suggest that future in vivo crystallography applications at XFELs will be another alternative to nano-crystallography. PMID:24914164

  7. Outrunning free radicals in room-temperature macromolecular crystallography

    PubMed Central

    Owen, Robin L.; Axford, Danny; Nettleship, Joanne E.; Owens, Raymond J.; Robinson, James I.; Morgan, Ann W.; Doré, Andrew S.; Lebon, Guillaume; Tate, Christopher G.; Fry, Elizabeth E.; Ren, Jingshan; Stuart, David I.; Evans, Gwyndaf

    2012-01-01

    A significant increase in the lifetime of room-temperature macromolecular crystals is reported through the use of a high-brilliance X-ray beam, reduced exposure times and a fast-readout detector. This is attributed to the ability to collect diffraction data before hydroxyl radicals can propagate through the crystal, fatally disrupting the lattice. Hydroxyl radicals are shown to be trapped in amorphous solutions at 100 K. The trend in crystal lifetime was observed in crystals of a soluble protein (immunoglobulin γ Fc receptor IIIa), a virus (bovine enterovirus serotype 2) and a membrane protein (human A2A adenosine G-protein coupled receptor). The observation of a similar effect in all three systems provides clear evidence for a common optimal strategy for room-temperature data collection and will inform the design of future synchrotron beamlines and detectors for macro­molecular crystallography. PMID:22751666

  8. Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser.

    PubMed

    Kupitz, Christopher; Basu, Shibom; Grotjohann, Ingo; Fromme, Raimund; Zatsepin, Nadia A; Rendek, Kimberly N; Hunter, Mark S; Shoeman, Robert L; White, Thomas A; Wang, Dingjie; James, Daniel; Yang, Jay-How; Cobb, Danielle E; Reeder, Brenda; Sierra, Raymond G; Liu, Haiguang; Barty, Anton; Aquila, Andrew L; Deponte, Daniel; Kirian, Richard A; Bari, Sadia; Bergkamp, Jesse J; Beyerlein, Kenneth R; Bogan, Michael J; Caleman, Carl; Chao, Tzu-Chiao; Conrad, Chelsie E; Davis, Katherine M; Fleckenstein, Holger; Galli, Lorenzo; Hau-Riege, Stefan P; Kassemeyer, Stephan; Laksmono, Hartawan; Liang, Mengning; Lomb, Lukas; Marchesini, Stefano; Martin, Andrew V; Messerschmidt, Marc; Milathianaki, Despina; Nass, Karol; Ros, Alexandra; Roy-Chowdhury, Shatabdi; Schmidt, Kevin; Seibert, Marvin; Steinbrener, Jan; Stellato, Francesco; Yan, Lifen; Yoon, Chunhong; Moore, Thomas A; Moore, Ana L; Pushkar, Yulia; Williams, Garth J; Boutet, Sébastien; Doak, R Bruce; Weierstall, Uwe; Frank, Matthias; Chapman, Henry N; Spence, John C H; Fromme, Petra

    2014-09-11

    Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth's oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the 'dangler' Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules. PMID:25043005

  9. Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser

    PubMed Central

    Kupitz, Christopher; Basu, Shibom; Grotjohann, Ingo; Fromme, Raimund; Zatsepin, Nadia A.; Rendek, Kimberly N.; Hunter, Mark S.; Shoeman, Robert L.; White, Thomas A.; Wang, Dingjie; James, Daniel; Yang, Jay-How; Cobb, Danielle E.; Reeder, Brenda; Sierra, Raymond G.; Liu, Haiguang; Barty, Anton; Aquila, Andrew L.; Deponte, Daniel; Kirian, Richard A.; Bari, Sadia; Bergkamp, Jesse J.; Beyerlein, Kenneth R.; Bogan, Michael J.; Caleman, Carl; Chao, Tzu-Chiao; Conrad, Chelsie E.; Davis, Katherine M.; Fleckenstein, Holger; Galli, Lorenzo; Hau-Riege, Stefan P.; Kassemeyer, Stephan; Laksmono, Hartawan; Liang, Mengning; Lomb, Lukas; Marchesini, Stefano; Martin, Andrew V.; Messerschmidt, Marc; Milathianaki, Despina; Nass, Karol; Ros, Alexandra; Roy-Chowdhury, Shatabdi; Schmidt, Kevin; Seibert, Marvin; Steinbrener, Jan; Stellato, Francesco; Yan, Lifen; Yoon, Chunhong; Moore, Thomas A.; Moore, Ana L.; Pushkar, Yulia; Williams, Garth J.; Boutet, Sébastien; Doak, R. Bruce; Weierstall, Uwe; Frank, Matthias; Chapman, Henry N.; Spence, John C. H.; Fromme, Petra

    2015-01-01

    Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth’s oxygenic atmosphere1. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed2 technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the ‘dangler’ Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies3,4. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules. PMID:25043005

  10. Structure of a photosynthetic reaction centre determined by serial femtosecond crystallography

    NASA Astrophysics Data System (ADS)

    Johansson, Linda C.; Arnlund, David; Katona, Gergely; White, Thomas A.; Barty, Anton; Deponte, Daniel P.; Shoeman, Robert L.; Wickstrand, Cecilia; Sharma, Amit; Williams, Garth J.; Aquila, Andrew; Bogan, Michael J.; Caleman, Carl; Davidsson, Jan; Doak, R. Bruce; Frank, Matthias; Fromme, Raimund; Galli, Lorenzo; Grotjohann, Ingo; Hunter, Mark S.; Kassemeyer, Stephan; Kirian, Richard A.; Kupitz, Christopher; Liang, Mengning; Lomb, Lukas; Malmerberg, Erik; Martin, Andrew V.; Messerschmidt, Marc; Nass, Karol; Redecke, Lars; Seibert, M. Marvin; Sjöhamn, Jennie; Steinbrener, Jan; Stellato, Francesco; Wang, Dingjie; Wahlgren, Weixaio Y.; Weierstall, Uwe; Westenhoff, Sebastian; Zatsepin, Nadia A.; Boutet, Sébastien; Spence, John C. H.; Schlichting, Ilme; Chapman, Henry N.; Fromme, Petra; Neutze, Richard

    2013-12-01

    Serial femtosecond crystallography is an X-ray free-electron-laser-based method with considerable potential to have an impact on challenging problems in structural biology. Here we present X-ray diffraction data recorded from microcrystals of the Blastochloris viridis photosynthetic reaction centre to 2.8 Å resolution and determine its serial femtosecond crystallography structure to 3.5 Å resolution. Although every microcrystal is exposed to a dose of 33 MGy, no signs of X-ray-induced radiation damage are visible in this integral membrane protein structure.

  11. Structure of a photosynthetic reaction centre determined by serial femtosecond crystallography

    PubMed Central

    Johansson, Linda C.; Arnlund, David; Katona, Gergely; White, Thomas A.; Barty, Anton; DePonte, Daniel P.; Shoeman, Robert L.; Wickstrand, Cecilia; Sharma, Amit; Williams, Garth J.; Aquila, Andrew; Bogan, Michael J.; Caleman, Carl; Davidsson, Jan; Doak, R Bruce; Frank, Matthias; Fromme, Raimund; Galli, Lorenzo; Grotjohann, Ingo; Hunter, Mark S.; Kassemeyer, Stephan; Kirian, Richard A.; Kupitz, Christopher; Liang, Mengning; Lomb, Lukas; Malmerberg, Erik; Martin, Andrew V.; Messerschmidt, Marc; Nass, Karol; Redecke, Lars; Seibert, M Marvin; Sjöhamn, Jennie; Steinbrener, Jan; Stellato, Francesco; Wang, Dingjie; Wahlgren, Weixaio Y.; Weierstall, Uwe; Westenhoff, Sebastian; Zatsepin, Nadia A.; Boutet, Sébastien; Spence, John C.H.; Schlichting, Ilme; Chapman, Henry N.; Fromme, Petra; Neutze, Richard

    2013-01-01

    Serial femtosecond crystallography is an X-ray free-electron-laser-based method with considerable potential to have an impact on challenging problems in structural biology. Here we present X-ray diffraction data recorded from microcrystals of the Blastochloris viridis photosynthetic reaction centre to 2.8 Å resolution and determine its serial femtosecond crystallography structure to 3.5 Å resolution. Although every microcrystal is exposed to a dose of 33 MGy, no signs of X-ray-induced radiation damage are visible in this integral membrane protein structure. PMID:24352554

  12. Perspectives and Pitfalls in Nucleic Acids Crystallography.

    PubMed

    Westhof, Eric

    2016-01-01

    X-ray crystallography offers precious and striking knowledge on biomolecular architectures. Although safeguards do exist to guarantee the accuracy of the structures deposited in databases, they are not always applied, leading to the spread of inaccurate data. The importance of validation reports in the publication process is emphasized. PMID:26227033

  13. AutoDrug: fully automated macromolecular crystallography workflows for fragment-based drug discovery

    PubMed Central

    Tsai, Yingssu; McPhillips, Scott E.; González, Ana; McPhillips, Timothy M.; Zinn, Daniel; Cohen, Aina E.; Feese, Michael D.; Bushnell, David; Tiefenbrunn, Theresa; Stout, C. David; Ludaescher, Bertram; Hedman, Britt; Hodgson, Keith O.; Soltis, S. Michael

    2013-01-01

    AutoDrug is software based upon the scientific workflow paradigm that integrates the Stanford Synchrotron Radiation Lightsource macromolecular crystallography beamlines and third-party processing software to automate the crystallo­graphy steps of the fragment-based drug-discovery process. AutoDrug screens a cassette of fragment-soaked crystals, selects crystals for data collection based on screening results and user-specified criteria and determines optimal data-collection strategies. It then collects and processes diffraction data, performs molecular replacement using provided models and detects electron density that is likely to arise from bound fragments. All processes are fully automated, i.e. are performed without user interaction or supervision. Samples can be screened in groups corresponding to particular proteins, crystal forms and/or soaking conditions. A single AutoDrug run is only limited by the capacity of the sample-storage dewar at the beamline: currently 288 samples. AutoDrug was developed in conjunction with RestFlow, a new scientific workflow-automation framework. RestFlow simplifies the design of AutoDrug by managing the flow of data and the organization of results and by orchestrating the execution of computational pipeline steps. It also simplifies the execution and interaction of third-party programs and the beamline-control system. Modeling AutoDrug as a scientific workflow enables multiple variants that meet the requirements of different user groups to be developed and supported. A workflow tailored to mimic the crystallography stages comprising the drug-discovery pipeline of CoCrystal Discovery Inc. has been deployed and successfully demonstrated. This workflow was run once on the same 96 samples that the group had examined manually and the workflow cycled successfully through all of the samples, collected data from the same samples that were selected manually and located the same peaks of unmodeled density in the resulting difference

  14. JBlulce Data Acquisition Software for Macromolecular Crystallography

    SciTech Connect

    2010-06-01

    JBlulce (Java Beam Line Universal Integrated Configuration Environment is a data acquisition software for macromolecular crystallography conforming user interface of the SSRL Blulce that has become a de-factor standard in the field. Besides this interface conformity, JBlulce is a unique system in terms of architecture, speec, capability and osftware implementation. It features only two software layers, the JBlulce clients and the EPICS servers, as compared to three layers present in Blulc and most of similar systems. This layers reduction provides a faster communication with hardware and an easier access to advanced hardware capabilities like on-the-fly scanning. Then JBlulc clients are designed to operate in parallel with the other beamline controls which streamlines the tasks performed by staff such as beamline preparation, maitenance, audting and user assistance. Another distinction is the deployment of multiple plugins that can be written in any programming languag thus involving more staff into the development. further on, JBlulce makes use of unified motion controls allowing for easy scanning and optimizing of any beamline component. Finally, the graphic interface is implemented in Java making full use of rich Java libraries and Jave IDE for debugging. to compare, Blulce user interface is implemented with aging Tcl/tk language providing very restricted capabilities. JBlulce makes full use of the industrial power and wide drivers selection of EPICS in controlling hardware; all hardware commuication is routed via multiple EPICS servers residing on local area network. JBlulce also includes several EPICS State Notation servers aimed at making hardware communication more robust. Besides using EPICS for controlling hardware, JBlulce extensively uses EPICS databases for efficien communications between multiple instances of JBlulce clients and JBlulce pplugins that can run in parallel on different computers. All of the above makes JBlulce one of the biggest and most

  15. JBlulce Data Acquisition Software for Macromolecular Crystallography

    Energy Science and Technology Software Center (ESTSC)

    2010-06-01

    JBlulce (Java Beam Line Universal Integrated Configuration Environment is a data acquisition software for macromolecular crystallography conforming user interface of the SSRL Blulce that has become a de-factor standard in the field. Besides this interface conformity, JBlulce is a unique system in terms of architecture, speec, capability and osftware implementation. It features only two software layers, the JBlulce clients and the EPICS servers, as compared to three layers present in Blulc and most of similarmore » systems. This layers reduction provides a faster communication with hardware and an easier access to advanced hardware capabilities like on-the-fly scanning. Then JBlulc clients are designed to operate in parallel with the other beamline controls which streamlines the tasks performed by staff such as beamline preparation, maitenance, audting and user assistance. Another distinction is the deployment of multiple plugins that can be written in any programming languag thus involving more staff into the development. further on, JBlulce makes use of unified motion controls allowing for easy scanning and optimizing of any beamline component. Finally, the graphic interface is implemented in Java making full use of rich Java libraries and Jave IDE for debugging. to compare, Blulce user interface is implemented with aging Tcl/tk language providing very restricted capabilities. JBlulce makes full use of the industrial power and wide drivers selection of EPICS in controlling hardware; all hardware commuication is routed via multiple EPICS servers residing on local area network. JBlulce also includes several EPICS State Notation servers aimed at making hardware communication more robust. Besides using EPICS for controlling hardware, JBlulce extensively uses EPICS databases for efficien communications between multiple instances of JBlulce clients and JBlulce pplugins that can run in parallel on different computers. All of the above makes JBlulce one of the biggest

  16. X-Ray Crystallography Reagent

    NASA Technical Reports Server (NTRS)

    Morrison, Dennis R. (Inventor); Mosier, Benjamin (Inventor)

    2003-01-01

    Microcapsules prepared by encapsulating an aqueous solution of a protein, drug or other bioactive substance inside a semi-permeable membrane by are disclosed. The microcapsules are formed by interfacial coacervation under conditions where the shear forces are limited to 0-100 dynes per square centimeter at the interface. By placing the microcapsules in a high osmotic dewatering solution. the protein solution is gradually made saturated and then supersaturated. and the controlled nucleation and crystallization of the protein is achieved. The crystal-filled microcapsules prepared by this method can be conveniently harvested and stored while keeping the encapsulated crystals in essentially pristine condition due to the rugged. protective membrane. Because the membrane components themselves are x-ray transparent, large crystal-containing microcapsules can be individually selected, mounted in x-ray capillary tubes and subjected to high energy x-ray diffraction studies to determine the 3-D smucture of the protein molecules. Certain embodiments of the microcapsules of the invention have composite polymeric outer membranes which are somewhat elastic, water insoluble, permeable only to water, salts, and low molecular weight molecules and are structurally stable in fluid shear forces typically encountered in the human vascular system.

  17. A novel inert crystal delivery medium for serial femtosecond crystallography

    PubMed Central

    Conrad, Chelsie E.; Basu, Shibom; James, Daniel; Wang, Dingjie; Schaffer, Alexander; Roy-Chowdhury, Shatabdi; Zatsepin, Nadia A.; Aquila, Andrew; Coe, Jesse; Gati, Cornelius; Hunter, Mark S.; Koglin, Jason E.; Kupitz, Christopher; Nelson, Garrett; Subramanian, Ganesh; White, Thomas A.; Zhao, Yun; Zook, James; Boutet, Sébastien; Cherezov, Vadim; Spence, John C. H.; Fromme, Raimund; Weierstall, Uwe; Fromme, Petra

    2015-01-01

    Serial femtosecond crystallography (SFX) has opened a new era in crystallo­graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes. PMID:26177184

  18. In-vacuum long-wavelength macromolecular crystallography

    PubMed Central

    Wagner, Armin; Duman, Ramona; Henderson, Keith; Mykhaylyk, Vitaliy

    2016-01-01

    Structure solution based on the weak anomalous signal from native (protein and DNA) crystals is increasingly being attempted as part of synchrotron experiments. Maximizing the measurable anomalous signal by collecting diffraction data at longer wavelengths presents a series of technical challenges caused by the increased absorption of X-rays and larger diffraction angles. A new beamline at Diamond Light Source has been built specifically for collecting data at wavelengths beyond the capability of other synchrotron macromolecular crystallography beamlines. Here, the theoretical considerations in support of the long-wavelength beamline are outlined and the in-vacuum design of the endstation is discussed, as well as other hardware features aimed at enhancing the accuracy of the diffraction data. The first commissioning results, representing the first in-vacuum protein structure solution, demonstrate the promising potential of the beamline. PMID:26960130

  19. Towards time-resolved serial crystallography in a microfluidic device

    PubMed Central

    Pawate, Ashtamurthy S.; Šrajer, Vukica; Schieferstein, Jeremy; Guha, Sudipto; Henning, Robert; Kosheleva, Irina; Schmidt, Marius; Ren, Zhong; Kenis, Paul J. A.; Perry, Sarah L.

    2015-01-01

    Serial methods for crystallography have the potential to enable dynamic structural studies of protein targets that have been resistant to single-crystal strategies. The use of serial data-collection strategies can circumvent challenges associated with radiation damage and repeated reaction initiation. This work utilizes a microfluidic crystallization platform for the serial time-resolved Laue diffraction analysis of macroscopic crystals of photoactive yellow protein (PYP). Reaction initiation was achieved via pulsed laser illumination, and the resultant electron-density difference maps clearly depict the expected pR1/pRE46Q and pR2/pRCW states at 10 µs and the pB1 intermediate at 1 ms. The strategies presented here have tremendous potential for extension to chemical triggering methods for reaction initiation and for extension to dynamic, multivariable analyses. PMID:26144226

  20. Synchrotron radiation macromolecular crystallography: science and spin-offs

    PubMed Central

    Helliwell, John R.; Mitchell, Edward P.

    2015-01-01

    A current overview of synchrotron radiation (SR) in macromolecular crystallography (MX) instrumentation, methods and applications is presented. Automation has been and remains a central development in the last decade, as have the rise of remote access and of industrial service provision. Results include a high number of Protein Data Bank depositions, with an increasing emphasis on the successful use of microcrystals. One future emphasis involves pushing the frontiers of using higher and lower photon energies. With the advent of X-ray free-electron lasers, closely linked to SR developments, the use of ever smaller samples such as nanocrystals, nanoclusters and single molecules is anticipated, as well as the opening up of femtosecond time-resolved diffraction structural studies. At SR sources, a very high-throughput assessment for the best crystal samples and the ability to tackle just a few micron and sub-micron crystals will become widespread. With higher speeds and larger detectors, diffraction data volumes are becoming long-term storage and archiving issues; the implications for today and the future are discussed. Together with the rise of the storage ring to its current pre-eminence in MX data provision, the growing tendency of central facility sites to offer other centralized facilities complementary to crystallography, such as cryo-electron microscopy and NMR, is a welcome development. PMID:25866664

  1. Synchrotron radiation macromolecular crystallography: science and spin-offs.

    PubMed

    Helliwell, John R; Mitchell, Edward P

    2015-03-01

    A current overview of synchrotron radiation (SR) in macromolecular crystallography (MX) instrumentation, methods and applications is presented. Automation has been and remains a central development in the last decade, as have the rise of remote access and of industrial service provision. Results include a high number of Protein Data Bank depositions, with an increasing emphasis on the successful use of microcrystals. One future emphasis involves pushing the frontiers of using higher and lower photon energies. With the advent of X-ray free-electron lasers, closely linked to SR developments, the use of ever smaller samples such as nanocrystals, nanoclusters and single molecules is anticipated, as well as the opening up of femtosecond time-resolved diffraction structural studies. At SR sources, a very high-throughput assessment for the best crystal samples and the ability to tackle just a few micron and sub-micron crystals will become widespread. With higher speeds and larger detectors, diffraction data volumes are becoming long-term storage and archiving issues; the implications for today and the future are discussed. Together with the rise of the storage ring to its current pre-eminence in MX data provision, the growing tendency of central facility sites to offer other centralized facilities complementary to crystallography, such as cryo-electron microscopy and NMR, is a welcome development. PMID:25866664

  2. New methodologies at PF AR-NW12A: the implementation of high-pressure macromolecular crystallography

    PubMed Central

    Chavas, Leonard Michel Gabriel; Nagae, Tadayuki; Yamada, Hiroyuki; Watanabe, Nobuhisa; Yamada, Yusuke; Hiraki, Masahiko; Matsugaki, Naohiro

    2013-01-01

    The macromolecular crystallography (MX) beamline AR-NW12A is evolving from its original design of high-throughput crystallography to a multi-purpose end-station. Among the various options to be implemented, great efforts were made in making available high-pressure MX (HPMX) at the beamline. High-pressure molecular biophysics is a developing field that attracts the interest of a constantly growing scientific community. A plethora of activities can benefit from high pressure, and investigations have been performed on its applicability to study multimeric complex assemblies, compressibility of proteins and their crystals, macromolecules originating from extremophiles, or even the trapping of higher-energy conformers for molecules of biological interest. Recent studies using HPMX showed structural hydrostatic-pressure-induced changes in proteins. The conformational modifications could explain the enzymatic mechanism differences between proteins of the same family, living at different environmental pressures, as well as the initial steps in the pressure-denaturation process that have been attributed to water penetration into the protein interior. To facilitate further HPMX, while allowing access to various individualized set-ups and experiments, the AR-NW12A sample environment has been revisited. Altogether, the newly added implementations will bring a fresh breath of life to AR-NW12A and allow the MX community to experiment in a larger set of fields related to structural biology. PMID:24121324

  3. The design of macromolecular crystallography diffraction experiments

    SciTech Connect

    Evans, Gwyndaf Axford, Danny; Owen, Robin L.

    2011-04-01

    Thoughts about the decisions made in designing macromolecular X-ray crystallography experiments at synchrotron beamlines are presented. The measurement of X-ray diffraction data from macromolecular crystals for the purpose of structure determination is the convergence of two processes: the preparation of diffraction-quality crystal samples on the one hand and the construction and optimization of an X-ray beamline and end station on the other. Like sample preparation, a macromolecular crystallography beamline is geared to obtaining the best possible diffraction measurements from crystals provided by the synchrotron user. This paper describes the thoughts behind an experiment that fully exploits both the sample and the beamline and how these map into everyday decisions that users can and should make when visiting a beamline with their most precious crystals.

  4. Graphical tools for macromolecular crystallography in PHENIX

    PubMed Central

    Echols, Nathaniel; Grosse-Kunstleve, Ralf W.; Afonine, Pavel V.; Bunkóczi, Gábor; Chen, Vincent B.; Headd, Jeffrey J.; McCoy, Airlie J.; Moriarty, Nigel W.; Read, Randy J.; Richardson, David C.; Richardson, Jane S.; Terwilliger, Thomas C.; Adams, Paul D.

    2012-01-01

    A new Python-based graphical user interface for the PHENIX suite of crystallography software is described. This interface unifies the command-line programs and their graphical displays, simplifying the development of new interfaces and avoiding duplication of function. With careful design, graphical interfaces can be displayed automatically, instead of being manually constructed. The resulting package is easily maintained and extended as new programs are added or modified. PMID:22675231

  5. Status of the crystallography beamlines at Elettra

    NASA Astrophysics Data System (ADS)

    Lausi, A.; Polentarutti, M.; Onesti, S.; Plaisier, J. R.; Busetto, E.; Bais, G.; Barba, L.; Cassetta, A.; Campi, G.; Lamba, D.; Pifferi, A.; Mande, S. C.; Sarma, D. D.; Sharma, S. M.; Paolucci, G.

    2015-03-01

    Elettra is one of the first 3rd-generation storage rings, recently upgraded to routinely operate in top-up mode at both 2.0 and 2.4 GeV. The facility hosts four dedicated beamlines for crystallography, two open to the users and two under construction, and expected to be ready for public use in 2015. In service since 1994, XRD1 is a general-purpose diffraction beamline. The light source for this wide (4-21 keV) energy range beamline is a permanent magnet wiggler. XRD1 covers experiments ranging from grazing incidence X-ray diffraction to macromolecular crystallography, from industrial applications of powder diffraction to X-ray phasing with long wavelengths. The bending magnet powder diffraction beamline MCX has been open to users since 2009, with a focus on microstructural investigations and studies under non-ambient conditions. A superconducting wiggler delivers a high photon flux to a new fully automated beamline dedicated to macromolecular crystallography and to a branch beamline hosting a high-pressure powder X-ray diffraction station (both currently under construction). Users of the latter experimental station will have access to a specialized sample preparation laboratory, shared with the SISSI infrared beamline. A high throughput crystallization platform equipped with an imaging system for the remote viewing, evaluation and scoring of the macromolecular crystallization experiments has also been established and is open to the user community.

  6. Microcrystal delivery by pulsed liquid droplet for serial femtosecond crystallography.

    PubMed

    Mafuné, Fumitaka; Miyajima, Ken; Tono, Kensuke; Takeda, Yoshihiro; Kohno, Jun Ya; Miyauchi, Naoya; Kobayashi, Jun; Joti, Yasumasa; Nango, Eriko; Iwata, So; Yabashi, Makina

    2016-04-01

    A liquid-droplet injector has been developed that delivers pristine microcrystals to an X-ray irradiation area for conducting serial femtosecond crystallography (SFX) with an X-ray free-electron laser (XFEL). By finely tuning the pulsed liquid droplets in time and space, a high hit rate of the XFEL pulses to microcrystals in the droplets was achieved for measurements using 5 µm tetragonal lysozyme crystals, which produced 4265 indexable diffraction images in about 30 min. The structure was determined at a resolution of 2.3 Å from <0.3 mg of protein. With further improvements such as reduction of the droplet size, liquid droplets have considerable potential as a crystal carrier for SFX with low sample consumption. PMID:27050131

  7. Optimizing high performance computing workflow for protein functional annotation.

    PubMed

    Stanberry, Larissa; Rekepalli, Bhanu; Liu, Yuan; Giblock, Paul; Higdon, Roger; Montague, Elizabeth; Broomall, William; Kolker, Natali; Kolker, Eugene

    2014-09-10

    Functional annotation of newly sequenced genomes is one of the major challenges in modern biology. With modern sequencing technologies, the protein sequence universe is rapidly expanding. Newly sequenced bacterial genomes alone contain over 7.5 million proteins. The rate of data generation has far surpassed that of protein annotation. The volume of protein data makes manual curation infeasible, whereas a high compute cost limits the utility of existing automated approaches. In this work, we present an improved and optmized automated workflow to enable large-scale protein annotation. The workflow uses high performance computing architectures and a low complexity classification algorithm to assign proteins into existing clusters of orthologous groups of proteins. On the basis of the Position-Specific Iterative Basic Local Alignment Search Tool the algorithm ensures at least 80% specificity and sensitivity of the resulting classifications. The workflow utilizes highly scalable parallel applications for classification and sequence alignment. Using Extreme Science and Engineering Discovery Environment supercomputers, the workflow processed 1,200,000 newly sequenced bacterial proteins. With the rapid expansion of the protein sequence universe, the proposed workflow will enable scientists to annotate big genome data. PMID:25313296

  8. The R-factor gap in macromolecular crystallography: an untapped potential for insights on accurate structures

    PubMed Central

    Holton, James M; Classen, Scott; Frankel, Kenneth A; Tainer, John A

    2014-01-01

    In macromolecular crystallography, the agreement between observed and predicted structure factors (Rcryst and Rfree) is seldom better than 20%. This is much larger than the estimate of experimental error (Rmerge). The difference between Rcryst and Rmerge is the R-factor gap. There is no such gap in small-molecule crystallography, for which calculated structure factors are generally considered more accurate than the experimental measurements. Perhaps the true noise level of macromolecular data is higher than expected? Or is the gap caused by inaccurate phases that trap refined models in local minima? By generating simulated diffraction patterns using the program MLFSOM, and including every conceivable source of experimental error, we show that neither is the case. Processing our simulated data yielded values that were indistinguishable from those of real data for all crystallographic statistics except the final Rcryst and Rfree. These values decreased to 3.8% and 5.5% for simulated data, suggesting that the reason for high R-factors in macromolecular crystallography is neither experimental error nor phase bias, but rather an underlying inadequacy in the models used to explain our observations. The present inability to accurately represent the entire macromolecule with both its flexibility and its protein-solvent interface may be improved by synergies between small-angle X-ray scattering, computational chemistry and crystallography. The exciting implication of our finding is that macromolecular data contain substantial hidden and untapped potential to resolve ambiguities in the true nature of the nanoscale, a task that the second century of crystallography promises to fulfill. Database Coordinates and structure factors for the real data have been submitted to the Protein Data Bank under accession 4tws. PMID:25040949

  9. Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography

    DOE PAGESBeta

    Mueller, C.; Marx, A.; Epp, S. W.; Zhong, Y.; Kuo, A.; Balo, A. R.; Soman, J.; Schotte, F.; Lemke, H. T.; Owen, R. L.; et al

    2015-08-18

    We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linacmore » Coherent Light Source (LCLS, Menlo Park, California, USA). As a result, the chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs.« less

  10. A brief history of macromolecular crystallography, illustrated by a family tree and its Nobel fruits.

    PubMed

    Jaskolski, Mariusz; Dauter, Zbigniew; Wlodawer, Alexander

    2014-09-01

    As a contribution to the celebration of the year 2014, declared by the United Nations to be 'The International Year of Crystallography', the FEBS Journal is dedicating this issue to papers showcasing the intimate union between macromolecular crystallography and structural biology, both in historical perspective and in current research. Instead of a formal editorial piece, by way of introduction, this review discusses the most important, often iconic, achievements of crystallographers that led to major advances in our understanding of the structure and function of biological macromolecules. We identified at least 42 scientists who received Nobel Prizes in Physics, Chemistry or Medicine for their contributions that included the use of X-rays or neutrons and crystallography, including 24 who made seminal discoveries in macromolecular sciences. Our spotlight is mostly, but not only, on the recipients of this most prestigious scientific honor, presented in approximately chronological order. As a summary of the review, we attempt to construct a genealogy tree of the principal lineages of protein crystallography, leading from the founding members to the present generation. PMID:24698025

  11. A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering

    PubMed Central

    Roedig, P.; Vartiainen, I.; Duman, R.; Panneerselvam, S.; Stübe, N.; Lorbeer, O.; Warmer, M.; Sutton, G.; Stuart, D. I.; Weckert, E.; David, C.; Wagner, A.; Meents, A.

    2015-01-01

    At low emittance synchrotron sources it has become possible to perform structure determinations from the measurement of multiple microcrystals which were previously considered too small for diffraction experiments. Conventional mounting techniques do not fulfill the requirements of these new experiments. They significantly contribute to background scattering and it is difficult to locate the crystals, making them incompatible with automated serial crystallography. We have developed a micro-fabricated sample holder from single crystalline silicon with micropores, which carries up to thousands of crystals and significantly reduces the background scattering level. For loading, the suspended microcrystals are pipetted onto the chip and excess mother liquor is subsequently soaked off through the micropores. Crystals larger than the pore size are retained and arrange themselves according to the micropore pattern. Using our chip we were able to collect 1.5 Å high resolution diffraction data from protein microcrystals with sizes of 4 micrometers and smaller. PMID:26022615

  12. A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering.

    PubMed

    Roedig, P; Vartiainen, I; Duman, R; Panneerselvam, S; Stübe, N; Lorbeer, O; Warmer, M; Sutton, G; Stuart, D I; Weckert, E; David, C; Wagner, A; Meents, A

    2015-01-01

    At low emittance synchrotron sources it has become possible to perform structure determinations from the measurement of multiple microcrystals which were previously considered too small for diffraction experiments. Conventional mounting techniques do not fulfill the requirements of these new experiments. They significantly contribute to background scattering and it is difficult to locate the crystals, making them incompatible with automated serial crystallography. We have developed a micro-fabricated sample holder from single crystalline silicon with micropores, which carries up to thousands of crystals and significantly reduces the background scattering level. For loading, the suspended microcrystals are pipetted onto the chip and excess mother liquor is subsequently soaked off through the micropores. Crystals larger than the pore size are retained and arrange themselves according to the micropore pattern. Using our chip we were able to collect 1.5 Å high resolution diffraction data from protein microcrystals with sizes of 4 micrometers and smaller. PMID:26022615

  13. Ceramic micro-injection molded nozzles for serial femtosecond crystallography sample delivery

    NASA Astrophysics Data System (ADS)

    Beyerlein, K. R.; Adriano, L.; Heymann, M.; Kirian, R.; Knoška, J.; Wilde, F.; Chapman, H. N.; Bajt, S.

    2015-12-01

    Serial femtosecond crystallography (SFX) using X-ray Free-Electron Lasers (XFELs) allows for room temperature protein structure determination without evidence of conventional radiation damage. In this method, a liquid suspension of protein microcrystals can be delivered to the X-ray beam in vacuum as a micro-jet, which replenishes the crystals at a rate that exceeds the current XFEL pulse repetition rate. Gas dynamic virtual nozzles produce the required micrometer-sized streams by the focusing action of a coaxial sheath gas and have been shown to be effective for SFX experiments. Here, we describe the design and characterization of such nozzles assembled from ceramic micro-injection molded outer gas-focusing capillaries. Trends of the emitted jet diameter and jet length as a function of supplied liquid and gas flow rates are measured by a fast imaging system. The observed trends are explained by derived relationships considering choked gas flow and liquid flow conservation. Finally, the performance of these nozzles in a SFX experiment is presented, including an analysis of the observed background.

  14. A new paradigm for macromolecular crystallography beamlines derived from high-pressure methodology and results

    PubMed Central

    Fourme, Roger; Girard, Eric; Dhaussy, Anne-Claire; Medjoubi, Kadda; Prangé, Thierry; Ascone, Isabella; Mezouar, Mohamed; Kahn, Richard

    2011-01-01

    Biological structures can now be investigated at high resolution by high-pressure X-ray macromolecular crystallography (HPMX). The number of HPMX studies is growing, with applications to polynucleotides, monomeric and multimeric proteins, complex assemblies and even a virus capsid. Investigations of the effects of pressure perturbation have encompassed elastic compression of the native state, study of proteins from extremophiles and trapping of higher-energy conformers that are often of biological interest; measurements of the compressibility of crystals and macromolecules were also performed. HPMX results were an incentive to investigate short and ultra-short wavelengths for standard biocrystallography. On cryocooled lysozyme crystals it was found that the data collection efficiency using 33 keV photons is increased with respect to 18 keV photons. This conclusion was extended from 33 keV down to 6.5 keV by exploiting previously published data. To be fully exploited, the potential of higher-energy photons requires detectors with a good efficiency. Accordingly, a new paradigm for MX beamlines was suggested, using conventional short and ultra-short wavelengths, aiming at the collection of very high accuracy data on crystals under standard conditions or under high pressure. The main elements of such beamlines are outlined. PMID:21169687

  15. Ceramic micro-injection molded nozzles for serial femtosecond crystallography sample delivery

    SciTech Connect

    Beyerlein, K. R.; Adriano, L.; Heymann, M.; Kirian, R.; Knoska, J.; Wilde, F.; Chapman, H. N.; Bajt, S.

    2015-12-08

    Serial femtosecond crystallography (SFX) using X-ray Free-Electron Lasers (XFELs) allows for room temperature protein structure determination without evidence of conventional radiation damage. In this method, a liquid suspension of protein microcrystals can be delivered to the X-ray beam in vacuum as a micro-jet, which replenishes the crystals at a rate that exceeds the current XFEL pulse repetition rate. Gas dynamic virtual nozzles produce the required micrometer-sized streams by the focusing action of a coaxial sheath gas and have been shown to be effective for SFX experiments. Here, we describe the design and characterization of such nozzles assembled from ceramic micro-injection molded outer gas-focusing capillaries. Trends of the emitted jet diameter and jet length as a function of supplied liquid and gas flow rates are measured by a fast imaging system. The observed trends are explained by derived relationships considering choked gas flow and liquidflow conservation. In conclusion, the performance of these nozzles in a SFX experiment is presented, including an analysis of the observed background.

  16. Ceramic micro-injection molded nozzles for serial femtosecond crystallography sample delivery

    DOE PAGESBeta

    Beyerlein, K. R.; Adriano, L.; Heymann, M.; Kirian, R.; Knoska, J.; Wilde, F.; Chapman, H. N.; Bajt, S.

    2015-12-08

    Serial femtosecond crystallography (SFX) using X-ray Free-Electron Lasers (XFELs) allows for room temperature protein structure determination without evidence of conventional radiation damage. In this method, a liquid suspension of protein microcrystals can be delivered to the X-ray beam in vacuum as a micro-jet, which replenishes the crystals at a rate that exceeds the current XFEL pulse repetition rate. Gas dynamic virtual nozzles produce the required micrometer-sized streams by the focusing action of a coaxial sheath gas and have been shown to be effective for SFX experiments. Here, we describe the design and characterization of such nozzles assembled from ceramic micro-injectionmore » molded outer gas-focusing capillaries. Trends of the emitted jet diameter and jet length as a function of supplied liquid and gas flow rates are measured by a fast imaging system. The observed trends are explained by derived relationships considering choked gas flow and liquidflow conservation. In conclusion, the performance of these nozzles in a SFX experiment is presented, including an analysis of the observed background.« less

  17. Ceramic micro-injection molded nozzles for serial femtosecond crystallography sample delivery

    SciTech Connect

    Beyerlein, K. R.; Heymann, M.; Kirian, R.; Adriano, L.; Bajt, S.; Knoška, J.; Wilde, F.; Chapman, H. N.

    2015-12-15

    Serial femtosecond crystallography (SFX) using X-ray Free-Electron Lasers (XFELs) allows for room temperature protein structure determination without evidence of conventional radiation damage. In this method, a liquid suspension of protein microcrystals can be delivered to the X-ray beam in vacuum as a micro-jet, which replenishes the crystals at a rate that exceeds the current XFEL pulse repetition rate. Gas dynamic virtual nozzles produce the required micrometer-sized streams by the focusing action of a coaxial sheath gas and have been shown to be effective for SFX experiments. Here, we describe the design and characterization of such nozzles assembled from ceramic micro-injection molded outer gas-focusing capillaries. Trends of the emitted jet diameter and jet length as a function of supplied liquid and gas flow rates are measured by a fast imaging system. The observed trends are explained by derived relationships considering choked gas flow and liquid flow conservation. Finally, the performance of these nozzles in a SFX experiment is presented, including an analysis of the observed background.

  18. A new paradigm for macromolecular crystallography beamlines derived from high-pressure methodology and results.

    PubMed

    Fourme, Roger; Girard, Eric; Dhaussy, Anne Claire; Medjoubi, Kadda; Prangé, Thierry; Ascone, Isabella; Mezouar, Mohamed; Kahn, Richard

    2011-01-01

    Biological structures can now be investigated at high resolution by high-pressure X-ray macromolecular crystallography (HPMX). The number of HPMX studies is growing, with applications to polynucleotides, monomeric and multimeric proteins, complex assemblies and even a virus capsid. Investigations of the effects of pressure perturbation have encompassed elastic compression of the native state, study of proteins from extremophiles and trapping of higher-energy conformers that are often of biological interest; measurements of the compressibility of crystals and macromolecules were also performed. HPMX results were an incentive to investigate short and ultra-short wavelengths for standard biocrystallography. On cryocooled lysozyme crystals it was found that the data collection efficiency using 33 keV photons is increased with respect to 18 keV photons. This conclusion was extended from 33 keV down to 6.5 keV by exploiting previously published data. To be fully exploited, the potential of higher-energy photons requires detectors with a good efficiency. Accordingly, a new paradigm for MX beamlines was suggested, using conventional short and ultra-short wavelengths, aiming at the collection of very high accuracy data on crystals under standard conditions or under high pressure. The main elements of such beamlines are outlined. PMID:21169687

  19. Morphological possibilities in general crystallography. Snow crystals.

    PubMed

    Janner, A

    2002-07-01

    Morphological features of snow crystals are analyzed on the basis of concepts of a general crystallography, where point groups of infinite order are possible. The observations are first formulated in a set of rules, leading to a macroscopic growth lattice and to continuous growth boundaries. Both are brought in connection with two-dimensional integral invertible transformations. Families of boundaries are considered, labeled by a set of indices restricted by selection rules and generalizing the law of rational indices. These properties are indicated graphically on a sample of 12 natural snow crystals. Their geometric and arithmetic properties are summarized in a table. PMID:12089456

  20. Statistical crystallography of surface micelle spacing

    NASA Technical Reports Server (NTRS)

    Noever, David A.

    1992-01-01

    The aggregation of the recently reported surface micelles of block polyelectrolytes is analyzed using techniques of statistical crystallography. A polygonal lattice (Voronoi mosaic) connects center-to-center points, yielding statistical agreement with crystallographic predictions; Aboav-Weaire's law and Lewis's law are verified. This protocol supplements the standard analysis of surface micelles leading to aggregation number determination and, when compared to numerical simulations, allows further insight into the random partitioning of surface films. In particular, agreement with Lewis's law has been linked to the geometric packing requirements of filling two-dimensional space which compete with (or balance) physical forces such as interfacial tension, electrostatic repulsion, and van der Waals attraction.

  1. Holographic Methods in X-ray Crystallography

    Energy Science and Technology Software Center (ESTSC)

    1995-07-28

    The holographic method makes use of partially modeled electron density and experimentally-measured structure factor amplitudes to recover electron density corresponding to the unmodeled part of a crystal structure. This paper describes a fast algorithm that makes it possible to apply the holographic method to sizable crystallographic problems. The algorithm uses positivity constraints on the electron density, and can incorporate a target electron density, making it similar to solvent flattening. Using both synthetic and experimental data,more » we assess the potential for applying the holographic method to macromolecular x-ray crystallography.« less

  2. Screening Ligands by X-ray crystallography.

    PubMed

    Davies, Douglas R

    2014-01-01

    X-ray crystallography is an invaluable technique in structure-based drug discovery, including fragment-based drug discovery, because it is the only technique that can provide a complete three dimensional readout of the interaction between the small molecule and its macromolecular target. X-ray diffraction (XRD) techniques can be employed as the sole method for conducting a screen of a fragment library, or it can be employed as the final technique in a screening campaign to confirm putative "hit" compounds identified by a variety of biochemical and/or biophysical screening techniques. Both approaches require an efficient technique to prepare dozens to hundreds of crystals for data collection, and a reproducible way to deliver ligands to the crystal. Here, a general method for screening cocktails of fragments is described. In cases where X-ray crystallography is employed as a method to verify putative hits, the cocktails of fragments described below would simply be replaced with single fragment solutions. PMID:24590727

  3. EIGER detector: application in macromolecular crystallography.

    PubMed

    Casanas, Arnau; Warshamanage, Rangana; Finke, Aaron D; Panepucci, Ezequiel; Olieric, Vincent; Nöll, Anne; Tampé, Robert; Brandstetter, Stefan; Förster, Andreas; Mueller, Marcus; Schulze-Briese, Clemens; Bunk, Oliver; Wang, Meitian

    2016-09-01

    The development of single-photon-counting detectors, such as the PILATUS, has been a major recent breakthrough in macromolecular crystallography, enabling noise-free detection and novel data-acquisition modes. The new EIGER detector features a pixel size of 75 × 75 µm, frame rates of up to 3000 Hz and a dead time as low as 3.8 µs. An EIGER 1M and EIGER 16M were tested on Swiss Light Source beamlines X10SA and X06SA for their application in macromolecular crystallography. The combination of fast frame rates and a very short dead time allows high-quality data acquisition in a shorter time. The ultrafine ϕ-slicing data-collection method is introduced and validated and its application in finding the optimal rotation angle, a suitable rotation speed and a sufficient X-ray dose are presented. An improvement of the data quality up to slicing at one tenth of the mosaicity has been observed, which is much finer than expected based on previous findings. The influence of key data-collection parameters on data quality is discussed. PMID:27599736

  4. The location of protein S8 and surrounding elements of 16S rRNA in the 70S ribosome from combined use of directed hydroxyl radical probing and X-ray crystallography.

    PubMed Central

    Lancaster, L; Culver, G M; Yusupova, G Z; Cate, J H; Yusupov, M M; Noller, H F

    2000-01-01

    Ribosomal protein S8, which is essential for the assembly of the central domain of 16S rRNA, is one of the most thoroughly studied RNA-binding proteins. To map its surrounding RNA in the ribosome, we carried out directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to nine different positions on the surface of protein S8 in 70S ribosomes. Hydroxyl radical-induced cleavage was observed near the classical S8-binding site in the 620 stem, and flanking the other S8-footprinted regions of the central domain at the three-helix junction near position 650 and the 825 and 860 stems. In addition, cleavage near the 5' terminus of 16S rRNA, in the 300 region of its 5' domain, and in the 1070 region of its 3'-major domain provide information about the proximity to S8 of RNA elements not directly involved in its binding. These data, along with previous footprinting and crosslinking results, allowed positioning of protein S8 and its surrounding RNA elements in a 7.8-A map of the Thermus thermophilus 70S ribosome. The resulting model is in close agreement with the extensive body of data from previous studies using protein-protein and protein-RNA crosslinking, chemical and enzymatic footprinting, and genetics. PMID:10836793

  5. Macromolecular Crystallography and Structural Biology Databases at NIST

    PubMed Central

    Gilliland, Gary L.

    2001-01-01

    In the late 1970s, macromolecular crystallography at NIST began with collaboration between NIST and NIH to establish a single-crystal neutron diffractometer. This instrument was constructed and employed to solve a number of crystal structures: bovine ribonuclease A, bovine-ribonuclease-uridine vanadate complex, and porcine insulin. In the mid 1980s a Biomolecular Structure Group was created establishing NIST capabilities in biomolecular singe-crystal x-ray diffraction. The group worked on a variety of structural problems until joining the NIST/UMBI Center for Advanced Research in Biotechnology (CARB) in 1987. Crystallographic studies at CARB were then focused on protein engineering efforts that included among others chymosin, subtilisin BPN', interleukin 1β, and glutathione S-transferase. Recently, the structural biology efforts have centered on enzymes in the chorismate metabolic pathways involved in amino acid biosynthesis and in structural genomics that involves determining the structures of “hypothetical” proteins to aid in assigning function. In addition to crystallographic studies, structural biology database activities began with the formal establishment of the Biological Macro-molecule Crystallization Database in 1989. Later, in 1997, NIST in partnership with Rutgers and UCSD formed the Research Collaboratory for Structural Bioinformatics that successfully acquired the Protein Data Bank. The NIST efforts in these activities have focused on data uniformity, establishing and maintaining the physical archive, and working with the NMR community. PMID:27500071

  6. Serial femtosecond crystallography opens new avenues for Structural Biology.

    PubMed

    Coe, Jesse; Fromme, Petra

    2016-01-01

    Free electron lasers (FELs) provide X-ray pulses in the femtosecond time domain with up to 10(12) higher photon flux than synchrotrons and open new avenues for the determination of difficult to crystallize proteins, like large complexes and human membrane proteins. While the X-ray pulses are so strong that they destroy any solid material, the crystals diffract before they are destroyed. The most successful application of FELs for biology has been the method of serial femtosecond crystallography (SFX) where nano or microcrystals are delivered to the FEL beam in a stream of their mother liquid at room temperature, which ensures the replenishment of the sample before the next X-ray pulse arrives. New injector technology allows also for the delivery of crystal in lipidic cubic phases or agarose, which reduces the sample amounts for an SFX data set by two orders of magnitude. Time-resolved SFX also allows for analysis of the dynamics of biomolecules, the proof of principle being recently shown for light-induced reactions in photosystem II and photoactive yellow protein. An SFX data sets consist of thousands of single crystal snapshots in random orientations, which can be analyzed now "on the fly" by data analysis programs specifically developed for SFX, but de-novo phasing is still a challenge, that might be overcome by two-color experiments or phasing by shape transforms. PMID:26786767

  7. X-Ray Crystallography: One Century of Nobel Prizes

    ERIC Educational Resources Information Center

    Galli, Simona

    2014-01-01

    In 2012, the United Nations General Assembly declared 2014 the International Year of Crystallography. Throughout the year 2014 and beyond, all the crystallographic associations and societies active all over the world are organizing events to attract the wider public toward crystallography and the numerous topics to which it is deeply interlinked.…

  8. Protein quantitation using various modes of high performance liquid chromatography.

    PubMed

    Grotefend, Sandra; Kaminski, Lukas; Wroblewitz, Stefanie; Deeb, Sami El; Kühn, Nancy; Reichl, Stephan; Limberger, Markus; Watt, Steven; Wätzig, Hermann

    2012-12-01

    Pharmaceuticals based on proteins (biologicals), such as monoclonal antibodies (mAb), attain more and more relevance since they were established as potent drugs in anticancer therapy or for the treatment of autoimmune based diseases. Due to their high efficiency it is essential to have accurate and precise methods for protein quantitation and the detection of protein aggregates, which in some cases may lead to adverse effects after application. Selectivity and precision of traditional protein quantification methods such as the Bradford assay or SDS-PAGE are insufficient for quality control (QC) purposes. In this work several HPLC separation modes, which can significantly improve these important parameters, were compared for their application in this field. High performance size exclusion (HP-SEC), strong anion exchange (SAX), weak cation exchange (WCX) as well as reversed phase chromatography are all already successfully applied in protein analysis. Good precision (SEC: <1.9%, SAX: <5%, RP: <2% and WCX: <3.5% - RSD% for peak areas day-to-day), high selectivity and low quantitation limits (<15μg/ml) for the model proteins ovalbumin, myoglobin and bovine serum albumin (BSA), respectively cytochrome c and lysozyme in the cation exchange mode, could be achieved. Consecutively, the four separation modes were compared to each other and to electrophoretic techniques in terms of precision, selectivity, analysis time, effort of sample and mobile phase preparation as well as separating capacity. Moreover, the analysis of an IgG1-type antibody was included in this study. PMID:22980318

  9. In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

    PubMed Central

    Jakobi, Arjen J.; Passon, Daniel M.; Knoops, Kèvin; Stellato, Francesco; Liang, Mengning; White, Thomas A.; Seine, Thomas; Messerschmidt, Marc; Chapman, Henry N.; Wilmanns, Matthias

    2016-01-01

    The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. The observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined. PMID:27006771

  10. In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

    DOE PAGESBeta

    Jakobi, Arjen J.; Passon, Daniel M.; Knoops, Kevin; Stellato, Francesco; Liang, Mengning; White, Thomas A.; Seine, Thomas; Messerschmidt, Marc; Chapman, Henry N.; Wilmanns, Matthias

    2016-03-01

    The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. Furthermore, the observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined.

  11. In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells.

    PubMed

    Jakobi, Arjen J; Passon, Daniel M; Knoops, Kèvin; Stellato, Francesco; Liang, Mengning; White, Thomas A; Seine, Thomas; Messerschmidt, Marc; Chapman, Henry N; Wilmanns, Matthias

    2016-03-01

    The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. The observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined. PMID:27006771

  12. Radiation damage to nucleoprotein complexes in macromolecular crystallography

    PubMed Central

    Bury, Charles; Garman, Elspeth F.; Ginn, Helen Mary; Ravelli, Raimond B. G.; Carmichael, Ian; Kneale, Geoff; McGeehan, John E.

    2015-01-01

    Significant progress has been made in macromolecular crystallography over recent years in both the understanding and mitigation of X-ray induced radiation damage when collecting diffraction data from crystalline proteins. In contrast, despite the large field that is productively engaged in the study of radiation chemistry of nucleic acids, particularly of DNA, there are currently very few X-ray crystallographic studies on radiation damage mechanisms in nucleic acids. Quantitative comparison of damage to protein and DNA crystals separately is challenging, but many of the issues are circumvented by studying pre-formed biological nucleoprotein complexes where direct comparison of each component can be made under the same controlled conditions. Here a model protein–DNA complex C.Esp1396I is employed to investigate specific damage mechanisms for protein and DNA in a biologically relevant complex over a large dose range (2.07–44.63 MGy). In order to allow a quantitative analysis of radiation damage sites from a complex series of macromolecular diffraction data, a computational method has been developed that is generally applicable to the field. Typical specific damage was observed for both the protein on particular amino acids and for the DNA on, for example, the cleavage of base-sugar N1—C and sugar-phosphate C—O bonds. Strikingly the DNA component was determined to be far more resistant to specific damage than the protein for the investigated dose range. At low doses the protein was observed to be susceptible to radiation damage while the DNA was far more resistant, damage only being observed at significantly higher doses. PMID:25723923

  13. Approaches to High-Performance Preparative Chromatography of Proteins

    NASA Astrophysics Data System (ADS)

    Sun, Yan; Liu, Fu-Feng; Shi, Qing-Hong

    Preparative liquid chromatography is widely used for the purification of chemical and biological substances. Different from high-performance liquid chromatography for the analysis of many different components at minimized sample loading, high-performance preparative chromatography is of much larger scale and should be of high resolution and high capacity at high operation speed and low to moderate pressure drop. There are various approaches to this end. For biochemical engineers, the traditional way is to model and optimize a purification process to make it exert its maximum capability. For high-performance separations, however, we need to improve chromatographic technology itself. We herein discuss four approaches in this review, mainly based on the recent studies in our group. The first is the development of high-performance matrices, because packing material is the central component of chromatography. Progress in the fabrication of superporous materials in both beaded and monolithic forms are reviewed. The second topic is the discovery and design of affinity ligands for proteins. In most chromatographic methods, proteins are separated based on their interactions with the ligands attached to the surface of porous media. A target-specific ligand can offer selective purification of desired proteins. Third, electrochromatography is discussed. An electric field applied to a chromatographic column can induce additional separation mechanisms besides chromatography, and result in electrokinetic transport of protein molecules and/or the fluid inside pores, thus leading to high-performance separations. Finally, expanded-bed adsorption is described for process integration to reduce separation steps and process time.

  14. Dietary protein level and performance of growing Baladi kids

    PubMed Central

    Abdelrahman, M. M.; Aljumaah, R. S.

    2014-01-01

    A study was conducted to evaluate the effect of feeding different levels of protein to black Baladi breed kids. Weanling Baladi kids (n=18; 75 to 90 days old) were selected and individually housed at our experimental farm. Kids were divided randomly to one of the three treatments for 12 weeks. The three dietary treatments were: T1: control ration, formulated according to NRC to cover the protein (level 1) and other nutrients requirements. T2: ration formulated to cover only 75% of protein (level 2) recommended by NRC. T3: control diet + 2.4 g undegradable methionine (Smartamine®)/day/kid (level 3). Feed intake, initial and monthly body weights were recorded. Blood samples were collected monthly and analyzed for metabolites and Co, Zn and Cu levels. Decreasing the dietary level of protein (T2) negatively affected (P<0.05) the total live weight gain, average daily gain and feed conversion ratio when compared with the control and T3 groups. Moreover, treatment, time and time × treatment caused a significant change on Co concentration in blood serum with higher value at the end of the experiment. Treatments had a significant effect (P<0.05) on blood serum cholesterol and protein levels. Undegradable methionine supplementation (T3) significantly increased longissimus dorsi weight, fat thickness and omental fat%. In conclusion, feeding Baladi kids below the NRC requirements of protein negatively affect the growth performance and feed efficiency. The recommended protein level by NRC for growing kids cover the requirements of growing black Baladi kids for maximum growth and productivity. PMID:27175130

  15. Alignment protocol for effective use of hard x-ray quad collimator for micro-crystallography

    NASA Astrophysics Data System (ADS)

    Xu, S.; Nagarajan, V.; Sanishvili, R.; Fischetti, R. F.

    2011-09-01

    In October 2009, a quad, mini-beam collimator was implemented at GM/CA CAT that allowed users to select between a 5, 10, or 20 micron mini-beam or a 300 micron scatter guard for macromolecular crystallography. Initial alignment of each pinhole to the optical axis of each path through the mini-beam collimator is performed under an optical microscope using an alignment jig. Next, the pre-aligned collimator and its kinematic mount are moved to the beamline and attached to a pair of high precision translation stages attached to an on-axis-visualization system for viewing the protein crystal under investigation. The collimator is aligned to the beam axis by two angular and two translational motions. The pitch and yaw adjustments are typically only done during initial installation, and therefore are not motorized. The horizontal and vertical positions are adjusted remotely with high precision translational stages. Final alignment of the collimator is achieved using several endstation components, namely, a YAG crystal at the sample position to visualize the mini-beam, a CCD detector to record an X-ray background image, and a PIN diode to record the mini-beam intensity. The alignment protocol and its opto-mechanical instrumentation design will be discussed in detail.

  16. The design of macromolecular crystallography diffraction experiments

    PubMed Central

    Evans, Gwyndaf; Axford, Danny; Owen, Robin L.

    2011-01-01

    The measurement of X-ray diffraction data from macro­molecular crystals for the purpose of structure determination is the convergence of two processes: the preparation of diffraction-quality crystal samples on the one hand and the construction and optimization of an X-ray beamline and end station on the other. Like sample preparation, a macromolecular crystallography beamline is geared to obtaining the best possible diffraction measurements from crystals provided by the synchrotron user. This paper describes the thoughts behind an experiment that fully exploits both the sample and the beamline and how these map into everyday decisions that users can and should make when visiting a beamline with their most precious crystals. PMID:21460444

  17. Crystallography of Alumina-YAG-Eutectic

    NASA Technical Reports Server (NTRS)

    Farmer, Serene C.; Sayir, Ali; Dickerson, Robert M.; Matson, Lawrence E.

    2000-01-01

    Multiple descriptions of the alumina-YAG eutectic crystallography appear in the ceramic literature. The orientation between two phases in a eutectic system has direct impact on residual stress, morphology, microstructural stability, and high temperature mechanical properties. A study to demonstrate that the different crystallographic relationships can be correlated with different growth constraints was undertaken. Fibers produced by Laser-Heated Float Zone (LHFZ) and Edge-defined Film-fed Growth (EFG) were examined. A map of the orientation relationship between Al2O3 and Y3Al5O12 and their relationship to the fiber growth axis as a function of pull rate are presented. Regions in which a single orientation predominates are identified.

  18. Fast fluorescence techniques for crystallography beamlines

    PubMed Central

    Stepanov, Sergey; Hilgart, Mark; Yoder, Derek W.; Makarov, Oleg; Becker, Michael; Sanishvili, Ruslan; Ogata, Craig M.; Venugopalan, Nagarajan; Aragão, David; Caffrey, Martin; Smith, Janet L.; Fischetti, Robert F.

    2011-01-01

    This paper reports on several developments of X-ray fluorescence techniques for macromolecular crystallography recently implemented at the National Institute of General Medical Sciences and National Cancer Institute beamlines at the Advanced Photon Source. These include (i) three-band on-the-fly energy scanning around absorption edges with adaptive positioning of the fine-step band calculated from a coarse pass; (ii) on-the-fly X-ray fluorescence rastering over rectangular domains for locating small and invisible crystals with a shuttle-scanning option for increased speed; (iii) fluorescence rastering over user-specified multi-segmented polygons; and (iv) automatic signal optimization for reduced radiation damage of samples. PMID:21808424

  19. The crystallography stations at the Alba synchrotron

    NASA Astrophysics Data System (ADS)

    Fauth, François; Boer, Roeland; Gil-Ortiz, Fernando; Popescu, Catalin; Vallcorba, Oriol; Peral, Inma; Fullà, Daniel; Benach, Jordi; Juanhuix, Jordi

    2015-08-01

    Alba is a 3rd-generation 3 GeV synchrotron facility with an emittance of 4.6nm·rad which has been operational since 2011 and has recently started top-up operation. Photons in a broad energy range of 0.08-80 keV are served to seven beamlines dedicated to a large variety of scientific fields. The portfolio includes two beamlines, XALOC and MSPD, fully dedicated to X-ray crystallography. BL13-XALOC is currently the only macromolecular crystallography beamline. The end-station includes a high-accuracy single-axis diffractometer with a removable minikappa stage, a sample-mounting robot and a large-area, photon-counting detector. The beamline optics, fed by an in-vacuum undulator, deliver a tunable photon beam between 5.5 and 22 keV. The beam size at the sample position can be adjusted by defocusing the mirrors in a range of 50-300μm in the horizontal direction and 5.5-300μm in the vertical direction. Beamline BL04-MSPD, which is fed by a superconducting wiggler, has two in-line end-stations. The first station is devoted to high-pressure/microdiffraction. It offers a μm beam in the range 20-50 keV, particularly suited for powder diffraction studies requiring a very small beam, e.g. mapping of cultural heritage samples and high-pressures studies. The second station is dedicated to high-resolution/high-throughput powder diffraction. It covers the 8-50 keV range and includes a heavy-duty 3-circle diffractometer equipped with a 13-channel multianalyzer detector with high-angular resolution ( FWHM) and a high-throughput, position-sensitive detector spanning in 2 range allowing millisecond data acquisitions.

  20. The collection of MicroED data for macromolecular crystallography.

    PubMed

    Shi, Dan; Nannenga, Brent L; de la Cruz, M Jason; Liu, Jinyang; Sawtelle, Steven; Calero, Guillermo; Reyes, Francis E; Hattne, Johan; Gonen, Tamir

    2016-05-01

    The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ∼10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals. PMID:27077331

  1. Characterization of wax as a potential diffraction intensity standard for macromolecular crystallography beamlines.

    PubMed

    Brandao-Neto, J; Thompson, S P; Lennie, A R; Ferreira, F F; Tang, C C

    2010-01-01

    A number of commercially available waxes in the form of thin disc samples have been investigated as possible diffraction intensity standards for macromolecular crystallography synchrotron beamlines. Synchrotron X-ray powder diffraction measurements show that beeswax offers the best performance of these waxes owing to its polycrystallinity. Crystallographic lattice parameters and diffraction intensities were examined between 281 and 309 K, and show stable and predictable thermal behaviour. Using an X-ray beam of known incident flux at lambda = 1 A, the diffraction power of two strong Bragg reflections for beeswax were quantified as a function of sample thickness and normalized to 10(10) photons s(-1). To demonstrate its feasibility as a diffraction intensity standard, test measurements were then performed on a new third-generation macromolecular crystallography synchrotron beamline. PMID:20029111

  2. X-ray crystallography over the past decade for novel drug discovery – where are we heading next?

    PubMed Central

    Zheng, Heping; Handing, Katarzyna B; Zimmerman, Matthew D; Shabalin, Ivan G; Almo, Steven C; Minor, Wladek

    2015-01-01

    Introduction Macromolecular X-ray crystallography has been the primary methodology for determining the three-dimensional structures of proteins, nucleic acids and viruses. Structural information has paved the way for structure-guided drug discovery and laid the foundations for structural bioinformatics. However, X-ray crystallography still has a few fundamental limitations, some of which may be overcome and complemented using emerging methods and technologies in other areas of structural biology. Areas covered This review describes how structural knowledge gained from X-ray crystallography has been used to advance other biophysical methods for structure determination (and vice versa). This article also covers current practices for integrating data generated by other biochemical and biophysical methods with those obtained from X-ray crystallography. Finally, the authors articulate their vision about how a combination of structural and biochemical/biophysical methods may improve our understanding of biological processes and interactions. Expert opinion X-ray crystallography has been, and will continue to serve as, the central source of experimental structural biology data used in the discovery of new drugs. However, other structural biology techniques are useful not only to overcome the major limitation of X-ray crystallography, but also to provide complementary structural data that is useful in drug discovery. The use of recent advancements in biochemical, spectroscopy and bioinformatics methods may revolutionize drug discovery, albeit only when these data are combined and analyzed with effective data management systems. Accurate and complete data management is crucial for developing experimental procedures that are robust and reproducible. PMID:26177814

  3. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    PubMed

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach. PMID:27498895

  4. Structure of the Angiotensin Receptor Revealed by Serial Femtosecond Crystallography

    SciTech Connect

    Zhang, Haitao; Unal, Hamiyet; Gati, Cornelius; Han, Gye Won; Liu, Wei; Zatsepin, Nadia A.; James, Daniel; Wang, Dingjie; Nelson, Garrett; Weierstall, Uwe; Sawaya, Michael R.; Xu, Qingping; Messerschmidt, Marc; Williams, Garth J.; Boutet, Sébastien; Yefanov, Oleksandr M.; White, Thomas A.; Wang, Chong; Ishchenko, Andrii; Tirupula, Kalyan C.; Desnoyer, Russell; Coe, Jesse; Conrad, Chelsie E.; Fromme, Petra; Stevens, Raymond C.; Katritch, Vsevolod; Karnik, Sadashiva S.; Cherezov, Vadim

    2015-05-07

    We report that angiotensin II type 1 receptor (AT1R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT1R blockers (ARBs), the structural basis for AT1R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT1R in complex with its selective antagonist ZD7155 at 2.9 Å resolution. The AT1R-ZD7155 complex structure revealed key structural features ofAT1R and critical interactions for ZD7155 binding. Finally, docking simulations of the clinically used ARBs into the AT1R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT1R structure-function relationship and structure-based drug design.

  5. Structure of the Angiotensin Receptor Revealed by Serial Femtosecond Crystallography

    DOE PAGESBeta

    Zhang, Haitao; Unal, Hamiyet; Gati, Cornelius; Han, Gye Won; Liu, Wei; Zatsepin, Nadia A.; James, Daniel; Wang, Dingjie; Nelson, Garrett; Weierstall, Uwe; et al

    2015-05-07

    We report that angiotensin II type 1 receptor (AT1R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT1R blockers (ARBs), the structural basis for AT1R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT1R in complex with its selective antagonist ZD7155 at 2.9 Å resolution. Themore » AT1R-ZD7155 complex structure revealed key structural features ofAT1R and critical interactions for ZD7155 binding. Finally, docking simulations of the clinically used ARBs into the AT1R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT1R structure-function relationship and structure-based drug design.« less

  6. Structure of the Angiotensin Receptor Revealed by Serial Femtosecond Crystallography

    PubMed Central

    Zhang, Haitao; Unal, Hamiyet; Gati, Cornelius; Han, Gye Won; Liu, Wei; Zatsepin, Nadia A.; James, Daniel; Wang, Dingjie; Nelson, Garrett; Weierstall, Uwe; Sawaya, Michael R.; Xu, Qingping; Messerschmidt, Marc; Williams, Garth J.; Boutet, Sébastien; Yefanov, Oleksandr M.; White, Thomas A.; Wang, Chong; Ishchenko, Andrii; Tirupula, Kalyan C.; Desnoyer, Russell; Coe, Jesse; Conrad, Chelsie E.; Fromme, Petra; Stevens, Raymond C.; Katritch, Vsevolod; Karnik, Sadashiva S.; Cherezov, Vadim

    2015-01-01

    SUMMARY Angiotensin II type 1 receptor (AT1R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT1R blockers (ARBs), the structural basis for AT1R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT1R in complex with its selective antagonist ZD7155 at 2.9 Å resolution. The AT1R-ZD7155 complex structure revealed key structural features of AT1R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT1R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT1R structure-function relationship and structure-based drug design. PMID:25913193

  7. Miniaturized kappa goniometer for macromolecular crystallography

    SciTech Connect

    Rosenbaum, G.; Westbrook, E. M.

    1997-07-01

    A goniometer with kappa geometry has been designed and built specifically for macromolecular crystallography. The main feature is a miniaturized kappa stage made possible by the small weight of specimen and specimen holder. The design goal was to: 1) eliminate interference between stage and area detector for specimen-to-detector distances of 100 mm and more; 2) minimize the sphere of confusion on expectation of dealing with very small crystals at third generation sources; 3) minimize the solid angle of shadow and inaccessible positioning of the sample due to interference of the stage with other objects in the sample area; 4) achieve a rotation speed of 10 degree/s at 0.5% constancy and 0.4 s acceleration time for 0.05 s exposures of 0.2 degree fine slice frames every 2 seconds, and 5) to achieve precise synchronization between rotation angle and shutter opening and closing. The kappa stage is mounted on a commercial high precision rotary table, designed for use in both horizontal and vertical orientation. This table provides the high precision rotation for data acquisition. The required crisp response and constant speed is delivered by a high output direct drive DC-motor, controlled by a closed-loop controller using feedback from a precision angular encoder. The kappa- and phi-motions are used for sample positioning only and are driven by miniature DC-motors equipped with integral encoders.

  8. Miniaturized kappa goniometer for macromolecular crystallography

    SciTech Connect

    Rosenbaum, G.; Westbrook, E.M.

    1997-07-01

    A goniometer with kappa geometry has been designed and built specifically for macromolecular crystallography. The main feature is a miniaturized kappa stage made possible by the small weight of specimen and specimen holder. The design goal was to: 1) eliminate interference between stage and area detector for specimen-to-detector distances of 100 mm and more; 2) minimize the sphere of confusion on expectation of dealing with very small crystals at third generation sources; 3) minimize the solid angle of shadow and inaccessible positioning of the sample due to interference of the stage with other objects in the sample area; 4) achieve a rotation speed of 10 degree/s at 0.5{percent} constancy and 0.4 s acceleration time for 0.05 s exposures of 0.2 degree fine slice frames every 2 seconds, and 5) to achieve precise synchronization between rotation angle and shutter opening and closing. The kappa stage is mounted on a commercial high precision rotary table, designed for use in both horizontal and vertical orientation. This table provides the high precision rotation for data acquisition. The required crisp response and constant speed is delivered by a high output direct drive DC-motor, controlled by a closed-loop controller using feedback from a precision angular encoder. The kappa- and phi-motions are used for sample positioning only and are driven by miniature DC-motors equipped with integral encoders.{copyright} {ital 1997 American Institute of Physics.}

  9. Phase Equilibria and Crystallography of Ceramic Oxides

    PubMed Central

    Wong-Ng, W.; Roth, R. S.; Vanderah, T. A.; McMurdie, H. F.

    2001-01-01

    Research in phase equilibria and crystallography has been a tradition in the Ceramics Division at National Bureau of Standards/National Institute of Standatrds and Technology (NBS/NIST) since the early thirties. In the early years, effort was concentrated in areas of Portland cement, ceramic glazes and glasses, instrument bearings, and battery materials. In the past 40 years, a large portion of the work was related to electronic materials, including ferroelectrics, piezoelectrics, ionic conductors, dielectrics, microwave dielectrics, and high-temperature superconductors. As a result of the phase equilibria studies, many new compounds have been discovered. Some of these discoveries have had a significant impact on US industry. Structure determinations of these new phases have often been carried out as a joint effort among NBS/NIST colleagues and also with outside collaborators using both single crystal and neutron and x-ray powder diffraction techniques. All phase equilibria diagrams were included in Phase Diagrams for Ceramists, which are collaborative publications between The American Ceramic Society (ACerS) and NBS/NIST. All x-ray powder diffraction patterns have been included in the Powder Diffraction File (PDF). This article gives a brief account of the history of the development of the phase equilibria and crystallographic research on ceramic oxides in the Ceramics Division. Represented systems, particularly electronic materials, are highlighted. PMID:27500068

  10. NMR crystallography: the use of chemical shifts

    NASA Astrophysics Data System (ADS)

    Harris, Robin K.

    2004-10-01

    Measurements of chemical shifts obtained from magic-angle spinning NMR spectra (together with quantum mechanical computations of shielding) can provide valuable information on crystallography. Examples are given of the determination of crystallographic asymmetric units, of molecular symmetry in the solid-state environment, and of crystallographic space group assignment. Measurements of full tensor components for 199Hg have given additional coordination information. The nature of intermolecular hydrogen bonding in cortisone acetate polymorphs and solvates is obtained from chemical shift information, also involving measurement of the full tensor parameters. The resulting data have been used as restraints, built into the computation algorithm, in the analysis of powder diffraction patterns to give full crystal structures. A combination of quantum mechanical computation of shielding and measurement of proton chemical shifts (obtained by high-speed MAS) leads to the determination of the position of a proton in an intermolecular hydrogen bond. A recently-developed computer program specifically based on crystallographic repetition has been shown to give acceptable results. Moreover, NMR chemical shifts can distinguish between static and dynamic disorder in crystalline materials and can be used to determine modes and rates of molecular exchange motion.

  11. Translation calibration of inverse-kappa goniometers in macromolecular crystallography

    PubMed Central

    Brockhauser, Sandor; White, Kristopher I.; McCarthy, Andrew A.; Ravelli, Raimond B. G.

    2011-01-01

    Precise and convenient crystal reorientation is of experimental importance in macromolecular crystallography (MX). The development of multi-axis goniometers, such as the ESRF/EMBL mini-κ, necessitates the corresponding development of calibration procedures that can be used for the setup, maintenance and troubleshooting of such devices. While traditional multi-axis goniometers require all rotation axes to intersect the unique point of the sample position, recently developed miniaturized instruments for sample reorientation in MX are not as restricted. However, the samples must always be re-centred following a change in orientation. To overcome this inconvenience and allow the use of multi-axis goniometers without the fundamental restriction of having all axes intersecting in the same point, an automatic translation correction protocol has been developed for such instruments. It requires precise information about the direction and location of the rotation axes. To measure and supply this information, a general, easy-to-perform translation calibration (TC) procedure has also been developed. The TC procedure is routinely performed on most MX beamlines at the ESRF and some results are presented for reference. PMID:21487180

  12. Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography

    SciTech Connect

    Mueller, C.; Marx, A.; Epp, S. W.; Zhong, Y.; Kuo, A.; Balo, A. R.; Soman, J.; Schotte, F.; Lemke, H. T.; Owen, R. L.; Pai, E. F.; Pearson, A. R.; Olson, J. S.; Anfinrud, P. A.; Ernst, O. P.; Miller, R. J. Dwayne

    2015-08-18

    We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linac Coherent Light Source (LCLS, Menlo Park, California, USA). As a result, the chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs.

  13. Crystallography & NMR system: A new software suite for macromolecular structure determination.

    PubMed

    Brünger, A T; Adams, P D; Clore, G M; DeLano, W L; Gros, P; Grosse-Kunstleve, R W; Jiang, J S; Kuszewski, J; Nilges, M; Pannu, N S; Read, R J; Rice, L M; Simonson, T; Warren, G L

    1998-09-01

    A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs, the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible. The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular structure determination by X-ray crystallography and solution NMR. PMID:9757107

  14. Implementation of remote monitoring and diffraction evaluation systems at the Photon Factory macromolecular crystallography beamlines

    PubMed Central

    Yamada, Yusuke; pHonda, Nobuo; Matsugaki, Naohiro; Igarashi, Noriyuki; Hiraki, Masahiko; Wakatsuki, Soichi

    2008-01-01

    Owing to recent advances in high-throughput technology in macromolecular crystallography beamlines, such as high-brilliant X-ray sources, high-speed readout detectors and robotics, the number of samples that can be examined in a single visit to the beamline has increased dramatically. In order to make these experiments more efficient, two functions, remote monitoring and diffraction image evaluation, have been implemented in the macromolecular crystallography beamlines at the Photon Factory (PF). Remote monitoring allows scientists to participate in the experiment by watching from their laboratories, without having to come to the beamline. Diffraction image evaluation makes experiments easier, especially when using the sample exchange robot. To implement these two functions, two independent clients have been developed that work specifically for remote monitoring and diffraction image evaluation. In the macromolecular crystallography beamlines at PF, beamline control is performed using STARS (simple transmission and retrieval system). The system adopts a client–server style in which client programs communicate with each other through a server process using the STARS protocol. This is an advantage of the extension of the system; implementation of these new functions required few modifications of the existing system. PMID:18421163

  15. Neutron diffractometer for bio-crystallography (BIX) with an imaging plate neutron detector

    SciTech Connect

    Niimura, Nobuo

    1994-12-31

    We have constructed a dedicated diffractometer for neutron crystallography in biology (BIX) on the JRR-3M reactor at JAERI (Japan Atomic Energy Research Institute). The diffraction intensity from a protein crystal is weaker than that from most inorganic materials. In order to overcome the intensity problem, an elastically bent silicon monochromator and a large area detector system were specially designed. A preliminary result of diffraction experiment using BIX has been reported. An imaging plate neutron detector has been developed and a feasibility experiment was carried out on BIX. Results are reported. An imaging plate neutron detector has been developed and a feasibility test was carried out using BIX.

  16. Protein crystal growth in a microgravity environment

    NASA Technical Reports Server (NTRS)

    Bugg, Charles E.

    1988-01-01

    Protein crystal growth is a major experimental problem and is the bottleneck in widespread applications of protein crystallography. Research efforts now being pursued and sponsored by NASA are making fundamental contributions to the understanding of the science of protein crystal growth. Microgravity environments offer the possibility of performing new types of experiments that may produce a better understanding of protein crystal growth processes and may permit growth environments that are more favorable for obtaining high quality protein crystals. A series of protein crystal growth experiments using the space shuttle was initiated. The first phase of these experiments was focused on the development of micro-methods for protein crystal growth by vapor diffusion techniques, using a space version of the hanging drop method. The preliminary space experiments were used to evolve prototype hardware that will form the basis for a more advanced system that can be used to evaluate effects of gravity on protein crystal growth.

  17. The kinetic dose limit in room-temperature time-resolved macromolecular crystallography

    PubMed Central

    Schmidt, M.; Šrajer, V.; Purwar, N.; Tripathi, S.

    2012-01-01

    Protein X-ray structures are determined with ionizing radiation that damages the protein at high X-ray doses. As a result, diffraction patterns deteriorate with the increased absorbed dose. Several strategies such as sample freezing or scavenging of X-ray-generated free radicals are currently employed to minimize this damage. However, little is known about how the absorbed X-ray dose affects time-resolved Laue data collected at physiological temperatures where the protein is fully functional in the crystal, and how the kinetic analysis of such data depends on the absorbed dose. Here, direct evidence for the impact of radiation damage on the function of a protein is presented using time-resolved macromolecular crystallography. The effect of radiation damage on the kinetic analysis of time-resolved X-ray data is also explored. PMID:22338689

  18. Discovery of Leukotriene A4 Hydrolase Inhibitors Using Metabolomics Biased Fragment Crystallography

    SciTech Connect

    Davies, D.; Mamat, B; Magnusson, O; Christensen, J; Haraldsson, M; Mishra, R; Pease, B; Hansen, E; Singh, J; et. al.

    2009-01-01

    We describe a novel fragment library termed fragments of life (FOL) for structure-based drug discovery. The FOL library includes natural small molecules of life, derivatives thereof, and biaryl protein architecture mimetics. The choice of fragments facilitates the interrogation of protein active sites, allosteric binding sites, and protein-protein interaction surfaces for fragment binding. We screened the FOL library against leukotriene A4 hydrolase (LTA4H) by X-ray crystallography. A diverse set of fragments including derivatives of resveratrol, nicotinamide, and indole were identified as efficient ligands for LTA4H. These fragments were elaborated in a small number of synthetic cycles into potent inhibitors of LTA4H representing multiple novel chemotypes for modulating leukotriene biosynthesis. Analysis of the fragment-bound structures also showed that the fragments comprehensively recapitulated key chemical features and binding modes of several reported LTA4H inhibitors.

  19. Using neutron protein crystallography to understand enzyme mechanisms

    SciTech Connect

    Glusker, Jenny P. Carrell, H. L.; Kovalevsky, Andrey Y.; Hanson, Leif; Fisher, S. Zoe; Mustyakimov, Marat; Mason, Sax; Forsyth, Trevor; Langan, Paul

    2010-11-01

    A description is given of the results of neutron diffraction studies of the structures of four different metal-ion complexes of deuterated d-xylose isomerase. A description is given of the results of neutron diffraction studies of the structures of four different metal-ion complexes of deuterated d-xylose isomerase. These represent four stages in the progression of the biochemical catalytic action of this enzyme. Analyses of the structural changes observed between the various three-dimensional structures lead to some insight into the mechanism of action of this enzyme.

  20. Simulation of modulated protein crystal structure and diffraction data in a supercell and in superspace

    SciTech Connect

    Lovelace, Jeffrey J.; Simone, Peter D.; Petříček, Václav; Borgstahl, Gloria E. O.

    2013-06-01

    A computer simulation was created for a modulated protein structure along with structure factors in a periodic supercell and in superspace for the purpose of developing and validating software modifications that will be used to solve and refine modulated protein crystals. The toolbox for computational protein crystallography is full of easy-to-use applications for the routine solution and refinement of periodic diffraction data sets and protein structures. There is a gap in the available software when it comes to aperiodic crystallographic data. Current protein crystallography software cannot handle modulated data, and small-molecule software for aperiodic crystallography cannot work with protein structures. To adapt software for modulated protein data requires training data to test and debug the changed software. Thus, a comprehensive training data set consisting of atomic positions with associated modulation functions and the modulated structure factors packaged as both a three-dimensional supercell and as a modulated structure in (3+1)D superspace has been created. The (3+1)D data were imported into Jana2006; this is the first time that this has been performed for protein data.

  1. Room-temperature macromolecular serial crystallography using synchrotron radiation.

    PubMed

    Stellato, Francesco; Oberthür, Dominik; Liang, Mengning; Bean, Richard; Gati, Cornelius; Yefanov, Oleksandr; Barty, Anton; Burkhardt, Anja; Fischer, Pontus; Galli, Lorenzo; Kirian, Richard A; Meyer, Jan; Panneerselvam, Saravanan; Yoon, Chun Hong; Chervinskii, Fedor; Speller, Emily; White, Thomas A; Betzel, Christian; Meents, Alke; Chapman, Henry N

    2014-07-01

    A new approach for collecting data from many hundreds of thousands of microcrystals using X-ray pulses from a free-electron laser has recently been developed. Referred to as serial crystallography, diffraction patterns are recorded at a constant rate as a suspension of protein crystals flows across the path of an X-ray beam. Events that by chance contain single-crystal diffraction patterns are retained, then indexed and merged to form a three-dimensional set of reflection intensities for structure determination. This approach relies upon several innovations: an intense X-ray beam; a fast detector system; a means to rapidly flow a suspension of crystals across the X-ray beam; and the computational infrastructure to process the large volume of data. Originally conceived for radiation-damage-free measurements with ultrafast X-ray pulses, the same methods can be employed with synchrotron radiation. As in powder diffraction, the averaging of thousands of observations per Bragg peak may improve the ratio of signal to noise of low-dose exposures. Here, it is shown that this paradigm can be implemented for room-temperature data collection using synchrotron radiation and exposure times of less than 3 ms. Using lysozyme microcrystals as a model system, over 40 000 single-crystal diffraction patterns were obtained and merged to produce a structural model that could be refined to 2.1 Å resolution. The resulting electron density is in excellent agreement with that obtained using standard X-ray data collection techniques. With further improvements the method is well suited for even shorter exposures at future and upgraded synchrotron radiation facilities that may deliver beams with 1000 times higher brightness than they currently produce. PMID:25075341

  2. Cleavage crystallography of liquid metal embrittled aluminum alloys

    NASA Technical Reports Server (NTRS)

    Reynolds, A. P.; Stoner, G. E.

    1991-01-01

    The crystallography of liquid metal-induced transgranular cleavage in six aluminum alloys having a variety of microstructures has been determined via Laue X-ray back reflection. The cleavage crystallography was independent of alloy microstructure, and the cleavage plane was 100-plane oriented in all cases. It was further determined that the cleavage crystallography was not influenced by alloy texture. Examination of the fracture surface indicated that there was not a unique direction of crack propagation. In addition, the existence of 100-plane cleavage on alloy 2024 fracture surfaces was inferred by comparison of secondary cleavage crack intersection geometry on the 2024 surfaces with the geometry of secondary cleavage crack intersections on the test alloys.

  3. Predicting the protein targets for athletic performance-enhancing substances

    PubMed Central

    2013-01-01

    Background The World Anti-Doping Agency (WADA) publishes the Prohibited List, a manually compiled international standard of substances and methods prohibited in-competition, out-of-competition and in particular sports. It would be ideal to be able to identify all substances that have one or more performance-enhancing pharmacological actions in an automated, fast and cost effective way. Here, we use experimental data derived from the ChEMBL database (~7,000,000 activity records for 1,300,000 compounds) to build a database model that takes into account both structure and experimental information, and use this database to predict both on-target and off-target interactions between these molecules and targets relevant to doping in sport. Results The ChEMBL database was screened and eight well populated categories of activities (Ki, Kd, EC50, ED50, activity, potency, inhibition and IC50) were used for a rule-based filtering process to define the labels “active” or “inactive”. The “active” compounds for each of the ChEMBL families were thereby defined and these populated our bioactivity-based filtered families. A structure-based clustering step was subsequently performed in order to split families with more than one distinct chemical scaffold. This produced refined families, whose members share both a common chemical scaffold and bioactivity against a common target in ChEMBL. Conclusions We have used the Parzen-Rosenblatt machine learning approach to test whether compounds in ChEMBL can be correctly predicted to belong to their appropriate refined families. Validation tests using the refined families gave a significant increase in predictivity compared with the filtered or with the original families. Out of 61,660 queries in our Monte Carlo cross-validation, belonging to 19,639 refined families, 41,300 (66.98%) had the parent family as the top prediction and 53,797 (87.25%) had the parent family in the top four hits. Having thus validated our approach, we used

  4. Applied Crystallography - Proceedings of the XVth Conference

    NASA Astrophysics Data System (ADS)

    Morawiec, H.; Ströż, D.

    1993-06-01

    The Table of Contents for the full book PDF is as follows: * Foreword * The International Centre for Diffraction Data and Its Future Developments * The Rietveld Method - A Historical Perspective * Real Structure in Quantitative Powder Diffraction Phase Analysis * Neutron Focusing Optics in Applied Crystallography * The Crystal Structures of Oxygen Deficient Rare Earth Oxides * Short-Range Order in Layer-Structured Ba1-xSrxBi2Nb2O9 Ferroelectrics * Radial Distribution Function as a Tool of Structural Studies on Noncrystalline Materials * Determination of Radial Distribution Function (RDF) of Electrodeposited Cu-Cd Alloys After Annealing * Spheres Packing as a Factor Describing the Local Environment and Structure Stability * X-Ray Stress Measurement of Samples Combined with Diffraction Line Analysis * Phase Stability and Martensitic Transformation in Cu-Zn and Cu-Zn-Al Single Crystals * Order, Defects, Precipitates and the Martensitic Transformation in β Cu-Zn-Al * Effect of γ Precipitates on the Martensitic Transformation in Cu-Zn-Al Alloys * Phase Transitions and Shape Memory Effect in a Thermomechanically Treated NiTi Alloy * Structure of Martensite and Bainite in CuAlMn Alloys * Glass-Ceramics * Mechanism of Texture Formation at the Rolling of Low Stacking Fault Energy Metals and Alloys * Shear Texture of Zinc and the Conditions of Its Occuring * The Development of Texture of ZnAlMg Sheets Depending on Deformation Geometry * Texture Stability of the D.S. NiAlMoCrTi Alloy After Heat Treatment * X-Ray Diffraction Method for Controlling of Texture Evolution in Layers * Texture and Lattice Imperfections Study of Some Low Alloyed Copper Alloys * Selected Examples of the Calculation of the Orientation Distribution Function for Low Crystal and Sample Symmetries * Automatical X-Ray Quantitative Phase Analysis * Application of a PC Computer for Crystallographic Calculations * Electron Diffraction Analysis using a Personal Computer * CA.R.INE Crystallography Version 2

  5. Protein crystal growth in space

    NASA Technical Reports Server (NTRS)

    Bugg, C. E.; Clifford, D. W.

    1987-01-01

    The advantages of protein crystallization in space, and the applications of protein crystallography to drug design, protein engineering, and the design of synthetic vaccines are examined. The steps involved in using protein crystallography to determine the three-dimensional structure of a protein are discussed. The growth chamber design and the hand-held apparatus developed for protein crystal growth by vapor diffusion techniques (hanging-drop method) are described; the experimental data from the four Shuttle missions are utilized to develop hardware for protein crystal growth in space and to evaluate the effects of gravity on protein crystal growth.

  6. Super-resolution biomolecular crystallography with low-resolution data.

    PubMed

    Schröder, Gunnar F; Levitt, Michael; Brunger, Axel T

    2010-04-22

    X-ray diffraction plays a pivotal role in the understanding of biological systems by revealing atomic structures of proteins, nucleic acids and their complexes, with much recent interest in very large assemblies like the ribosome. As crystals of such large assemblies often diffract weakly (resolution worse than 4 A), we need methods that work at such low resolution. In macromolecular assemblies, some of the components may be known at high resolution, whereas others are unknown: current refinement methods fail as they require a high-resolution starting structure for the entire complex. Determining the structure of such complexes, which are often of key biological importance, should be possible in principle as the number of independent diffraction intensities at a resolution better than 5 A generally exceeds the number of degrees of freedom. Here we introduce a method that adds specific information from known homologous structures but allows global and local deformations of these homology models. Our approach uses the observation that local protein structure tends to be conserved as sequence and function evolve. Cross-validation with R(free) (the free R-factor) determines the optimum deformation and influence of the homology model. For test cases at 3.5-5 A resolution with known structures at high resolution, our method gives significant improvements over conventional refinement in the model as monitored by coordinate accuracy, the definition of secondary structure and the quality of electron density maps. For re-refinements of a representative set of 19 low-resolution crystal structures from the Protein Data Bank, we find similar improvements. Thus, a structure derived from low-resolution diffraction data can have quality similar to a high-resolution structure. Our method is applicable to the study of weakly diffracting crystals using X-ray micro-diffraction as well as data from new X-ray light sources. Use of homology information is not restricted to X

  7. Macromolecular crystallography beamline X25 at the NSLS

    PubMed Central

    Héroux, Annie; Allaire, Marc; Buono, Richard; Cowan, Matthew L.; Dvorak, Joseph; Flaks, Leon; LaMarra, Steven; Myers, Stuart F.; Orville, Allen M.; Robinson, Howard H.; Roessler, Christian G.; Schneider, Dieter K.; Shea-McCarthy, Grace; Skinner, John M.; Skinner, Michael; Soares, Alexei S.; Sweet, Robert M.; Berman, Lonny E.

    2014-01-01

    Beamline X25 at the NSLS is one of the five beamlines dedicated to macromolecular crystallography operated by the Brookhaven National Laboratory Macromolecular Crystallography Research Resource group. This mini-gap insertion-device beamline has seen constant upgrades for the last seven years in order to achieve mini-beam capability down to 20 µm × 20 µm. All major components beginning with the radiation source, and continuing along the beamline and its experimental hutch, have changed to produce a state-of-the-art facility for the scientific community. PMID:24763654

  8. Recent Major Improvements to the ALS Sector 5 MacromolecularCrystallography Beamlines

    SciTech Connect

    Morton, Simon A.; Glossinger, James; Smith-Baumann, Alexis; McKean, John P.; Trame, Christine; Dickert, Jeff; Rozales, Anthony; Dauz,Azer; Taylor, John; Zwart, Petrus; Duarte, Robert; Padmore, Howard; McDermott, Gerry; Adams, Paul

    2007-07-01

    Although the Advanced Light Source (ALS) was initially conceived primarily as a low energy (1.9GeV) 3rd generation source of VUV and soft x-ray radiation it was realized very early in the development of the facility that a multipole wiggler source coupled with high quality, (brightness preserving), optics would result in a beamline whose performance across the optimal energy range (5-15keV) for macromolecular crystallography (MX) would be comparable to, or even exceed, that of many existing crystallography beamlines at higher energy facilities. Hence, starting in 1996, a suite of three beamlines, branching off a single wiggler source, was constructed, which together formed the ALS Macromolecular Crystallography Facility. From the outset this facility was designed to cater equally to the needs of both academic and industrial users with a heavy emphasis placed on the development and introduction of high throughput crystallographic tools, techniques, and facilities--such as large area CCD detectors, robotic sample handling and automounting facilities, a service crystallography program, and a tightly integrated, centralized, and highly automated beamline control environment for users. This facility was immediately successful, with the primary Multiwavelength Anomalous Diffraction beamline (5.0.2) in particular rapidly becoming one of the foremost crystallographic facilities in the US--responsible for structures such as the 70S ribosome. This success in-turn triggered enormous growth of the ALS macromolecular crystallography community and spurred the development of five additional ALS MX beamlines all utilizing the newly developed superconducting bending magnets ('superbends') as sources. However in the years since the original Sector 5.0 beamlines were built the performance demands of macromolecular crystallography users have become ever more exacting; with growing emphasis placed on studying larger complexes, more difficult structures, weakly diffracting or smaller

  9. Crystallography: Resolution beyond the diffraction limit

    NASA Astrophysics Data System (ADS)

    Shen, Jian-Ren

    2016-02-01

    A method has been devised that extends the resolution of X-ray crystal structures beyond the diffraction limit. This might help to improve the visualization of structures of proteins that form 'poorly diffracting' crystals. See Letter p.202

  10. Combining electron crystallography and X-ray crystallography to study the MlotiK1 cyclic nucleotide-regulated potassium channel

    PubMed Central

    Clayton, Gina M.; Aller, Steve G.; Wang, Jimin; Unger, Vinzenz; Morais-Cabral, João H.

    2010-01-01

    We have recently reported the X-ray structure of the cyclic nucleotide regulated potassium channel, MlotiK1. Here we describe the application of both electron and X-ray crystallography to obtain high quality crystals. We suggest that the combined application of these techniques provides a useful strategy for membrane protein structure determination. We also present negative stain projection and cryo-data projection maps. These maps provide new insights about the properties of the MlotiK1 channel. In particular, a comparison of a 9 Å cryo-data projection with calculated model maps strongly suggests that there is a very weak interaction between the pore and the S1-S4 domains of this 6 TM tetrameric cation channel and that the S1-S4 domains can adopt multiple orientations relative to the pore. PMID:19545635

  11. Tinker Toys, Crystallography, and the Introductory Mineralogy Course

    ERIC Educational Resources Information Center

    Buseck, Peter R.

    1970-01-01

    Describes the use of Tinker Toys to construct three dimensional models of crystals useful in illustrating many concepts of crystallography. Space lattices representing all of the Bravais types can be constructed. Also discusses the use of appropriate models to demonstrate the various symmetry operations. Bibliography. (LC)

  12. Models as an Aid to Courses in Crystallography and Mineralogy.

    ERIC Educational Resources Information Center

    Brady, K. T.

    1983-01-01

    Three models used in teaching crystallography/mineralogy at the University of Technology (Papua, New Guinea) are described. These include stereographic projection model, optical indicatrix models for Istropic/Anisotropic minerals, and model showing effect of anisotropic minerals under crossed polars. Photographs of the models are also included.…

  13. Two-Dimensional Crystallography Introduced by the Sprinkler Watering Problem

    ERIC Educational Resources Information Center

    De Toro, Jose A.; Calvo, Gabriel F.; Muniz, Pablo

    2012-01-01

    The problem of optimizing the number of circular sprinklers watering large fields is used to introduce, from a purely elementary geometrical perspective, some basic concepts in crystallography and comment on a few size effects in condensed matter physics. We examine square and hexagonal lattices to build a function describing the, so-called, dry…

  14. Using Two-Dimensional Colloidal Crystals to Understand Crystallography

    ERIC Educational Resources Information Center

    Bosse, Stephanie A.; Loening, Nikolaus M.

    2008-01-01

    X-ray crystallography is an essential technique for modern chemistry and biochemistry, but it is infrequently encountered by undergraduate students owing to lack of access to equipment, the time-scale for generating diffraction-quality molecular crystals, and the level of mathematics involved in analyzing the resulting diffraction patterns.…

  15. Performance of young calves fed soybean protein replacers.

    PubMed

    Akinyele, I O; Harshbarger, K E

    1983-04-01

    In two 12-wk trials we evaluated soybean protein in calf milk replacers. In trial 1, twelve 5-day-old Holstein calves were allotted randomly to three milk replacers. The diets contained 26% crude protein. In trial 2, 18 calves were used and the diet contained 30% crude protein. In trial 1 period 1 (10 to 15 days), mean coefficients of apparent digestibility (%) for milk protein, soy protein concentrate, and full fat soy flour replacers were dry matter 92.0, 70.0, 71.0; protein 90.1, 56.6, 61.3; fat 88.9, 55.0, 53.2;; and ash 85.8, 62.5, 61.2. Average daily nitrogen retentions were 10.8, -1.24, and -.7 g. Results in period 2 (30 to 35 days) for digestibilities and nitrogen retention showed considerable improvement over those in period 1. In trial 2 period 1, mean coefficients of apparent digestibility (%) for milk protein, soy protein concentrate, and defatted soy flour replacers were dry matter 87.5, 66.6, 47.9; protein 80.5, 57.2, 28.5; fat 81.5, 55.9, 33.5; and ash 83.8, 58.9, 48.4. Average daily nitrogen retentions were 10.8, 2.9, and -3.6 g. Results in period 2 for digestibilities and nitrogen retention also showed considerable improvement. Calves are able to use soy based replacers more effectively with age. PMID:6682865

  16. Developing advanced x-ray scattering methods combined with crystallography and computation

    PubMed Central

    Perry, J. Jefferson P.; Tainer, John A.

    2013-01-01

    The extensive use of small angle x-ray scattering (SAXS) over the last few years is rapidly providing new insights into protein interactions, complex formation and conformational states in solution. This SAXS methodology allows for detailed biophysical quantification of samples of interest. Initial analyses provide a judgment of sample quality, revealing the potential presence of aggregation, the overall extent of folding or disorder, the radius of gyration, maximum particle dimensions and oligomerization state. Structural characterizations include ab initio approaches from SAXS data alone, and when combined with previously determined crystal/NMR, atomistic modeling can further enhance structural solutions and assess validity. This combination can provide definitions of architectures, spatial organizations of protein domains within a complex, including those not determined by crystallography or NMR, as well as defining key conformational states of a protein interaction. SAXS is not generally constrained by macromolecule size, and the rapid collection of data in a 96-well plate format provides methods to screen sample conditions. This includes screening for co-factors, substrates, differing protein or nucleotide partners or small molecule inhibitors, to more fully characterize the variations within assembly states and key conformational changes. Such analyses may be useful for screening constructs and conditions to determine those most likely to promote crystal growth of a complex under study. Moreover, these high throughput structural determinations can be leveraged to define how polymorphisms affect assembly formations and activities. This is in addition to potentially providing architectural characterizations of complexes and interactions for systems biology-based research, and distinctions in assemblies and interactions in comparative genomics. Thus, SAXS combined with crystallography/NMR and computation provides a unique set of tools that should be considered

  17. Structural Studies on Membrane Proteins and Biological Macromolecular Assemblies in Japan

    NASA Astrophysics Data System (ADS)

    Tsukihara, Tomitake

    Structural studies on membrane proteins have been performed at atomic level by both three-dimensional X-ray crystallography and two-dimensional electron crystallography in Japan as in Europe and Unites States. More than 13 membrane protein structures were elucidate by X-ray method in our country, and seven membrane protein structures were determined by cryo-electron microscopic method developed by Fujiyoshi of Kyoto University. Extensive crystallographic studies on calcium pump and cytochrome c oxidase elucidated their functional mechanisms at atomic level. Structure and switching mechanism of a flagellum were studied by X-ray and electron microscopic methods. Vault structure exhibiting D39 symmetry was determined by X-ray method.

  18. Fluorescent Approaches to High Throughput Crystallography

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Forsythe, Elizabeth; Achari, Aniruddha

    2006-01-01

    We have shown that by covalently modifying a subpopulation, less than or equal to 1%, of a macromolecule with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification, and the presence of the probe at low concentrations does not affect the X-ray data quality or the crystallization behavior. The presence of the trace fluorescent label gives a number of advantages when used with high throughput crystallizations. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination crystals show up as bright objects against a dark background. Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Brightly fluorescent crystals are readily found against less bright precipitated phases, which under white light illumination may obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries as the protein or protein structures is all that shows up. Fluorescence intensity is a faster search parameter, whether visually or by automated methods, than looking for crystalline features. We are now testing the use of high fluorescence intensity regions, in the absence of clear crystalline features or "hits", as a means for determining potential lead conditions. A working hypothesis is that kinetics leading to non-structured phases may overwhelm and trap more slowly formed ordered assemblies, which subsequently show up as regions of brighter fluorescence intensity. Preliminary experiments with test proteins have resulted in the extraction of a number of crystallization conditions from screening outcomes based solely on the presence of bright fluorescent regions. Subsequent experiments will test this approach using a wider

  19. Which Strategy for a Protein Crystallization Project?

    NASA Technical Reports Server (NTRS)

    Kundrot, Craig E.

    2003-01-01

    The three-dimensional, atomic-resolution protein structures produced by X-ray crystallography over the past 50+ years have led to tremendous chemical understanding of fundamental biochemical processes. The pace of discovery in protein crystallography has increased greatly with advances in molecular biology, crystallization techniques, cryo-crystallography, area detectors, synchrotrons and computing. While the methods used to produce single, well-ordered crystals have also evolved over the years in response to increased understanding and advancing technology, crystallization strategies continue to be rooted in trial-and-error approaches. This review summarizes the current approaches in protein crystallization and surveys the first results to emerge from the structural genomics efforts.

  20. Automatic processing of macromolecular crystallography X-ray diffraction data at the ESRF.

    PubMed

    Monaco, Stéphanie; Gordon, Elspeth; Bowler, Matthew W; Delagenière, Solange; Guijarro, Matias; Spruce, Darren; Svensson, Olof; McSweeney, Sean M; McCarthy, Andrew A; Leonard, Gordon; Nanao, Max H

    2013-06-01

    The development of automated high-intensity macromolecular crystallography (MX) beamlines at synchrotron facilities has resulted in a remarkable increase in sample throughput. Developments in X-ray detector technology now mean that complete X-ray diffraction datasets can be collected in less than one minute. Such high-speed collection, and the volumes of data that it produces, often make it difficult for even the most experienced users to cope with the deluge. However, the careful reduction of data during experimental sessions is often necessary for the success of a particular project or as an aid in decision making for subsequent experiments. Automated data reduction pipelines provide a fast and reliable alternative to user-initiated processing at the beamline. In order to provide such a pipeline for the MX user community of the European Synchrotron Radiation Facility (ESRF), a system for the rapid automatic processing of MX diffraction data from single and multiple positions on a single or multiple crystals has been developed. Standard integration and data analysis programs have been incorporated into the ESRF data collection, storage and computing environment, with the final results stored and displayed in an intuitive manner in the ISPyB (information system for protein crystallography beamlines) database, from which they are also available for download. In some cases, experimental phase information can be automatically determined from the processed data. Here, the system is described in detail. PMID:23682196

  1. Automatic processing of macromolecular crystallography X-ray diffraction data at the ESRF

    PubMed Central

    Monaco, Stéphanie; Gordon, Elspeth; Bowler, Matthew W.; Delagenière, Solange; Guijarro, Matias; Spruce, Darren; Svensson, Olof; McSweeney, Sean M.; McCarthy, Andrew A.; Leonard, Gordon; Nanao, Max H.

    2013-01-01

    The development of automated high-intensity macromolecular crystallography (MX) beamlines at synchrotron facilities has resulted in a remarkable increase in sample throughput. Developments in X-ray detector technology now mean that complete X-ray diffraction datasets can be collected in less than one minute. Such high-speed collection, and the volumes of data that it produces, often make it difficult for even the most experienced users to cope with the deluge. However, the careful reduction of data during experimental sessions is often necessary for the success of a particular project or as an aid in decision making for subsequent experiments. Automated data reduction pipelines provide a fast and reliable alternative to user-initiated processing at the beamline. In order to provide such a pipeline for the MX user community of the European Synchrotron Radiation Facility (ESRF), a system for the rapid automatic processing of MX diffraction data from single and multiple positions on a single or multiple crystals has been developed. Standard integration and data analysis programs have been incorporated into the ESRF data collection, storage and computing environment, with the final results stored and displayed in an intuitive manner in the ISPyB (information system for protein crystallography beamlines) database, from which they are also available for download. In some cases, experimental phase information can be automatically determined from the processed data. Here, the system is described in detail. PMID:23682196

  2. The Influence of Early Protein Energy Malnutrition on Subsequent Behavior and Intellectual Performance.

    ERIC Educational Resources Information Center

    Gupta, Sarita

    1990-01-01

    Protein-energy malnutrition in early childhood, as seen in many developing countries, influences subsequent behavior and intellectual performance. These impairments are associated with further reduction in fine motor skills and academic performance. (Author)

  3. Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography.

    PubMed

    Keedy, Daniel A; Kenner, Lillian R; Warkentin, Matthew; Woldeyes, Rahel A; Hopkins, Jesse B; Thompson, Michael C; Brewster, Aaron S; Van Benschoten, Andrew H; Baxter, Elizabeth L; Uervirojnangkoorn, Monarin; McPhillips, Scott E; Song, Jinhu; Alonso-Mori, Roberto; Holton, James M; Weis, William I; Brunger, Axel T; Soltis, S Michael; Lemke, Henrik; Gonzalez, Ana; Sauter, Nicholas K; Cohen, Aina E; van den Bedem, Henry; Thorne, Robert E; Fraser, James S

    2015-01-01

    Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180--240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function. PMID:26422513

  4. Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography

    DOE PAGESBeta

    Keedy, Daniel A.; Kenner, Lillian R.; Warkentin, Matthew; Woldeyes, Rahel A.; Hopkins, Jesse B.; Thompson, Michael C.; Brewster, Aaron S.; Van Benschoten, Andrew H.; Baxter, Elizabeth L.; Uervirojnangkoorn, Monarin; et al

    2015-09-30

    Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences ofmore » these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.« less

  5. Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography

    PubMed Central

    Keedy, Daniel A; Kenner, Lillian R; Warkentin, Matthew; Woldeyes, Rahel A; Hopkins, Jesse B; Thompson, Michael C; Brewster, Aaron S; Van Benschoten, Andrew H; Baxter, Elizabeth L; Uervirojnangkoorn, Monarin; McPhillips, Scott E; Song, Jinhu; Alonso-Mori, Roberto; Holton, James M; Weis, William I; Brunger, Axel T; Soltis, S Michael; Lemke, Henrik; Gonzalez, Ana; Sauter, Nicholas K; Cohen, Aina E; van den Bedem, Henry; Thorne, Robert E; Fraser, James S

    2015-01-01

    Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function. DOI: http://dx.doi.org/10.7554/eLife.07574.001 PMID:26422513

  6. Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography

    SciTech Connect

    Keedy, Daniel A.; Kenner, Lillian R.; Warkentin, Matthew; Woldeyes, Rahel A.; Hopkins, Jesse B.; Thompson, Michael C.; Brewster, Aaron S.; Van Benschoten, Andrew H.; Baxter, Elizabeth L.; Uervirojnangkoorn, Monarin; McPhillips, Scott E.; Song, Jinhu; Alonso-Mori, Roberto; Holton, James M.; Weis, William I.; Brunger, Axel T.; Soltis, S. Michael; Lemke, Henrik; Gonzalez, Ana; Sauter, Nicholas K.; Cohen, Aina E.; van den Bedem, Henry; Thorne, Robert E.; Fraser, James S.

    2015-09-30

    Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.

  7. PRIGo: a new multi-axis goniometer for macromolecular crystallography

    PubMed Central

    Waltersperger, Sandro; Olieric, Vincent; Pradervand, Claude; Glettig, Wayne; Salathe, Marco; Fuchs, Martin R.; Curtin, Adrian; Wang, Xiaoqiang; Ebner, Simon; Panepucci, Ezequiel; Weinert, Tobias; Schulze-Briese, Clemens; Wang, Meitian

    2015-01-01

    The Parallel Robotics Inspired Goniometer (PRIGo) is a novel compact and high-precision goniometer providing an alternative to (mini-)kappa, traditional three-circle goniometers and Eulerian cradles used for sample reorientation in macromolecular crystallography. Based on a combination of serial and parallel kinematics, PRIGo emulates an arc. It is mounted on an air-bearing stage for rotation around ω and consists of four linear positioners working synchronously to achieve x, y, z translations and χ rotation (0–90°), followed by a ϕ stage (0–360°) for rotation around the sample holder axis. Owing to the use of piezo linear positioners and active correction, PRIGo features spheres of confusion of <1 µm, <7 µm and <10 µm for ω, χ and ϕ, respectively, and is therefore very well suited for micro-crystallography. PRIGo enables optimal strategies for both native and experimental phasing crystallographic data collection. Herein, PRIGo hardware and software, its calibration, as well as applications in macromolecular crystallography are described. PMID:26134792

  8. The macromolecular crystallography facility at the advanced light source

    NASA Astrophysics Data System (ADS)

    Earnest, Thomas; Padmore, Howard; Cork, Carl; Behrsing, Rolf; Kim, Sung-Hou

    1996-10-01

    Synchrotron radiation offers several advantages over the use of rotating anode sources for biological crystallography, which allow for the collection of higher-resolution data, substantially more rapid data collection, phasing by multiwavelength anomalous diffraction (MAD) techniques, and time-resolved experiments using polychromatic radiation (Laue diffraction). The use of synchrotron radiation is often necessary to record useful data from crystals which diffract weakly or have very large unit cells. The high brightness and stability characteristics of the advanced light source (ALS) at Lawrence Berkeley National Laboratory, along with the low emittance and long straight sections to accommodate insertion devices present in third generation synchrotrons like the ALS, lead to several advantages in the field of macromolecular crystallography. We are presently constructing a macromolecular crystallography facility at the ALS which is optimized for user-friendliness and high-throughput data collection, with advanced capabilities for MAD and Laue experiments. The X-rays will be directed to three branchlines. A well-equipped support lab will be available for biochemistry, crystal mounting and sample storage, as well as computer hardware and software available, along with staff support, allowing for the complete processing of data on site.

  9. Crystallography and spin-crossover. A view of breathing materials.

    PubMed

    Guionneau, Philippe

    2014-01-14

    The spin-crossover phenomenon (SCO) is a fascinating field that potentially concerns any material containing a (d(4)-d(7)) transition metal complex finding therefore an echo in as diverse research fields as chemistry, physics, biology and geology. Particularly, molecular and coordination-polymers SCO solids are thoroughly investigated since their bistability promises new routes towards a large panel of potential applications including smart pigments, optical switches or memory devices. Notwithstanding these motivating applicative targets, numerous fundamental aspects of SCO are still debated. Among them, the investigation of the structure-property relationships is unfailingly at the heart of the SCO research field. All the facets of the richness of the structural behaviors shown by SCO compounds are only revealed when exploring the whole sample scales -from atomic to macroscopic- all the external stimuli-temperature, pressure, light and any combinations and derived perturbations- and the various forms of the SCO compounds in the solid state -crystalline powders, single-crystals, poorly crystalline or nano-sized particles. Crystallography allows investigating all these aspects of SCO solids. In the past few years, crystallography has certainly been in a significant phase of development pushing the frontiers of investigations, in particular thanks to the progress in X-ray diffraction techniques. The encounter between SCO materials and crystallography is captivating, taking advantages from each other. In this paper, a personal account mainly based on our recent results provides perspectives and new approaches that should be developed in the investigation of SCO materials. PMID:24201509

  10. High-Throughput Crystallography: Reliable and Efficient Identification of Fragment Hits.

    PubMed

    Schiebel, Johannes; Krimmer, Stefan G; Röwer, Karine; Knörlein, Anna; Wang, Xiaojie; Park, Ah Young; Stieler, Martin; Ehrmann, Frederik R; Fu, Kan; Radeva, Nedyalka; Krug, Michael; Huschmann, Franziska U; Glöckner, Steffen; Weiss, Manfred S; Mueller, Uwe; Klebe, Gerhard; Heine, Andreas

    2016-08-01

    Today the identification of lead structures for drug development often starts from small fragment-like molecules raising the chances to find compounds that successfully pass clinical trials. At the heart of the screening for fragments binding to a specific target, crystallography delivers structural information essential for subsequent drug design. While it is common to search for bound ligands in electron densities calculated directly after an initial refinement cycle, we raise the important question whether this strategy is viable for fragments characterized by low affinities. Here, we describe and provide a collection of high-quality diffraction data obtained from 364 protein crystals treated with diverse fragments. Subsequent data analysis showed that ∼25% of all hits would have been missed without further refining the resulting structures. To enable fast and reliable hit identification, we have designed an automated refinement pipeline that will inspire the development of optimized tools facilitating the successful application of fragment-based methods. PMID:27452405

  11. Structural disorder: a tool for housekeeping proteins performing tissue-specific interactions.

    PubMed

    Banerjee, Sanghita; De, Rajat K

    2016-09-01

    An interaction between a pair of proteins unique for a particular tissue is denoted as a tissue-specific interaction (TSI). Tissue-specific (TS) proteins always perform TSIs with a limited number of interacting partners. However, it has been claimed that housekeeping (HK) proteins frequently take part in TSIs. This is actually an unusual phenomenon. How a single HK protein mediates TSIs - remains an interesting yet an unsolved question. We have hypothesized that HK proteins have attained a high degree of structural flexibility to modulate TSIs efficiently. We have observed that HK proteins are selected to be intrinsically disordered compared to TS proteins. Therefore, the purposeful adaptation of structural disorder brings out special advantages for HK proteins compared to TS proteins. We have demonstrated that TSIs may play vital roles in shaping the molecular adaptation of disordered regions within HK proteins. We also have noticed that HK proteins, mediating a huge number of TSIs, have a greater portion of their interacting interfaces overlapped with the adjacent disordered segment. Moreover, these HK proteins, mediating TSIs, preferably adapt single domain (SD). We have concluded that HK proteins adapt a high degree of structural flexibility to mediate TSIs. Besides, having a SD along with structural flexibility is more economic than maintaining multiple domains with a rigid structure. This assists them in attaining various structural conformations upon binding to their partners, thereby designing an economically optimum molecular system. PMID:26375894

  12. Protein crystallization - is it rocket science?

    PubMed

    DeLucas, L J.

    2001-07-01

    Fueled by initial space shuttle results, the National Aeronautics and Space Administration (NASA) has been supporting fundamental studies of macromolecular crystal growth since 1985. The majority of this research is directed at understanding the relationship between experimental variables and important crystal characteristics. The program has resulted in new methods and technology that will benefit the crystallography community's effort to meet the ever-increasing demand for protein structural information. Microgravity crystallization results indicate a potential impact on structural biology's more challenging problems, as soon as long-duration experiments can be performed on the International Space Station. PMID:11445465

  13. Preparation of Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

    PubMed Central

    Liu, Wei; Ishchenko, Andrii; Cherezov, Vadim

    2014-01-01

    We have recently established a procedure for serial femtosecond crystallography in lipidic cubic phase (LCP-SFX) for protein structure determination at X-ray free electron lasers (XFELs). LCP-SFX uses the gel-like lipidic cubic phase (LCP) as a matrix for growth and delivery of membrane protein microcrystals for crystallographic data collection. LCP is a liquid-crystalline mesophase, composed of lipids and water. It provides a membrane-mimicking environment that stabilizes membrane proteins and supports their crystallization. Here we describe detailed procedures for the preparation and characterization of microcrystals for LCP-SFX applications. The advantages of LCP-SFX over traditional crystallographic methods include the capability of collecting room temperature high-resolution data with minimal effects of radiation damage from sub-10 µm crystals of membrane and soluble proteins that are difficult to crystallize, while eliminating the need for crystal harvesting and cryo-cooling. Compared to SFX methods for microcrystals in solution using liquid injectors, LCP-SFX reduces protein consumption by 2–3 orders of magnitude for data collection at currently available XFELs. The whole procedure typically takes 3–5 days, including the time required for crystals to grow. PMID:25122522

  14. Protein Crystal Growth With the Aid of Microfluidics

    NASA Technical Reports Server (NTRS)

    vanderWoerd, Mark

    2003-01-01

    Protein crystallography is one of three well-known methods to obtain the structure of proteins. A major rate limiting step in protein crystallography is protein crystal nucleation and growth, which is still largely a process conducted by trial-and-error methods. Many attempts have been made to improve protein crystal growth by performing growth in microgravity. Although the use of microgravity appears to improve crystal quality in some attempts, this method has been inefficient because several reasons: we lack a fundamental understanding of macromolecular crystal growth in general and of the influence of microgravity in particular, we have to start with crystal growth conditions in microgravity based on conditions on the ground and finally the hardware does not allow for experimental iteration without reloading samples on the ground. To partially accommodate the disadvantages of the current hardware, we have used microfluidic technology (Lab-on-a-Chip devices) to design the concept of a more efficient crystallization device, suitable for use on the International Space Station and in high-throughput applications on the ground. The concept and properties of microfluidics, the application design process, and the advances in protein crystal growth hardware will be discussed in this presentation. Some examples of proteins crystallized in the new hardware will be discussed, including the differences between conventional crystallization versus crystallization in microfluidics.

  15. Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography.

    PubMed

    Leuthold, Mila M; Koromyslova, Anna D; Singh, Bishal K; Hansman, Grant S

    2016-01-01

    The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions. The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains. PMID:27167457

  16. Crystallography of rare galactic honeycomb structure near supernova 1987a

    NASA Technical Reports Server (NTRS)

    Noever, David A.

    1994-01-01

    Near supernova 1987a, the rare honeycomb structure of 20-30 galactic bubbles measures 30 x 90 light years. Its remarkable regularity in bubble size suggests a single-event origin which may correlate with the nearby supernova. To test the honeycomb's regularity in shape and size, the formalism of statistical crystallography is developed here for bubble sideness. The standard size-shape relations (Lewis's law, Desch's law, and Aboav-Weaire's law) govern area, perimeter and nearest neighbor shapes. Taken together, they predict a highly non-equilibrium structure for the galactic honeycomb which evolves as a bimodal shape distribution without dominant bubble perimeter energy.

  17. Biophysical Highlights from 54 Years of Macromolecular Crystallography

    PubMed Central

    Richardson, Jane S.; Richardson, David C.

    2014-01-01

    The United Nations has declared 2014 the International Year of Crystallography, and in commemoration, this review features a selection of 54 notable macromolecular crystal structures that have illuminated the field of biophysics in the 54 years since the first excitement of the myoglobin and hemoglobin structures in 1960. Chronological by publication of the earliest solved structure, each illustrated entry briefly describes key concepts or methods new at the time and key later work leveraged by knowledge of the three-dimensional atomic structure. PMID:24507592

  18. JBluIce-EPICS control system for macromolecular crystallography.

    SciTech Connect

    Stepanov, S.; Makarov, O.; Hilgart, M.; Pothineni, S.; Urakhchin, A.; Devarapalli, S.; Yoder, D.; Becker, M.; Ogata, C.; Sanishvili, R.; Nagarajan, V.; Smith, J. L.; Fischetti, R. F.

    2011-01-01

    The trio of macromolecular crystallography beamlines constructed by the General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) in Sector 23 of the Advanced Photon Source (APS) have been in growing demand owing to their outstanding beam quality and capacity to measure data from crystals of only a few micrometres in size. To take full advantage of the state-of-the-art mechanical and optical design of these beamlines, a significant effort has been devoted to designing fast, convenient, intuitive and robust beamline controls that could easily accommodate new beamline developments. The GM/CA-CAT beamline controls are based on the power of EPICS for distributed hardware control, the rich Java graphical user interface of Eclipse RCP and the task-oriented philosophy as well as the look and feel of the successful SSRL BluIce graphical user interface for crystallography. These beamline controls feature a minimum number of software layers, the wide use of plug-ins that can be written in any language and unified motion controls that allow on-the-fly scanning and optimization of any beamline component. This paper describes the ways in which BluIce was combined with EPICS and converted into the Java-based JBluIce, discusses the solutions aimed at streamlining and speeding up operations and gives an overview of the tools that are provided by this new open-source control system for facilitating crystallographic experiments, especially in the field of microcrystallography.

  19. Serial femtosecond crystallography: A revolution in structural biology.

    PubMed

    Martin-Garcia, Jose M; Conrad, Chelsie E; Coe, Jesse; Roy-Chowdhury, Shatabdi; Fromme, Petra

    2016-07-15

    Macromolecular crystallography at synchrotron sources has proven to be the most influential method within structural biology, producing thousands of structures since its inception. While its utility has been instrumental in progressing our knowledge of structures of molecules, it suffers from limitations such as the need for large, well-diffracting crystals, and radiation damage that can hamper native structural determination. The recent advent of X-ray free electron lasers (XFELs) and their implementation in the emerging field of serial femtosecond crystallography (SFX) has given rise to a remarkable expansion upon existing crystallographic constraints, allowing structural biologists access to previously restricted scientific territory. SFX relies on exceptionally brilliant, micro-focused X-ray pulses, which are femtoseconds in duration, to probe nano/micrometer sized crystals in a serial fashion. This results in data sets comprised of individual snapshots, each capturing Bragg diffraction of single crystals in random orientations prior to their subsequent destruction. Thus structural elucidation while avoiding radiation damage, even at room temperature, can now be achieved. This emerging field has cultivated new methods for nanocrystallogenesis, sample delivery, and data processing. Opportunities and challenges within SFX are reviewed herein. PMID:27143509

  20. A beamline for macromolecular crystallography at the Advanced Light Source

    SciTech Connect

    Padmore, H.A.; Earnest, T.; Kim, S.H.; Thompson, A.C.; Robinson, A.L.

    1994-08-01

    A beamline for macromolecular crystallography has been designed for the ALS. The source will be a 37-pole wiggler with a, 2-T on-axis peak field. The wiggler will illuminate three beamlines, each accepting 3 mrad of horizontal aperture. The central beamline will primarily be used for multiple-wavelength anomalous dispersion measurements in the wavelength range from 4 to 0.9 {angstrom}. The beamline optics will comprise a double-crystal monochromator with a collimating pre-mirror and a double-focusing mirror after the monochromator. The two side stations will be used for fixed-wavelength experiments within the wavelength range from 1.5 to 0.95 {angstrom}. The optics will consist of a conventional vertically focusing cylindrical mirror followed by an asymmetrically cut curved-crystal monochromator. This paper presents details of the optimization of the wiggler source for crystallography, gives a description of the beamline configuration, and discusses the reasons for the choices made.

  1. Serial crystallography on in vivo grown microcrystals using synchrotron radiation

    PubMed Central

    Gati, Cornelius; Bourenkov, Gleb; Klinge, Marco; Rehders, Dirk; Stellato, Francesco; Oberthür, Dominik; Yefanov, Oleksandr; Sommer, Benjamin P.; Mogk, Stefan; Duszenko, Michael; Betzel, Christian; Schneider, Thomas R.; Chapman, Henry N.; Redecke, Lars

    2014-01-01

    Crystal structure determinations of biological macromolecules are limited by the availability of sufficiently sized crystals and by the fact that crystal quality deteriorates during data collection owing to radiation damage. Exploiting a micrometre-sized X-ray beam, high-precision diffractometry and shutterless data acquisition with a pixel-array detector, a strategy for collecting data from many micrometre-sized crystals presented to an X-ray beam in a vitrified suspension is demonstrated. By combining diffraction data from 80 Trypanosoma brucei procathepsin B crystals with an average volume of 9 µm3, a complete data set to 3.0 Å resolution has been assembled. The data allowed the refinement of a structural model that is consistent with that previously obtained using free-electron laser radiation, providing mutual validation. Further improvements of the serial synchrotron crystallography technique and its combination with serial femtosecond crystallography are discussed that may allow the determination of high-resolution structures of micrometre-sized crystals. PMID:25075324

  2. X-ray crystallography and biological metal centers: is seeing believing?

    SciTech Connect

    Sommerhalter, M.; Lieberman, R.L.; Rosenzweig, A.C.

    2010-03-08

    Metalloenzyme crystal structures have a major impact on our understanding of biological metal centers. They are often the starting point for mechanistic and computational studies and inspire synthetic modeling chemistry. The strengths and limitations of X-ray crystallography in determining properties of biological metal centers and their corresponding ligand spheres are explored through examples, including ribonucleotide reductase R2 and particulate methane monooxygenase. Protein crystal structures locate metal ions within a protein fold and reveal the identities and coordination geometries of amino acid ligands. Data collection strategies that exploit the anomalous scattering effect of metal ions can establish metal ion identity. The quality of crystallographic data, particularly the resolution, determines the level of detail that can be extracted from a protein crystal structure. Complementary spectroscopic techniques can provide crucial information regarding the redox state of the metal center as well as the presence, type, and protonation state of exogenous ligands. The final result of the crystallographic characterization of a metalloenzyme is a model based on crystallographic data, supported by information from biophysical and modeling studies, influenced by sample handling, and interpreted carefully by the crystallographer.

  3. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7

    PubMed Central

    Langer, Gerrit G; Cohen, Serge X; Lamzin, Victor S; Perrakis, Anastassis

    2008-01-01

    ARP/wARP is a software suite to build macromolecular models in X-ray crystallography electron density maps. Structural genomics initiatives and the study of complex macromolecular assemblies and membrane proteins all rely on advanced methods for 3D structure determination. ARP/wARP meets these needs by providing the tools to obtain a macromolecular model automatically, with a reproducible computational procedure. ARP/wARP 7.0 tackles several tasks: iterative protein model building including a high-level decision-making control module; fast construction of the secondary structure of a protein; building flexible loops in alternate conformations; fully automated placement of ligands, including a choice of the best fitting ligand from a “cocktail”; and finding ordered water molecules. All protocols are easy to handle by a non-expert user through a graphical user interface or a command line. The time required is typically a few minutes although iterative model building may take a few hours. PMID:18600222

  4. Mapping the Accessible Conformational Landscape of an Insect Carboxylesterase Using Conformational Ensemble Analysis and Kinetic Crystallography.

    PubMed

    Correy, Galen J; Carr, Paul D; Meirelles, Tamara; Mabbitt, Peter D; Fraser, Nicholas J; Weik, Martin; Jackson, Colin J

    2016-06-01

    The proper function of enzymes often depends upon their efficient interconversion between particular conformational sub-states on a free-energy landscape. Experimentally characterizing these sub-states is challenging, which has limited our understanding of the role of protein dynamics in many enzymes. Here, we have used a combination of kinetic crystallography and detailed analysis of crystallographic protein ensembles to map the accessible conformational landscape of an insect carboxylesterase (LcαE7) as it traverses all steps in its catalytic cycle. LcαE7 is of special interest because of its evolving role in organophosphate insecticide resistance. Our results reveal that a dynamically coupled network of residues extends from the substrate-binding site to a surface loop. Interestingly, the coupling of this network that is apparent in the apoenzyme appears to be reduced in the phosphorylated enzyme intermediate. Altogether, the results of this work highlight the importance of protein dynamics to enzyme function and the evolution of new activity. PMID:27210287

  5. The structural biology center at the APS: an integrated user facility for macromolecular crystallography

    NASA Astrophysics Data System (ADS)

    Rosenbaum, G.; Westbrook, E. M.

    1997-07-01

    The Structural Biology Center (SBC) has developed and operates a sector (undulator and bending magnet) of the APS as a user facility for macromolecular crystallography. Crystallographically determined structures of proteins, nucleic acids and their complexes with proteins, viruses, and complexes between macromolecules and small ligands have become of central importance in molecular and cellular biology. Major design goals were to make the extremely high brilliance of the APS available for brilliance limited studies, and to achieve a high throughput of less demanding studies, as well as optimization for MAS-phasing. Crystal samples will include extremely small crystals, crystals with large unit cells (viruses, ribosomes, etc.) and ensembles of closely similar crystal structures for drug design, protein engineering, etc. Data are recorded on a 3000×3000 pixel CCD-area detector (optionally on image plates). The x-ray optics of both beamlines has been designed to produce a highly demagnified image of the source in order to match the focal size with the sizes of the sample and the resolution element of the detector. Vertical focusing is achieved by a flat, cylindrically bent mirror. Horizontal focusing is achieved by sagitally bending the second crystal of the double crystal monochromator. Monochromatic fluxes of 1.3*1013 ph/s into focal sizes of 0.08 mm (horizontal)×0.04 mm (vertical) FWHM (flux density 3.5*1015 ph/s/mm2) have been recorded.

  6. Scalable Fabrication of Electrospun Nanofibrous Membranes Functionalized with Citric Acid for High-Performance Protein Adsorption.

    PubMed

    Fu, Qiuxia; Wang, Xueqin; Si, Yang; Liu, Lifang; Yu, Jianyong; Ding, Bin

    2016-05-11

    Fabricating protein adsorbents with high adsorption capacity and appreciable throughput is extremely important and highly desired for the separation and purification of protein products in the biomedical and pharmaceutical industries, yet still remains a great challenge. Herein, we demonstrate the synthesis of a novel protein adsorbent by in situ functionalizing eletrospun ethylene-vinyl alcohol (EVOH) nanofibrous membranes (NFM) with critic acid (CCA). Taking advantage of the merits of large specific surface area, highly tortuous open-porous structure, abundant active carboxyl groups introduced by CCA, superior chemical stability, and robust mechanical strength, the obtained CCA-grafted EVOH NFM (EVOH-CCA NFM) present an excellent integrated protein (take lysozyme as the model protein) adsorption performance with a high capacity of 284 mg g(-1), short equilibrium time of 6 h, ease of elution, and good reusability. Meanwhile, the adsorption performance of EVOH-CCA NFM can be optimized by regulating buffer pH, ionic strength, and initial concentration of protein solutions. More importantly, a dynamic binding efficiency of 250 mg g(-1) can be achieved driven solely by the gravity of protein solution, which matches well with the demands of the high yield and energy conservation in the actual protein purification process. Furthermore, the resultant EVOH-CCA NFM also possess unique selectivity for positively charged proteins which was confirmed by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Significantly, the successful synthesis of such intriguing and economic EVOH-CCA NFM may provide a promising candidate for the next generation of protein adsorbents for rapid, massive, and cost-effective separation and purification of proteins. PMID:27111287

  7. Effect of cleaning agents and additives on Protein A ligand degradation and chromatography performance.

    PubMed

    Yang, Lihua; Harding, Jason D; Ivanov, Alexander V; Ramasubramanyan, Natarajan; Dong, Diane D

    2015-03-13

    Protein A chromatography, employing the recombinant Protein A ligand, is widely used as a capture step for antibody and Fc-fusion proteins manufacture. Protein A ligands in these matrices are susceptible to degradation/loss when exposed to cleaning agents such as sodium hydroxide, resulting in loss of capacity on reuse. In this study, MabSelect Protein A ligand and MabSelect SuRe Protein A ligand were chosen to evaluate the impact of alkaline cleaning solutions on the ligands and the packed columns. The Protein A ligands alone and the Protein A columns were incubated or cycled in different concentrations of sodium hydroxide solutions with and without additives, respectively. Ligand integrity (degradation) and ligand function (binding affinity) were studied using SDS-PAGE and customized Biacore technology, surface plasma resonance (SPR) and were successfully correlated with column performance measurement in terms of static binding capacity (SBC), dynamic binding capacity (DBC) and recovery as a function of exposure to cleaning agents with and without additives. The findings and the methodology presented in this study are not only able to determine appropriate cleaning conditions for Protein A chromatography, but also provided tools to enable systematic and rapid study of the cleaning solutions and conditions. PMID:25680549

  8. HPTLC-aptastaining - Innovative protein detection system for high-performance thin-layer chromatography.

    PubMed

    Morschheuser, Lena; Wessels, Hauke; Pille, Christina; Fischer, Judith; Hünniger, Tim; Fischer, Markus; Paschke-Kratzin, Angelika; Rohn, Sascha

    2016-01-01

    Protein analysis using high-performance thin-layer chromatography (HPTLC) is not commonly used but can complement traditional electrophoretic and mass spectrometric approaches in a unique way. Due to various detection protocols and possibilities for hyphenation, HPTLC protein analysis is a promising alternative for e.g., investigating posttranslational modifications. This study exemplarily focused on the investigation of lysozyme, an enzyme which is occurring in eggs and technologically added to foods and beverages such as wine. The detection of lysozyme is mandatory, as it might trigger allergenic reactions in sensitive individuals. To underline the advantages of HPTLC in protein analysis, the development of innovative, highly specific staining protocols leads to improved sensitivity for protein detection on HPTLC plates in comparison to universal protein derivatization reagents. This study aimed at developing a detection methodology for HPTLC separated proteins using aptamers. Due to their affinity and specificity towards a wide range of targets, an aptamer based staining procedure on HPTLC (HPTLC-aptastaining) will enable manifold analytical possibilities. Besides the proof of its applicability for the very first time, (i) aptamer-based staining of proteins is applicable on different stationary phase materials and (ii) furthermore, it can be used as an approach for a semi-quantitative estimation of protein concentrations. PMID:27220270

  9. HPTLC-aptastaining – Innovative protein detection system for high-performance thin-layer chromatography

    NASA Astrophysics Data System (ADS)

    Morschheuser, Lena; Wessels, Hauke; Pille, Christina; Fischer, Judith; Hünniger, Tim; Fischer, Markus; Paschke-Kratzin, Angelika; Rohn, Sascha

    2016-05-01

    Protein analysis using high-performance thin-layer chromatography (HPTLC) is not commonly used but can complement traditional electrophoretic and mass spectrometric approaches in a unique way. Due to various detection protocols and possibilities for hyphenation, HPTLC protein analysis is a promising alternative for e.g., investigating posttranslational modifications. This study exemplarily focused on the investigation of lysozyme, an enzyme which is occurring in eggs and technologically added to foods and beverages such as wine. The detection of lysozyme is mandatory, as it might trigger allergenic reactions in sensitive individuals. To underline the advantages of HPTLC in protein analysis, the development of innovative, highly specific staining protocols leads to improved sensitivity for protein detection on HPTLC plates in comparison to universal protein derivatization reagents. This study aimed at developing a detection methodology for HPTLC separated proteins using aptamers. Due to their affinity and specificity towards a wide range of targets, an aptamer based staining procedure on HPTLC (HPTLC-aptastaining) will enable manifold analytical possibilities. Besides the proof of its applicability for the very first time, (i) aptamer-based staining of proteins is applicable on different stationary phase materials and (ii) furthermore, it can be used as an approach for a semi-quantitative estimation of protein concentrations.

  10. HPTLC-aptastaining – Innovative protein detection system for high-performance thin-layer chromatography

    PubMed Central

    Morschheuser, Lena; Wessels, Hauke; Pille, Christina; Fischer, Judith; Hünniger, Tim; Fischer, Markus; Paschke-Kratzin, Angelika; Rohn, Sascha

    2016-01-01

    Protein analysis using high-performance thin-layer chromatography (HPTLC) is not commonly used but can complement traditional electrophoretic and mass spectrometric approaches in a unique way. Due to various detection protocols and possibilities for hyphenation, HPTLC protein analysis is a promising alternative for e.g., investigating posttranslational modifications. This study exemplarily focused on the investigation of lysozyme, an enzyme which is occurring in eggs and technologically added to foods and beverages such as wine. The detection of lysozyme is mandatory, as it might trigger allergenic reactions in sensitive individuals. To underline the advantages of HPTLC in protein analysis, the development of innovative, highly specific staining protocols leads to improved sensitivity for protein detection on HPTLC plates in comparison to universal protein derivatization reagents. This study aimed at developing a detection methodology for HPTLC separated proteins using aptamers. Due to their affinity and specificity towards a wide range of targets, an aptamer based staining procedure on HPTLC (HPTLC-aptastaining) will enable manifold analytical possibilities. Besides the proof of its applicability for the very first time, (i) aptamer-based staining of proteins is applicable on different stationary phase materials and (ii) furthermore, it can be used as an approach for a semi-quantitative estimation of protein concentrations. PMID:27220270

  11. Advance chromatin extraction improves capture performance of protein A affinity chromatography.

    PubMed

    Nian, Rui; Zhang, Wei; Tan, Lihan; Lee, Jeremy; Bi, Xeuzhi; Yang, Yuansheng; Gan, Hui Theng; Gagnon, Pete

    2016-01-29

    Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise. Protein A ligand leakage from columns loaded with chromatin-extracted harvest was half the level observed on protein A columns loaded with non-extracted harvest. Columns loaded with chromatin-extracted harvest were cleaned more effectively by 50-100mM NaOH than columns loaded with non-extracted harvest that were cleaned with 250-500mM NaOH. Two protein A media with IgG capacities in excess of 50g/L were loaded with chromatin-extracted harvest, washed with 2.0M NaCl before elution, and the eluted IgG fraction titrated to pH 5.5 before microfiltration. Host protein contamination in the filtrate was reduced to <1ppm, DNA to <1ppb, protein A leakage to 0.5ppm, and aggregates to 1.0%. Caprylic acid and allantoin were both reduced below 5ppm. Step recovery of IgG was 99.4%. Addition of a single polishing step reduced residual protein A beneath the level of detection and aggregates to <0.1%. Overall process recovery including chromatin extraction was 90%. PMID:26774119

  12. Applications of multigrain crystallography for the study of post-perovskite microstructures

    NASA Astrophysics Data System (ADS)

    Merkel, S.; Langrand, C.; Hilairet, N.; Rosa, A. D.

    2015-12-01

    Silicate post-perovskite is found at the Earth's core-mantle boundary, 2900 km below the Earth's surface. Understanding the mechanical properties of this phase is important for constraining the dynamics of this region. It is, however, a multiscale problem with post-perovskite defects driving the behavior of single-crystals, themselves parts of polycrystals, polycrystals plastically deforming due to mantle convection. Here, we wish to address one aspect of this multiscale problem by analyzing the behavior of individual grains of post-perovskite inside a polycrystalline aggregate. Post-perovskite is not stable under ambient pressures and should be studied under the pressures of the Earth's core-mantle boundary: 135 GPa, We use diamond anvil cells to generate such pressure conditions and study grains of post-perovskite using multigrain crystallography, in-situ, at pressures above 1 megabar. Experiment was performed at the ID11 beamline of the ESRF on a 80 μm diameter and 20 μm thickness post-perovskite sample to identify grains of post-perovskite and follow their behavior inside a polycrystalline aggregate. Diamond anvil cells allow experiments under extreme conditions but access to sample diffraction and sample rotation are limited. Such limitation in sample access can be a problem for multigrain crystallography. Hence, in this presentation, we will investigate the fine details of such analysis. In particular, we will focus on evaluating the proportion of grains that can be indexed successfully and how grains can be localized within the sample. Eventually, these results will allow us to follow the orientation and position of hundreds of grains inside a polycrystalline aggregate undergoing physical processes such as plastic deformation or phase transformations.

  13. Effect of whey protein hydrolysate on performance and recovery of top-class orienteering runners.

    PubMed

    Hansen, Mette; Bangsbo, Jens; Jensen, Jørgen; Bibby, Bo Martin; Madsen, Klavs

    2015-04-01

    This trial aimed to examine the effect of whey protein hydrolysate intake before and after exercise sessions on endurance performance and recovery in elite orienteers during a training camp. Eighteen elite orienteers participated in a randomized controlled intervention trial during a 1-week training camp (13 exercise sessions). Half of the runners (PRO-CHO) ingested a protein drink before (0.3 g kg(-1)) and a protein-carbohydrate drink after (0.3 g protein kg(-1) and 1 g carbohydrate kg(-1)) each exercise session. The others ingested energy and time-matched carbohydrate drinks (CHO). A 4-km run-test with 20 control points was performed before and on the last day of the intervention. Blood and saliva were obtained in the mornings, before and after run-tests, and after the last training session. During the intervention, questionnaires were fulfilled regarding psychological sense of performance capacity and motivation. PRO-CHO and not CHO improved performance in the 4-km run-test (interaction p < .05). An increase in serum creatine kinase was observed during the week, which was greater in CHO than PRO-CHO (interaction p < .01). Lactate dehydrogenase (p < .001) and cortisol (p = .057) increased during the week, but the change did not differ between groups. Reduction in sense of performance capacity during the intervention was greater in CHO (p < .05) than PRO-CHO. In conclusion, ingestion of whey protein hydrolysate before and after each exercise session improves performance and reduces markers of muscle damage during a strenuous 1-week training camp. The results indicate that protein supplementation in conjunction with each exercise session facilitates the recovery from strenuous training in elite orienteers. PMID:25029703

  14. Perspectives on protein crystallisation

    SciTech Connect

    Ochi, T.; Stojanoff, V.; Bolanos-Garcia, V.M.; Moreno, A.

    2009-12-11

    This final part on 'perspectives' is focused on new strategies that can be used to crystallise proteins and improve the crystal quality of macromolecular complexes using any of the methods reviewed in this focused issue. Some advantages and disadvantages, limitations, and plausible applications to high-resolution X-ray crystallography are discussed.

  15. Energy optimization of a regular macromolecular crystallography beamline for ultra-high-resolution crystallography

    SciTech Connect

    Rosenbaum, Gerd; Ginell, Stephan L.; Chen, Julian C.-H.

    2015-01-01

    In this study, a practical method for operating existing undulator synchrotron beamlines at photon energies considerably higher than their standard operating range is described and applied at beamline 19-ID of the Structural Biology Center at the Advanced Photon Source enabling operation at 30 keV. Adjustments to the undulator spectrum were critical to enhance the 30 keV flux while reducing the lower- and higher-energy harmonic contamination. A Pd-coated mirror and Al attenuators acted as effective low- and high-bandpass filters. The resulting flux at 30 keV, although significantly lower than with X-ray optics designed and optimized for this energy, allowed for accurate data collection on crystals of the small protein crambin to 0.38 Å resolution.

  16. Energy optimization of a regular macromolecular crystallography beamline for ultra-high-resolution crystallography

    DOE PAGESBeta

    Rosenbaum, Gerd; Ginell, Stephan L.; Chen, Julian C.-H.

    2015-01-01

    In this study, a practical method for operating existing undulator synchrotron beamlines at photon energies considerably higher than their standard operating range is described and applied at beamline 19-ID of the Structural Biology Center at the Advanced Photon Source enabling operation at 30 keV. Adjustments to the undulator spectrum were critical to enhance the 30 keV flux while reducing the lower- and higher-energy harmonic contamination. A Pd-coated mirror and Al attenuators acted as effective low- and high-bandpass filters. The resulting flux at 30 keV, although significantly lower than with X-ray optics designed and optimized for this energy, allowed for accuratemore » data collection on crystals of the small protein crambin to 0.38 Å resolution.« less

  17. A history of experimental phasing in macromolecular crystallography

    PubMed Central

    Isaacs, Neil

    2016-01-01

    It was just over a century ago that W. L. Bragg published a paper describing the first crystal structures to be determined using X-ray diffraction data. These structures were obtained from considerations of X-ray diffraction (Bragg equation), crystallography (crystal lattices and symmetry) and the scattering power of different atoms. Although W. H. Bragg proposed soon afterwards, in 1915, that the periodic electron density in crystals could be analysed using Fourier transforms, it took some decades before experimental phasing methods were developed. Many scientists contributed to this development and this paper presents the author’s own perspective on this history. There will be other perspectives, so what follows is a history, rather than the history, of experimental phasing. PMID:26960116

  18. Nonequilibrium phase transitions in cuprates observed by ultrafast electron crystallography.

    PubMed

    Gedik, Nuh; Yang, Ding-Shyue; Logvenov, Gennady; Bozovic, Ivan; Zewail, Ahmed H

    2007-04-20

    Nonequilibrium phase transitions, which are defined by the formation of macroscopic transient domains, are optically dark and cannot be observed through conventional temperature- or pressure-change studies. We have directly determined the structural dynamics of such a nonequilibrium phase transition in a cuprate superconductor. Ultrafast electron crystallography with the use of a tilted optical geometry technique afforded the necessary atomic-scale spatial and temporal resolutions. The observed transient behavior displays a notable "structural isosbestic" point and a threshold effect for the dependence of c-axis expansion (Deltac) on fluence (F), with Deltac/F = 0.02 angstrom/(millijoule per square centimeter). This threshold for photon doping occurs at approximately 0.12 photons per copper site, which is unexpectedly close to the density (per site) of chemically doped carriers needed to induce superconductivity. PMID:17446397

  19. Data processing pipeline for serial femtosecond crystallography at SACLA1

    PubMed Central

    Nakane, Takanori; Joti, Yasumasa; Tono, Kensuke; Yabashi, Makina; Nango, Eriko; Iwata, So; Ishitani, Ryuichiro; Nureki, Osamu

    2016-01-01

    A data processing pipeline for serial femtosecond crystallography at SACLA was developed, based on Cheetah [Barty et al. (2014). J. Appl. Cryst.47, 1118–1131] and CrystFEL [White et al. (2016). J. Appl. Cryst.49, 680–689]. The original programs were adapted for data acquisition through the SACLA API, thread and inter-node parallelization, and efficient image handling. The pipeline consists of two stages: The first, online stage can analyse all images in real time, with a latency of less than a few seconds, to provide feedback on hit rate and detector saturation. The second, offline stage converts hit images into HDF5 files and runs CrystFEL for indexing and integration. The size of the filtered compressed output is comparable to that of a synchrotron data set. The pipeline enables real-time feedback and rapid structure solution during beamtime. PMID:27275146

  20. The Mn4Ca photosynthetic water-oxidation catalyst studied by simultaneous X-ray spectroscopy and crystallography using an X-ray free-electron laser

    PubMed Central

    Tran, Rosalie; Kern, Jan; Hattne, Johan; Koroidov, Sergey; Hellmich, Julia; Alonso-Mori, Roberto; Sauter, Nicholas K.; Bergmann, Uwe; Messinger, Johannes; Zouni, Athina; Yano, Junko; Yachandra, Vittal K.

    2014-01-01

    The structure of photosystem II and the catalytic intermediate states of the Mn4CaO5 cluster involved in water oxidation have been studied intensively over the past several years. An understanding of the sequential chemistry of light absorption and the mechanism of water oxidation, however, requires a new approach beyond the conventional steady-state crystallography and X-ray spectroscopy at cryogenic temperatures. In this report, we present the preliminary progress using an X-ray free-electron laser to determine simultaneously the light-induced protein dynamics via crystallography and the local chemistry that occurs at the catalytic centre using X-ray spectroscopy under functional conditions at room temperature. PMID:24914152

  1. JBluIce–EPICS control system for macromolecular crystallography

    PubMed Central

    Stepanov, Sergey; Makarov, Oleg; Hilgart, Mark; Pothineni, Sudhir Babu; Urakhchin, Alex; Devarapalli, Satish; Yoder, Derek; Becker, Michael; Ogata, Craig; Sanishvili, Ruslan; Venugopalan, Nagarajan; Smith, Janet L.; Fischetti, Robert F.

    2011-01-01

    The trio of macromolecular crystallography beamlines constructed by the General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) in Sector 23 of the Advanced Photon Source (APS) have been in growing demand owing to their outstanding beam quality and capacity to measure data from crystals of only a few micrometres in size. To take full advantage of the state-of-the-art mechanical and optical design of these beamlines, a significant effort has been devoted to designing fast, convenient, intuitive and robust beamline controls that could easily accommodate new beamline developments. The GM/CA-CAT beamline controls are based on the power of EPICS for distributed hardware control, the rich Java graphical user interface of Eclipse RCP and the task-oriented philosophy as well as the look and feel of the successful SSRL BluIce graphical user interface for crystallography. These beamline controls feature a minimum number of software layers, the wide use of plug-ins that can be written in any language and unified motion controls that allow on-the-fly scanning and optimization of any beamline com­ponent. This paper describes the ways in which BluIce was combined with EPICS and converted into the Java-based JBluIce, discusses the solutions aimed at streamlining and speeding up operations and gives an overview of the tools that are provided by this new open-source control system for facilitating crystallo­graphic experiments, especially in the field of microcrystallo­graphy. PMID:21358048

  2. Assessment of microcrystal quality by transmission electron microscopy for efficient serial femtosecond crystallography.

    PubMed

    Barnes, Christopher O; Kovaleva, Elena G; Fu, Xiaofeng; Stevenson, Hilary P; Brewster, Aaron S; DePonte, Daniel P; Baxter, Elizabeth L; Cohen, Aina E; Calero, Guillermo

    2016-07-15

    Serial femtosecond crystallography (SFX) employing high-intensity X-ray free-electron laser (XFEL) sources has enabled structural studies on microcrystalline protein samples at non-cryogenic temperatures. However, the identification and optimization of conditions that produce well diffracting microcrystals remains an experimental challenge. Here, we report parallel SFX and transmission electron microscopy (TEM) experiments using fragmented microcrystals of wild type (WT) homoprotocatechuate 2,3-dioxygenase (HPCD) and an active site variant (H200Q). Despite identical crystallization conditions and morphology, as well as similar crystal size and density, the indexing efficiency of the diffraction data collected using the H200Q variant sample was over 7-fold higher compared to the diffraction results obtained using the WT sample. TEM analysis revealed an abundance of protein aggregates, crystal conglomerates and a smaller population of highly ordered lattices in the WT sample as compared to the H200Q variant sample. While not reported herein, the 1.75 Å resolution structure of the H200Q variant was determined from ∼16 min of beam time, demonstrating the utility of TEM analysis in evaluating sample monodispersity and lattice quality, parameters critical to the efficiency of SFX experiments. PMID:26944553

  3. Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography

    DOE PAGESBeta

    Nogly, Przemyslaw; Panneels, Valerie; Nelson, Garrett; Gati, Cornelius; Kimura, Tetsunari; Milne, Christopher; Milathianaki, Despina; Kubo, Minoru; Wu, Wenting; Conrad, Chelsie; et al

    2016-08-22

    Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1 ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1 mg per collected time point. Accumulation of M intermediate within themore » crystal lattice is confirmed by time-resolved visible absorption spectroscopy. Furthermore, this study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.« less

  4. The linkage between ribosomal crystallography, metal ions, heteropolytungstates and functional flexibility

    NASA Astrophysics Data System (ADS)

    Bashan, Anat; Yonath, Ada

    2008-11-01

    Crystallography of ribosomes, the universal cell nucleoprotein assemblies facilitating the translation of the genetic-code into proteins, met with severe problems owing to the large size, complex structure, inherent flexibility and high conformational variability of the ribosome. For the case of the small ribosomal subunit, which caused extreme difficulties, post-crystallization treatment by minute amounts of a heteropolytungstate cluster allowed structure determination at atomic resolution. This cluster played a dual role: providing anomalous phasing power and dramatically increased the resolution, by stabilization of a selected functional conformation. Thus, four out of the fourteen clusters that bind to each of the crystallized small subunits are attached to a specific ribosomal protein in a fashion that may control a significant component of the subunit internal flexibility, by "gluing" symmetrical related subunits. Here, we highlight basic issues in the relationship between metal ions and macromolecules and present common traits controlling in the interactions between polymetalates and various macromolecules, which may be extended towards the exploitation of polymetalates for therapeutical treatment.

  5. Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography.

    PubMed

    Nogly, Przemyslaw; Panneels, Valerie; Nelson, Garrett; Gati, Cornelius; Kimura, Tetsunari; Milne, Christopher; Milathianaki, Despina; Kubo, Minoru; Wu, Wenting; Conrad, Chelsie; Coe, Jesse; Bean, Richard; Zhao, Yun; Båth, Petra; Dods, Robert; Harimoorthy, Rajiv; Beyerlein, Kenneth R; Rheinberger, Jan; James, Daniel; DePonte, Daniel; Li, Chufeng; Sala, Leonardo; Williams, Garth J; Hunter, Mark S; Koglin, Jason E; Berntsen, Peter; Nango, Eriko; Iwata, So; Chapman, Henry N; Fromme, Petra; Frank, Matthias; Abela, Rafael; Boutet, Sébastien; Barty, Anton; White, Thomas A; Weierstall, Uwe; Spence, John; Neutze, Richard; Schertler, Gebhard; Standfuss, Jörg

    2016-01-01

    Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1 ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1 mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX. PMID:27545823

  6. Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography

    PubMed Central

    Nogly, Przemyslaw; Panneels, Valerie; Nelson, Garrett; Gati, Cornelius; Kimura, Tetsunari; Milne, Christopher; Milathianaki, Despina; Kubo, Minoru; Wu, Wenting; Conrad, Chelsie; Coe, Jesse; Bean, Richard; Zhao, Yun; Båth, Petra; Dods, Robert; Harimoorthy, Rajiv; Beyerlein, Kenneth R.; Rheinberger, Jan; James, Daniel; DePonte, Daniel; Li, Chufeng; Sala, Leonardo; Williams, Garth J.; Hunter, Mark S.; Koglin, Jason E.; Berntsen, Peter; Nango, Eriko; Iwata, So; Chapman, Henry N.; Fromme, Petra; Frank, Matthias; Abela, Rafael; Boutet, Sébastien; Barty, Anton; White, Thomas A.; Weierstall, Uwe; Spence, John; Neutze, Richard; Schertler, Gebhard; Standfuss, Jörg

    2016-01-01

    Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1 ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1 mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX. PMID:27545823

  7. Designing a diverse high-quality library for crystallography-based FBDD screening.

    PubMed

    Tounge, Brett A; Parker, Michael H

    2011-01-01

    A well-chosen set of fragments is able to cover a large chemical space using a small number of compounds. The actual size and makeup of the fragment set is dependent on the screening method since each technique has its own practical limits in terms of the number of compounds that can be screened and requirements for compound solubility. In this chapter, an overview of the general requirements for a fragment library is presented for different screening platforms. In the case of the FBDD work at Johnson & Johnson Pharmaceutical Research and Development, L.L.C., our main screening technology is X-ray crystallography. Since every soaked protein crystal needs to be diffracted and a protein structure determined to delineate if a fragment binds, the size of our initial screening library cannot be a rate-limiting factor. For this reason, we have chosen 900 as the appropriate primary fragment library size. To choose the best set, we have developed our own mix of simple property ("Rule of 3") and "bad" substructure filtering. While this gets one a long way in terms of limiting the fragment pool, there are still tens of thousands of compounds to choose from after this initial step. Many of the choices left at this stage are not drug-like, so we have developed an FBDD Score to help select a 900-compound set. The details of this score and the filtering are presented. PMID:21371585

  8. Crystal structure of the TLDc domain of oxidation resistance protein 2 from zebrafish.

    PubMed

    Blaise, Mickaël; Alsarraf, Husam M A B; Wong, Jaslyn E M M; Midtgaard, Søren Roi; Laroche, Fabrice; Schack, Lotte; Spaink, Herman; Stougaard, Jens; Thirup, Søren

    2012-06-01

    The oxidation resistance proteins (OXR) help to protect eukaryotes from reactive oxygen species. The sole C-terminal domain of the OXR, named TLDc is sufficient to perform this function. However, the mechanism by which oxidation resistance occurs is poorly understood. We present here the crystal structure of the TLDc domain of the oxidation resistance protein 2 from zebrafish. The structure was determined by X-ray crystallography to atomic resolution (0.97Å) and adopts an overall globular shape. Two antiparallel β-sheets form a central β-sandwich, surrounded by two helices and two one-turn helices. The fold shares low structural similarity to known structures. PMID:22434723

  9. Cerebellar protein expression in three different mouse strains and their relevance for motor performance.

    PubMed

    Pollak, Daniela; Weitzdoerfer, Rachel; Yang, Yae-Won; Prast, Helmut; Hoeger, Harald; Lubec, Gert

    2005-01-01

    The present study uses a proteomic approach to link motor function to cerebellar protein expression in 129X1/SvJ, C57BL/6J and nNOS WT mice. Poor performance on the Rota rod, the standard test for motor coordination, was detected in 129X1/SvJ mice. No gross impairments of neurological, cognitive and behavioural functions were observed. Identification and quantification of 48 proteins revealed reduced expression of calbindin, septin 5 and syntaxin binding protein 1 in 129X1/SvJ. In nNos WT glucose-6-phosphate 1 dehydrogenase X was decreased whereas dihydropyrimidinase-related protein-4 was increased. In C57BL/6J stress-70 protein, alpha enolase, NAD-dependent deacetylase sirtuin 2, septin 2, dihydropyrimidinase-related protein-2 and brain derived neurotrophic factor showed elevated levels. Neurological examination, Rota rod test, Morris Water Maze, Multiple-T-Maze, Open field and Elevated plus-maze were employed to study motor, cognitive and behavioural function. Mice were sacrificed and cerebellar tissue was homogenized. Proteins were extracted and separated on two-dimensional gel electrophoresis with subsequent in-gel digestion followed by mass spectrometrical analysis of peptides (MALDI-TOF/TOF-TOF). Quantification of spots was carried out by specific software. A strong association of impaired motor function with altered cerebellar protein expression of calbindin, septin 5 and syntaxin binding protein 1in 129X1/SvJ was observed and is in agreement with previous observations of motor deficiencies in a calbindin knock-out mouse. These results have to be taken into account when using 129X1/SvJ for biochemical, toxicological or gene targeting experiments as well as when studying the above-mentioned proteins or corresponding pathways and cascades in this mouse strain. PMID:15567512

  10. Ribosomal crystallography: peptide bond formation and its inhibition.

    PubMed

    Bashan, Anat; Zarivach, Raz; Schluenzen, Frank; Agmon, Ilana; Harms, Joerg; Auerbach, Tamar; Baram, David; Berisio, Rita; Bartels, Heike; Hansen, Harly A S; Fucini, Paola; Wilson, Daniel; Peretz, Moshe; Kessler, Maggie; Yonath, Ada

    2003-09-01

    Ribosomes, the universal cellular organelles catalyzing the translation of genetic code into proteins, are protein/RNA assemblies, of a molecular weight 2.5 mega Daltons or higher. They are built of two subunits that associate for performing protein biosynthesis. The large subunit creates the peptide bond and provides the path for emerging proteins. The small has key roles in initiating the process and controlling its fidelity. Crystallographic studies on complexes of the small and the large eubacterial ribosomal subunits with substrate analogs, antibiotics, and inhibitors confirmed that the ribosomal RNA governs most of its activities, and indicated that the main catalytic contribution of the ribosome is the precise positioning and alignment of its substrates, the tRNA molecules. A symmetry-related region of a significant size, containing about two hundred nucleotides, was revealed in all known structures of the large ribosomal subunit, despite the asymmetric nature of the ribosome. The symmetry rotation axis, identified in the middle of the peptide-bond formation site, coincides with the bond connecting the tRNA double-helical features with its single-stranded 3' end, which is the moiety carrying the amino acids. This thus implies sovereign movements of tRNA features and suggests that tRNA translocation involves a rotatory motion within the ribosomal active site. This motion is guided and anchored by ribosomal nucleotides belonging to the active site walls, and results in geometry suitable for peptide-bond formation with no significant rearrangements. The sole geometrical requirement for this proposed mechanism is that the initial P-site tRNA adopts the flipped orientation. The rotatory motion is the major component of unified machinery for peptide-bond formation, translocation, and nascent protein progression, since its spiral nature ensures the entrance of the nascent peptide into the ribosomal exit tunnel. This tunnel, assumed to be a passive path for the

  11. Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids

    PubMed Central

    Hesse, Almut

    2016-01-01

    Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports. PMID:27559481

  12. Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids.

    PubMed

    Hesse, Almut; Weller, Michael G

    2016-01-01

    Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports. PMID:27559481

  13. Encapsulation performance of proteins and traditional materials for spray dried flavors.

    PubMed

    Charve, Joséphine; Reineccius, Gary A

    2009-03-25

    The objective of this study was to evaluate the potential of selected proteins as alternative materials for flavor encapsulation by spray drying. Two traditional materials (gum acacia and modified starch) and three proteins (sodium caseinate, whey and soy protein isolates) were used at different infeed solid levels; test compounds included (R)-(+)-limonene and three alpha,beta-unsaturated aldehydes ((E)-2-hexenal, (E)-cinnamaldehyde, citral). The primary criteria for performance were flavor retention during drying and protection against losses during storage. Limonene oxidation and nonenzymatic browning were investigated as two possible deterioration routes. Overall, higher infeed solids improved retention during drying and limited flavor losses (aldehydes and limonene) during storage in traditional materials only. The materials giving the highest flavor retention during drying were gum acacia (94%), modified starch (88%) and whey protein isolate (87%). Gum acacia provided the highest retention of aldehydes during storage (37 to 58%) after 28 days at 40 degrees C but did not afford good protection against limonene oxidation. Oppositely, protein materials effectively limited limonene oxidation (>70% retained). Nonenzymatic browning was observed for all powders prepared with proteins, especially whey protein isolate, whereas no browning occurred with traditional materials. PMID:19231860

  14. A high-performance liquid chromatography method for determining transition metal content in proteins.

    PubMed

    Atanassova, Anelia; Lam, Robert; Zamble, Deborah B

    2004-12-01

    Transition metals are common components of cellular proteins and the detailed study of metalloproteins necessitates the identification and quantification of bound metal ions. Screening for metals is also an informative step in the initial characterization of the numerous unknown and unclassified proteins now coming through the proteomic pipeline. We have developed a high-performance liquid chromatography method for the quantitative determination of the most prevalent biological transition metals: manganese, iron, cobalt, nickel, copper, and zinc. The method is accurate and simple and can be adapted for automated high-throughput studies. The metal analysis involves acid hydrolysis to release the metal ions into solution, followed by ion separation on a mixed-bead ion-exchange column and absorbance detection after postcolumn derivatization with the metallochromic indicator 4-(2-pyridylazo)resorcinol. The potential interferences by common components of protein solutions were investigated. The metal content of a variety of metalloproteins was analyzed and the data were compared to data obtained from inductively coupled plasma-atomic emission spectroscopy. The sensitivity of the assay allows for the detection of 0.1-0.8 nmol, depending on the metal. The amount of protein required is governed by the size of the protein and the fraction of protein with metal bound. For routine analysis 50 microg was used but for many proteins 10 microg would be sufficient. The advantages, disadvantages, and possible applications of this method are discussed. PMID:15519577

  15. A comparison of protein A chromatographic stationary phases: performance characteristics for monoclonal antibody purification.

    PubMed

    Liu, Zhuo; Mostafa, Sigma S; Shukla, Abhinav A

    2015-01-01

    Protein A chromatography remains the dominant capture step used during the downstream purification of monoclonal antibodies (mAbs). With the recent expiry of the Repligen patent on recombinant Protein A, a variety of new Protein A resins have been introduced in the market. Given productivity limitations during downstream processing that have come into sharper focus with the recent increase in cell culture titers for mAbs, the selection of an appropriate Protein A resin has direct implications on the overall process economics of mAb production. The performance of seven different Protein A chromatographic resins was compared with respect to static binding capacity and dynamic binding capacity as a function of flow rate. This data was translated into a comparison of productivity (g mAb purified per unit resin volume per unit time) for the seven stationary phases. In addition, elution pH and host cell protein impurity levels after product capture on each of these resins were determined. The current article provides an effective methodology and dataset for the selection of the optimal Protein A chromatographic resin. PMID:24823474

  16. Atomic-level Snapshot Catches Protein Motor in Action

    SciTech Connect

    2009-01-01

    Using a state-of-the-art protein crystallography beamline at Berkeley Labs Advanced Light Source, researchers have captured a critical action shapshot of an enzyme that is vital to the survival of all biological cells.

  17. Lipid Monolayer and Sparse Matrix Screening for Growing Two-Dimensional Crystals for Electron Crystallography: Methods and Examples

    PubMed Central

    Yeager, Mark; Dryden, Kelly A.; Ganser-Pornillos, Barbie K.

    2014-01-01

    Electron microscopy provides an efficient method for rapidly assessing whether a solution of macromolecules is homogeneous and monodisperse. If the macromolecules can be induced to form two-dimensional crystals that are a single layer in thickness, then electron crystallography of frozen-hydrated crystals has the potential of achieving three-dimensional density maps at sub-nanometer or even atomic resolution. Here we describe the lipid monolayer and sparse matrix screening methods for growing two-dimensional crystals and present successful applications to soluble macromolecular complexes: carboxysome shell proteins and HIV CA, respectively. Since it is common to express recombinant proteins with poly-His tags for purification by metal affinity chromatography, the monolayer technique using bulk lipids doped with Ni2+ lipids has the potential for broad application. Likewise, the sparse matrix method uses screening conditions for three-dimensional crystallization and is therefore of broad applicability. PMID:23132079

  18. High performance protein microarrays based on glycidyl methacrylate-modified polyethylene terephthalate plastic substrate.

    PubMed

    Liu, Yingshuai; Li, Chang Ming; Hu, Weihua; Lu, Zhisong

    2009-01-15

    There is a great challenge to immobilize high density of probe molecules for high performance protein microarrays, and this is achieved in this work by using polyethylene terephthalate (PET) plastic substrate onto which glycidyl methacrylate (GMA) photopolymer is grafted under mild conditions to introduce high density of epoxy groups for covalent immobilization of proteins. The poly(GMA)-grafted PET (PGMA-PET) surface was characterized with atomic force microscope (AFM) and attenuated total reflectance Fourier transform infra-red (ATR-FTIR) spectroscopy. For high density of protein immobilization and good quality of microspots, experiments were conducted to optimize the printing buffer, and an optimal buffer was found out to be PBS with 10% glycerol+0.003% triton X-100. According to the studies of loading capacity and immobilization kinetics, the optimal protein probe concentration and incubation time for the efficient immobilization are 200 microg mL(-1) and 8h, respectively. The performance of the PGMA-PET-based protein microarrays is evaluated with sandwich immunoassay using rat IgG and anti-rat IgG as model proteins, demonstrating a limit of detection (LOD) of 10 pg mL(-1) and a dynamic range of five orders of magnitude which are better than or very comparable with the reported or commercially available immunoassays, while providing a high-throughput approach. The work renders a simple and economic method to manufacture high performance protein microarrays and is expected to have great potentials in broad applications related to clinic diagnosis, drug discovery and proteomic research. PMID:19064107

  19. CHARACTERIZATION OF DRUG INTERACTIONS WITH SERUM PROTEINS BY USING HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe; Barnaby, Omar; Jackson, Abby; Yoo, Michelle J.; Papastavros, Efthimia; Pfaunmiller, Erika; Sobansky, Matt; Tong, Zenghan

    2011-01-01

    The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding. PMID:21395530

  20. Getting More From Less: Correlated Single-Crystal Spectroscopy and X-ray Crystallography at the NSLS (456th Brookhaven Lecture)

    SciTech Connect

    Orville, Allen

    2010-04-21

    By integrating different techniques to collect complementary data at beam line X26C of the National Syncrotron Light Source (NSLS), Allen Orville and his colleagues of the Macromolecular Crystallography Research Resource are providing new insights into the structures of macromolecules. During the 456th Brookhaven Lecture, on Wednesday, April 21st, Orville will describe his approach and his findings in a talk entitled “Getting More From Less: Correlated Singe-Crystal Spectroscopy and X-Ray Crystallography at the NSLS,” beginning at 4 p.m. in Berkner Hall. Refreshments will be offered before and after the lecture. During his talk, Orville will discuss his field of protein crystallography, reviewing its landmark discoveries and explaining new ways of using the NSLS and, in the future, the NSLS-II, to build on those discoveries. Allen Orville took his Ph.D. in biology from the University of Minnesota in 1997. After completing a postdoc with the Howard Hughes Medical Institute at the University of Oregon, 1997-2000, he began an assistant professor at the Georgia Institute of Technology. Orville joined BNL's Biology Department in 2006 as an associate biophysicist and was promoted to biophysicist in 2008.

  1. Architectural plasticity of AMPK revealed by electron microscopy and X-ray crystallography

    PubMed Central

    Ouyang, Yan; Zhu, Li; Li, Yifang; Guo, Miaomiao; Liu, Yang; Cheng, Jin; Zhao, Jing; Wu, Yi

    2016-01-01

    Mammalian AMP-activated protein kinase (AMPK) acts as an important sensor of cellular energy homeostasis related with AMP/ADP to ATP ratio. The overall architecture of AMPK has been determined in either homotrimer or monomer form by electron microscopy (EM) and X-ray crystallography successively. Accordingly proposed models have consistently revealed a key role of the α subunit linker in sensing adenosine nucleoside binding on the γ subunit and mediating allosteric regulation of kinase domain (KD) activity, whereas there are vital differences in orienting N-terminus of α subunit and locating carbohydrate-binding module (CBM) of β subunit. Given that Mg2+, an indispensable cofactor of AMPK was present in the EM sample preparation buffer however absent when forming crystals, here we carried out further reconstructions without Mg2+ to expectably inspect if this ion may contribute to this difference. However, no essential alteration has been found in this study compared to our early work. Further analyses indicate that the intra-molecular movement of the KD and CBM are most likely due to the flexible linkage of the disordered linkers with the rest portion as well as a contribution from the plasticity in the inter-molecular assembly mode, which might ulteriorly reveal an architectural complication of AMPK. PMID:27063142

  2. Sequential substitution of K(+) bound to Na(+),K(+)-ATPase visualized by X-ray crystallography.

    PubMed

    Ogawa, Haruo; Cornelius, Flemming; Hirata, Ayami; Toyoshima, Chikashi

    2015-01-01

    Na(+),K(+)-ATPase transfers three Na(+) from the cytoplasm into the extracellular medium and two K(+) in the opposite direction per ATP hydrolysed. The binding and release of Na(+) and K(+) are all thought to occur sequentially. Here we demonstrate by X-ray crystallography of the ATPase in E2·MgF4(2-)·2K(+), a state analogous to E2·Pi·2K(+), combined with isotopic measurements, that the substitution of the two K(+) with congeners in the extracellular medium indeed occurs at different rates, substantially faster at site II. An analysis of thermal movements of protein atoms in the crystal shows that the M3-M4E helix pair opens and closes the ion pathway leading to the extracellular medium, allowing K(+) at site II to be substituted first. Taken together, these results indicate that site I K(+) is the first cation to bind to the empty cation-binding sites after releasing three Na(+). PMID:26258479

  3. Micro-Crystallography Developments at GM/CA-CAT at the APS

    SciTech Connect

    Xu Shenglan; Makarov, Oleg; Benn, Rich; Yoder, Derek W.; Stepanov, Sergey; Becker, Michael; Corcoran, Stephen; Hilgart, Mark; Nagarajan, Venugopalan; Ogata, Craig M.; Pothineni, Sudhir; Sanishvili, Ruslan; Fischetti, Robert F.; Smith, Janet L.

    2010-06-23

    Recently, several important structures have been solved using micro-crystallographic techniques that previously could not have been solved with conventional crystallography. At GM/CA-CAT we continue to develop micro-crystallographic capabilities for difficult problems such as small crystals of large macromolecular complexes or membrane proteins grown in the lipidic cubic phase. This paper will describe three major upgrades to our arsenal of tools, 'mini-beam' collimators, active beamstop, and an improved goniostat. Our 'mini-beam' collimators have evolved to a new triple-collimator fabricated from molybdenum as a uni-body. This has significantly improved the robustness, ease of initial alignment, and reduction of background. More recently, two prototypes of a quad-collimator have been developed and fabricated to provide a selection of mini-beams of 5, 10, 20 {mu}m and a 300 {mu}m scatter-guard on a single body. The smaller beams and samples have increased the demand on the tolerances of our goniostat. To meet these challenges we have designed and implemented a goniostat with a 1-micron peak-to-peak sphere of confusion. This is a significant improvement over the previous 6 micron sphere of confusion of the commercially available air-bearing and XY stages. Finally, an 'active beamstop' has been constructed. This will provide non-invasive, real time feedback at the sample during data collection.

  4. Architectural plasticity of AMPK revealed by electron microscopy and X-ray crystallography.

    PubMed

    Ouyang, Yan; Zhu, Li; Li, Yifang; Guo, Miaomiao; Liu, Yang; Cheng, Jin; Zhao, Jing; Wu, Yi

    2016-01-01

    Mammalian AMP-activated protein kinase (AMPK) acts as an important sensor of cellular energy homeostasis related with AMP/ADP to ATP ratio. The overall architecture of AMPK has been determined in either homotrimer or monomer form by electron microscopy (EM) and X-ray crystallography successively. Accordingly proposed models have consistently revealed a key role of the α subunit linker in sensing adenosine nucleoside binding on the γ subunit and mediating allosteric regulation of kinase domain (KD) activity, whereas there are vital differences in orienting N-terminus of α subunit and locating carbohydrate-binding module (CBM) of β subunit. Given that Mg(2+), an indispensable cofactor of AMPK was present in the EM sample preparation buffer however absent when forming crystals, here we carried out further reconstructions without Mg(2+) to expectably inspect if this ion may contribute to this difference. However, no essential alteration has been found in this study compared to our early work. Further analyses indicate that the intra-molecular movement of the KD and CBM are most likely due to the flexible linkage of the disordered linkers with the rest portion as well as a contribution from the plasticity in the inter-molecular assembly mode, which might ulteriorly reveal an architectural complication of AMPK. PMID:27063142

  5. Crystallography Open Database – an open-access collection of crystal structures

    PubMed Central

    Gražulis, Saulius; Chateigner, Daniel; Downs, Robert T.; Yokochi, A. F. T.; Quirós, Miguel; Lutterotti, Luca; Manakova, Elena; Butkus, Justas; Moeck, Peter; Le Bail, Armel

    2009-01-01

    The Crystallography Open Database (COD), which is a project that aims to gather all available inorganic, metal–organic and small organic molecule structural data in one database, is described. The database adopts an open-access model. The COD currently contains ∼80 000 entries in crystallographic information file format, with nearly full coverage of the International Union of Crystallography publications, and is growing in size and quality. PMID:22477773

  6. Effect of salmon protein hydrolysate and spray-dried plasma protein on growth performance of weanling pigs.

    PubMed

    Tucker, J L; Naranjo, V D; Bidner, T D; Southern, L L

    2011-05-01

    Two experiments, each consisting of 2 trials, were conducted to determine the effect of salmon protein hydrolysate (SPH) and spray-dried plasma protein (SDPP) fed during the first week postweaning and their subsequent effect on the growth performance of weanling pigs. Pigs were fed in a 3-phase feeding program with durations of 7 d for phase 1 in both Exp. 1 and 2; 14 or 15 d for phase 2 in Exp. 1 and 2, respectively; and 7 or 8 d for phase 3 in Exp. 1 and 2, respectively. Dietary treatments were fed only during phase 1, whereas the same diet was fed to all pigs in phases 2 and 3. Pigs were blocked by initial BW and sex, and littermates were balanced across treatments. Data from the 2 trials within each experiment were combined and analyzed together; no treatment × trial interactions (P > 0.10) were observed. In Exp. 1, a total of 324 weanling pigs (10 replications of 5 or 6 pigs per pen) with an average initial BW of 6.4 ± 1.3 kg were assigned to 1) a control diet with no SPH or SDPP, 2) 1.5% SPH, 3) 3.0% SPH, 4) 1.5% SDPP, 5) 3.0% SDPP, or 6) 1.5% SPH + 1.5% SDPP. Experiment 2 was similar to Exp. 1, but red blood cells were removed from all diets to reduce diet complexity. In Exp. 2, weanling pigs (n = 320, 14 replications of 5 or 6 pigs per pen) with an average initial BW of 5.4 ± 1.2 kg were assigned to 1) a control diet with no SPH or SDPP, 2) 1.5% SPH, 3) 1.5% SDPP, or 4) 1.5% SPH + 1.5% SDPP. Three batches of SPH were used, and each batch was analyzed for AA composition. In Exp. 1, the inclusion of SDPP or SPH during phase 1 did not affect (P > 0.10) ADG, ADFI, or G:F compared with those of pigs fed the control diet. No carryover effects on growth performance were observed in any of the subsequent phases. Overall, G:F was greater (P = 0.08) in pigs fed the 1.5% diets compared with those fed the 3.0% diets. In Exp. 2, no differences (P > 0.10) were observed in ADG, ADFI, or G:F among pigs fed the SPH or SDPP diets compared with those of pigs fed the

  7. Assessment of the effect of methionine supplementation and inclusion of hydrolyzed wheat protein in milk protein-based milk replacers on the performance of intensively fed Holstein calves.

    PubMed

    Castro, J J; Hwang, G H; Saito, A; Vermeire, D A; Drackley, J K

    2016-08-01

    The objectives of this study were to compare 2 milk replacers containing only milk proteins with or without supplemental Met, and to compare a milk replacer containing hydrolyzed wheat protein at 4.5% of dry matter (DM) and supplemental Lys and Met against the 2 all-milk-protein formulas, by assessing their effect on the growth performance, efficiency, and plasma urea nitrogen of pre-weaning Holstein calves. Thus, 57 Holstein calves were allotted to the following 3 treatments: (1) a skim milk plus whey protein concentrate-based milk replacer (SMWP) containing about 2.6% Lys and 0.6% Met on a DM basis; (2) SMWP + M based on skim milk and whey proteins, containing about 2.6% Lys, and supplemental Met to reach 0.9% on a DM basis; and (3) a skim milk plus whey protein concentrate plus 4.5% of the DM as hydrolyzed wheat protein based milk replacer (HWP + LM) where the wheat protein replaced 50% of the whey protein concentrate, and also contained supplemental Lys and Met to match the profile of SMWP + M (i.e., Lys 2.6 and Met 0.9% on DM basis). No difference in any of the responses was observed by supplementing the milk protein based formula with Met or when hydrolyzed wheat protein was added to the formula. Results indicate that (1) a milk replacer based on skim milk protein and whey protein with a Lys concentration of ~2.6% does not benefit from Met supplementation, and (2) milk replacer containing 4.5% of the DM as hydrolyzed wheat protein and supplemented with Lys and Met can support the same growth performance as milk protein-based formulas. PMID:27179863

  8. Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography.

    PubMed

    Roessler, Christian G; Agarwal, Rakhi; Allaire, Marc; Alonso-Mori, Roberto; Andi, Babak; Bachega, José F R; Bommer, Martin; Brewster, Aaron S; Browne, Michael C; Chatterjee, Ruchira; Cho, Eunsun; Cohen, Aina E; Cowan, Matthew; Datwani, Sammy; Davidson, Victor L; Defever, Jim; Eaton, Brent; Ellson, Richard; Feng, Yiping; Ghislain, Lucien P; Glownia, James M; Han, Guangye; Hattne, Johan; Hellmich, Julia; Héroux, Annie; Ibrahim, Mohamed; Kern, Jan; Kuczewski, Anthony; Lemke, Henrik T; Liu, Pinghua; Majlof, Lars; McClintock, William M; Myers, Stuart; Nelsen, Silke; Olechno, Joe; Orville, Allen M; Sauter, Nicholas K; Soares, Alexei S; Soltis, S Michael; Song, Heng; Stearns, Richard G; Tran, Rosalie; Tsai, Yingssu; Uervirojnangkoorn, Monarin; Wilmot, Carrie M; Yachandra, Vittal; Yano, Junko; Yukl, Erik T; Zhu, Diling; Zouni, Athina

    2016-04-01

    X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples. PMID:26996959

  9. Holographic LEED: A direct method for surface crystallography

    NASA Astrophysics Data System (ADS)

    Vamvakas, John Athanasios

    Since 1960's Low Energy Electron Diffraction (LEED) has been one of the most reliable methods for surface crystallography. It has solved hundreds of structures over the past 20-25 years and continues to be a powerful tool in the hands of crystallographers. Yet, the main disadvantage of the method is the fact that it is very time consuming. The programs that do the multiple scattering calculations can run literally for days! The key part of the method is the initial "guess" of a structure that will be close the one being seeked. A wrong guess would lead to huge amounts of wasted time and effort. We suggest a direct method that can give us a pretty good idea of the structure under determination. We call this method of ours: Holographic LEED (h-LEED) because it is based on the ideas of Dennis Gabor, the inventor of holography. The 3D images h-LEED reconstructs from LEED diffraction patterns can be reliably used to initialize LEED thus reducing the annoying computation time as well as the effort required by the crystallographer. We show that h-LEED produces good images for p(2× 2) reconstruction of adsorbed atoms by testing it on two adsorption systems: O/Ni(001) and K/Ni(001). The images were reconstructed from both diffuse LEED patterns from disordered adsorbates and superstructure Bragg spots from ordered adsorbates.

  10. Identification of rogue datasets in serial crystallography1

    PubMed Central

    Assmann, Greta; Brehm, Wolfgang; Diederichs, Kay

    2016-01-01

    Advances in beamline optics, detectors and X-ray sources allow new techniques of crystallographic data collection. In serial crystallography, a large number of partial datasets from crystals of small volume are measured. Merging of datasets from different crystals in order to enhance data completeness and accuracy is only valid if the crystals are isomorphous, i.e. sufficiently similar in cell parameters, unit-cell contents and molecular structure. Identification and exclusion of non-isomorphous datasets is therefore indispensable and must be done by means of suitable indicators. To identify rogue datasets, the influence of each dataset on CC1/2 [Karplus & Diederichs (2012 ▸). Science, 336, 1030–1033], the correlation coefficient between pairs of intensities averaged in two randomly assigned subsets of observations, is evaluated. The presented method employs a precise calculation of CC1/2 that avoids the random assignment, and instead of using an overall CC1/2, an average over resolution shells is employed to obtain sensible results. The selection procedure was verified by measuring the correlation of observed (merged) intensities and intensities calculated from a model. It is found that inclusion and merging of non-isomorphous datasets may bias the refined model towards those datasets, and measures to reduce this effect are suggested. PMID:27275144

  11. Protein separation in carousel multicolumn setup. Performance analysis and experimental validation.

    PubMed

    Gorczyca, Rafał; Marek, Wojciech; Bochenek, Roman; Piątkowski, Wojciech; Antos, Dorota

    2016-08-19

    To overcome limitations of periodic separations of proteins in batch chromatographic columns Carousel Multi-Column Setup (CMS) has been recently suggested and theoretically analyzed in a previous study (R. Bochenek, W. Marek, W. Piątkowski, D. Antos, J. Chromatogr. A, 1301 (2013) 60-72). In this system, feed and mobile phase streams are subsequently delivered through parallel columns to mimic their countercurrent movement with respect to the fluid flow. All fluxes in the system are synchronized to ensure continuous feed delivery, which however causes reduction in the size of the operating window compared to batchwise-operating systems. In this study to improve the performance of CMS, additional process variables have been considered, such as the flow rate gradient and feed concentration. Though altering both variables allowed improving the separation selectivity and extending the operating window, the feed concentration appeared to be the most influential parameter affecting the process performance. Moreover, a procedure for practical realization of protein separations in CMS has been developed, including hints about the process design, configuration of columns and detectors, and use of pumps. As the case study, the separation of a ternary mixture of proteins, i.e., cytochrome C, lysozyme and immunoglobulin G, on hydrophobic interaction columns was used. A target product was a protein with intermediate adsorption strength that was isolated out of a more and less strongly adsorbed compound. PMID:27443251

  12. The New York Consortium on Membrane Protein Structure (NYCOMPS): a high-throughput platform for structural genomics of integral membrane proteins

    PubMed Central

    Love, James; Mancia, Filippo; Shapiro, Lawrence; Punta, Marco; Rost, Burkhard; Girvin, Mark; Wang, Da-Neng; Zhou, Ming; Hunt, John F.; Szyperski, Thomas; Gouaux, Eric; MacKinnon, Roderick; McDermott, Ann; Honig, Barry; Inouye, Masayori; Montelione, Gaetano

    2011-01-01

    The New York Consortium on Membrane Protein Structure (NYCOMPS) was formed to accelerate the acquisition of structural information on membrane proteins by applying a structural genomics approach. NY-COMPS comprises a bioinformatics group, a centralized facility operating a high-throughput cloning and screening pipeline, a set of associated wet labs that perform high-level protein production and structure determination by x-ray crystallography and NMR, and a set of investigators focused on methods development. In the first three years of operation, the NYCOMPS pipeline has so far produced and screened 7,250 expression constructs for 8,045 target proteins. Approximately 600 of these verified targets were scaled up to levels required for structural studies, so far yielding 24 membrane protein crystals. Here we describe the overall structure of NYCOMPS and provide details on the high-throughput pipeline. PMID:20690043

  13. Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals

    DOE PAGESBeta

    Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Lyubimov, Artem Y.; Hattne, Johan; Brewster, Aaron S.; Sauter, Nicholas K.; Brunger, Axel T.; Weis, William I.

    2015-03-17

    There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as themore » resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited.« less

  14. Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals

    DOE PAGESBeta

    Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Lyubimov, Artem Y.; Hattne, Johan; Brewster, Aaron S.; Sauter, Nicholas K.; Brunger, Axel T.; Weis, William I.

    2015-03-17

    There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as themore » resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. In conclusion, these developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited.« less

  15. Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals

    SciTech Connect

    Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Lyubimov, Artem Y.; Hattne, Johan; Brewster, Aaron S.; Sauter, Nicholas K.; Brunger, Axel T.; Weis, William I.

    2015-03-17

    There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as the resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited.

  16. Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals

    SciTech Connect

    Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Lyubimov, Artem Y.; Hattne, Johan; Brewster, Aaron S.; Sauter, Nicholas K.; Brunger, Axel T.; Weis, William I.

    2015-03-17

    There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as the resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. In conclusion, these developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited.

  17. Native State Mass Spectrometry, Surface Plasmon Resonance, and X-ray Crystallography Correlate Strongly as a Fragment Screening Combination.

    PubMed

    Woods, Lucy A; Dolezal, Olan; Ren, Bin; Ryan, John H; Peat, Thomas S; Poulsen, Sally-Ann

    2016-03-10

    Fragment-based drug discovery (FBDD) is contingent on the development of analytical methods to identify weak protein-fragment noncovalent interactions. Herein we have combined an underutilized fragment screening method, native state mass spectrometry, together with two proven and popular fragment screening methods, surface plasmon resonance and X-ray crystallography, in a fragment screening campaign against human carbonic anhydrase II (CA II). In an initial fragment screen against a 720-member fragment library (the "CSIRO Fragment Library") seven CA II binding fragments, including a selection of nonclassical CA II binding chemotypes, were identified. A further 70 compounds that comprised the initial hit chemotypes were subsequently sourced from the full CSIRO compound collection and screened. The fragment results were extremely well correlated across the three methods. Our findings demonstrate that there is a tremendous opportunity to apply native state mass spectrometry as a complementary fragment screening method to accelerate drug discovery. PMID:26882437

  18. Which strategy for a protein crystallization project?

    PubMed

    Kundrot, C E

    2004-03-01

    The three-dimensional, atomic-resolution protein structures produced by X-ray crystallography over the past 50+ years have led to tremendous chemical understanding of fundamental biochemical processes. The pace of discovery in protein crystallography has increased greatly with advances in molecular biology, crystallization techniques, cryocrystallography, area detectors, synchrotrons and computing. While the methods used to produce single, well-ordered crystals have also evolved over the years in response to increased understanding and advancing technology, crystallization strategies continue to be rooted in trial-and-error approaches. This review summarizes the current approaches in protein crystallization and surveys the first results to emerge from the structural genomics efforts. PMID:15004692

  19. Which strategy for a protein crystallization project?

    NASA Technical Reports Server (NTRS)

    Kundrot, C. E.

    2004-01-01

    The three-dimensional, atomic-resolution protein structures produced by X-ray crystallography over the past 50+ years have led to tremendous chemical understanding of fundamental biochemical processes. The pace of discovery in protein crystallography has increased greatly with advances in molecular biology, crystallization techniques, cryocrystallography, area detectors, synchrotrons and computing. While the methods used to produce single, well-ordered crystals have also evolved over the years in response to increased understanding and advancing technology, crystallization strategies continue to be rooted in trial-and-error approaches. This review summarizes the current approaches in protein crystallization and surveys the first results to emerge from the structural genomics efforts.

  20. Caspase-3 binds diverse P4 residues in peptides as revealed by crystallography and structural modeling.

    SciTech Connect

    Fang, Bin; Fu, Guoxing; Agniswamy, Johnson; Harrison, Robert W.; Weber, Irene T.

    2009-03-31

    Caspase-3 recognition of various P4 residues in its numerous protein substrates was investigated by crystallography, kinetics, and calculations on model complexes. Asp is the most frequent P4 residue in peptide substrates, although a wide variety of P4 residues are found in the cellular proteins cleaved by caspase-3. The binding of peptidic inhibitors with hydrophobic P4 residues, or no P4 residue, is illustrated by crystal structures of caspase-3 complexes with Ac-IEPD-Cho, Ac-WEHD-Cho, Ac-YVAD-Cho, and Boc-D(OMe)-Fmk at resolutions of 1.9-2.6 {angstrom}. The P4 residues formed favorable hydrophobic interactions in two separate hydrophobic regions of the binding site. The side chains of P4 Ile and Tyr form hydrophobic interactions with caspase-3 residues Trp206 and Trp214 within a non-polar pocket of the S4 subsite, while P4 Trp interacts with Phe250 and Phe252 that can also form the S5 subsite. These interactions of hydrophobic P4 residues are distinct from those for polar P4 Asp, which indicates the adaptability of caspase-3 for binding diverse P4 residues. The predicted trends in peptide binding from molecular models had high correlation with experimental values for peptide inhibitors. Analysis of structural models for the binding of 20 different amino acids at P4 in the aldehyde peptide Ac-XEVD-Cho suggested that the majority of hydrophilic P4 residues interact with Phe250, while hydrophobic residues interact with Trp206, Phe250, and Trp214. Overall, the S4 pocket of caspase-3 exhibits flexible adaptation for different residues and the new structures and models, especially for hydrophobic P4 residues, will be helpful for the design of caspase-3 based drugs.

  1. Effects crude protein levels on female Nile tilapia (Oreochromis niloticus) reproductive performance parameters.

    PubMed

    de Oliveira, Marinez Moraes; Ribeiro, Tainá; Orlando, Tamira Maria; de Oliveira, Dênio Garcia Silva; Drumond, Mariana Martins; de Freitas, Rilke Tadeu Fonseca; Rosa, Priscila Vieira

    2014-11-10

    The goal of the present work was to study the reproductive performance of Nile Tilapia (Oreochromis niloticus) female broodstock fed diets containing different levels of crude protein (CP). Two hundred and forty Nile tilapia (O. niloticus) were used at an average age of 30 months, with 180 females and 60 males. The broodstock were lodged separately in masonry tanks with continuous water flow. The females were stocked in thirty tanks with dimensions of 8 m(3) in a completely randomized design consisting of five treatments and six replications. The treatments consisted of five diets with different levels of CP (32, 34, 36, 38 and 40%) and with digestible energy per gram of protein of 9.5 kg of feed. The crude protein (CP) levels positively influenced (p<0.05) reproductive parameters (female relative and absolute fecundity, egg diameter, fasting larvae survival capacity), the somatic indexes (gonadosomatic (GSI), hepatosomatic (HIS), viscerosomatic (VSI)), total plasma protein, albumin and triglycerides. There were no significant differences (p>0.05) observed with regard to spawning weight and female weight. The reproductive parameters studied in the present research indicate that diets formulated with 38% CP with digestible energy per gram of CP of 9.5 were the best diets for tilapia females during the reproductive period. PMID:25201770

  2. Diverse, high-quality test set for the validation of protein-ligand docking performance.

    PubMed

    Hartshorn, Michael J; Verdonk, Marcel L; Chessari, Gianni; Brewerton, Suzanne C; Mooij, Wijnand T M; Mortenson, Paul N; Murray, Christopher W

    2007-02-22

    A procedure for analyzing and classifying publicly available crystal structures has been developed. It has been used to identify high-resolution protein-ligand complexes that can be assessed by reconstructing the electron density for the ligand using the deposited structure factors. The complexes have been clustered according to the protein sequences, and clusters have been discarded if they do not represent proteins thought to be of direct interest to the pharmaceutical or agrochemical industry. Rules have been used to exclude complexes containing non-drug-like ligands. One complex from each cluster has been selected where a structure of sufficient quality was available. The final Astex diverse set contains 85 diverse, relevant protein-ligand complexes, which have been prepared in a format suitable for docking and are to be made freely available to the entire research community (http://www.ccdc.cam.ac.uk). The performance of the docking program GOLD against the new set is assessed using a variety of protocols. Relatively unbiased protocols give success rates of approximately 80% for redocking into native structures, but it is possible to get success rates of over 90% with some protocols. PMID:17300160

  3. CHARACTERIZATION OF DRUG-PROTEIN INTERACTIONS IN BLOOD USING HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Hage, David S.; Jackson, Abby; Sobansky, Matt; Schiel, John E.; Yoo, Michelle J.; Joseph, K. S.

    2009-01-01

    The binding of drugs with proteins in blood, serum or plasma is an important process in determining the activity, distribution, rate of excretion, and toxicity of drugs in the body. High-performance affinity chromatography (HPAC) has received a great deal of interest as a means for studying these interactions. This review examines the various techniques that have been used in HPAC to examine drug-protein binding and discusses the types of information that can be obtained through this approach. A comparison of these techniques with traditional methods for binding studies (e.g., equilibrium dialysis and ultrafiltration) will also be presented. The use of HPAC with specific serum proteins and binding agents will then be discussed, including human serum albumin and α1-acid glycoprotein. Several examples from the literature are provided to illustrate the applications of such research. Recent developments in this field are also described, such as the use of improved immobilization techniques, new data analysis methods, techniques for working for directly with complex biological samples, and work with immobilized lipoproteins. The relative advantages and limitations of the methods that are described will be considered and the possible use of these techniques in the high-throughput screening or characterization of drug-protein binding will be discussed. PMID:19278006

  4. High-Performance Affinity Chromatography: Applications in Drug-Protein Binding Studies and Personalized Medicine.

    PubMed

    Li, Zhao; Beeram, Sandya R; Bi, Cong; Suresh, D; Zheng, Xiwei; Hage, David S

    2016-01-01

    The binding of drugs with proteins and other agents in serum is of interest in personalized medicine because this process can affect the dosage and action of drugs. The extent of this binding may also vary with a given disease state. These interactions may involve serum proteins, such as human serum albumin or α1-acid glycoprotein, or other agents, such as lipoproteins. High-performance affinity chromatography (HPAC) is a tool that has received increasing interest as a means for studying these interactions. This review discusses the general principles of HPAC and the various approaches that have been used in this technique to examine drug-protein binding and in work related to personalized medicine. These approaches include frontal analysis and zonal elution, as well as peak decay analysis, ultrafast affinity extraction, and chromatographic immunoassays. The operation of each method is described and examples of applications for these techniques are provided. The type of information that can be obtained by these methods is also discussed, as related to the analysis of drug-protein binding and the study of clinical or pharmaceutical samples. PMID:26827600

  5. Performance characteristics needed for protein crystal diffraction x-ray detectors.

    SciTech Connect

    Westbrook, E. M.

    1999-09-21

    During the 1990's, macromolecular crystallography became progressively more dependent on synchrotrons X-ray sources for diffraction data collection. Detectors of this diffraction data at synchrotrons beamlines have evolved over the decade, from film to image phosphor plates, and then to CCD systems. These changes have been driven by the data quality and quantity improvements each newer detector technology provided. The improvements have been significant. It is likely that newer detector technologies will be adopted at synchrotron beamlines for crystallographic diffraction data collection in the future, but these technologies will have to compete with existing CCD detector systems which are already excellent and are getting incrementally better in terms of size, speed, efficiency, and resolving power. Detector development for this application at synchrotrons must concentrate on making systems which are bigger and faster than CCDs and which can capture weak data more efficiently. And there is a need for excellent detectors which are less expensive than CCD systems.

  6. Nucleic acid crystallography: a view from the nucleic acid database.

    PubMed

    Berman, H M; Gelbin, A; Westbrook, J

    1996-01-01

    What are the future directions of the field of nucleic acid crystallography? Although there have been many duplex structures determined, the sample is still relatively small. This is especially true if one wants to derive enough information about the relationships between sequence and structure. Indeed, there are data for all the possible 10 dimer steps, but for some steps it is very limited. If the structural code resides in trimers or tetrad steps then there is simply not enough data to do meaningful statistical analyses. So the first direction that needs to be explored is the determination of more structures with more varied sequences. The other noticeable thing about the data is the shortness of the strands. While it is probably true that attempts to crystallize very long sequences will not meet with success, the idea of crystallizing sequences engineered to fit together via sticky ends such as has been done for the CAP-DNA complex (Schultz et al., 1990) should give data about the behavior of much longer stretches of DNA. The question of the effects of environment on the structure of DNA continues to be a very important one to address since DNA is rarely alone. The preliminary data we have analysed from the current sample shows that the conformation of some steps are very sensitive to packing type. Numerous studies of the hydration around DNA shows that there is a real synergy between the hydration structure and the base conformation. More data will allow further quantitation of these observations. RNA structure is the next very exciting frontier. The emerging structures of duplexes with internal loops, the two hammerhead ribozyme structures and the group I intron ribozyme have given us a glimpse of the complexity and elegance of this class of molecules. With the technology now in place to allow the determination of the structures of these molecules, the expectation is that now we will see a large increase in the number of these structures in the NDB. PMID

  7. Internal crystallography and thermal history of natural gold alloys

    NASA Astrophysics Data System (ADS)

    Hough, R.; Cleverley, J. S.

    2011-12-01

    New studies of gold are revealing how metallography is a key component of our understanding of the deposition of precious alloys in primary ore systems. Alluvial gold nuggets once thought to be secondary in origin have now been shown to be the erosional residue of hypogene systems, i.e. primary. This has been achieved through analysis of the internal crystallography using electron back scattered diffraction of large area ion beam polished gold samples. Comparisons of the microstructure are also being made with experiments on gold alloys with the same Ag contents where real time heating and in-situ microstructure mapping reveal the structures are of high temperature origin. A new frontier in gold analysis in both hypogene and supergene systems is the nano domain. In hypogene settings gold at all scales can be metallic and particulate as has been directly observed in refractory ores, or the so called "invisible gold" in pyrite and arsenopyrite. Such nanoparticulate and colloidal transport of gold is a viable mechanism of dispersing the gold during weathering of ore deposits. These gold nanoparticles, long known about in materials sciences and manufacturing have now been seen in these natural environments. Such colloids are also likely to play an important role in gold transport in hydrothermal deposits. The regularly heterogeneous distribution, trace concentration and nanoparticulate grain size of metallic gold in all ore systems has made it difficult for direct observation. Yet, it is critical to be able to establish a broad view of the microstructural/microchemical residence of the actual gold in a given sample. New generation element mapping tools now allow us to 'see' this invisible gold component for the first time and to probe its chemistry and controls on deposition. These studies have the potential to provide a new approach and view of the formation, deposition and provenance history of the metal in all gold deposits.

  8. Hagfish slime threads as a biomimetic model for high performance protein fibres.

    PubMed

    Fudge, Douglas S; Hillis, Sonja; Levy, Nimrod; Gosline, John M

    2010-09-01

    Textile manufacturing is one of the largest industries in the world, and synthetic fibres represent two-thirds of the global textile market. Synthetic fibres are manufactured from petroleum-based feedstocks, which are becoming increasingly expensive as demand for finite petroleum reserves continues to rise. For the last three decades, spider silks have been held up as a model that could inspire the production of protein fibres exhibiting high performance and ecological sustainability, but unfortunately, artificial spider silks have yet to fulfil this promise. Previous work on the biomechanics of protein fibres from the slime of hagfishes suggests that these fibres might be a superior biomimetic model to spider silks. Based on the fact that the proteins within these 'slime threads' adopt conformations that are similar to those in spider silks when they are stretched, we hypothesized that draw processing of slime threads should yield fibres that are comparable to spider dragline silk in their mechanical performance. Here we show that draw-processed slime threads are indeed exceptionally strong and tough. We also show that post-drawing steps such as annealing, dehydration and covalent cross-linking can dramatically improve the long-term dimensional stability of the threads. The data presented here suggest that hagfish slime threads are a model that should be pursued in the quest to produce fibres that are ecologically sustainable and economically viable. PMID:20729569

  9. Growth performance and carcase quality in broiler chickens fed on bacterial protein grown on natural gas.

    PubMed

    Øverland, M; Schøyen, H F; Skrede, A

    2010-10-01

    1. The effects of increasing concentrations (0, 40, 80 or 120 g/kg) of bacterial protein meal (BPM) and bacterial protein autolysate (BPA) grown on natural gas on growth performance and carcase quality in broiler chickens were examined. 2. Adding BPM to diets reduced feed intake and improved gain: feed from 0 to 21 d and overall to 35 d, but did not significantly affect weight gain compared to the soybean meal based control diet. 3. Increasing concentrations of BPA significantly reduced growth rate, feed intake, gain: feed, carcase weight and dressing percentage, but significantly increased carcase dry matter, fat and energy content. 4. Adding BPM to diets had no effect on viscosity of diets and jejunal digesta, and minor effects on litter quality, whereas BPA increased the viscosity of diets and jejunal digesta, improved litter quality at 21 d, but decreased litter quality at 32 d. 5. To conclude, broiler chickens performed better on a BPM product with intact proteins than on an autolysate with ruptured cell walls and a high content of free amino acids and low molecular-weight peptides. PMID:21058073

  10. Performance of Protein-Ligand Force Fields for the Flavodoxin-Flavin Mononucleotide System.

    PubMed

    Robertson, Michael J; Tirado-Rives, Julian; Jorgensen, William L

    2016-08-01

    The ability to accurately perform molecular dynamics and free energy perturbation calculations for protein-ligand systems is of broad interest to the biophysical and pharmaceutical sciences. In this work, several popular force fields are evaluated for reproducing experimental properties of the flavodoxin/flavin mononucleotide system. Calculated (3)J couplings from molecular dynamics simulations probing φ and χ1 dihedral angles are compared to over 1000 experimental measurements. Free energy perturbation calculations were also executed between different protein mutants for comparison with experimental data for relative free energies of binding. Newer versions of popular protein force fields reproduced (3)J backbone and side chain couplings with good accuracy, with RMSD values near or below one hertz in most cases. OPLS-AA/M paired with CM5 charges for the ligand performed particularly well, both for the (3)J couplings and FEP results, with a mean unsigned error for relative free energies of binding of 0.36 kcal/mol. PMID:27441982

  11. Effects of dietary protein concentration on performance and nutrient digestibility in Pekin ducks during aflatoxicosis.

    PubMed

    Chen, X; Murdoch, R; Zhang, Q; Shafer, D J; Applegate, T J

    2016-04-01

    A 14-d study was conducted to determine the impact of dietary crude protein concentration on performance, serum biochemistry, and nutrient digestive functions in Pekin ducklings during aflatoxicosis. A total of 144 male Pekin ducklings were randomly allotted to 4 dietary treatments arranged in a 2×2 factorial with 2 crude protein (CP) (20 and 24% on an analyzed basis) with or without 0.2 mg/kg aflatoxin B1 (AFB1) (0.21 mg/kg analyzed). The AFB1 reduced BW gain, feed intake, and breast muscle weight by 33 to 43% (P<0.0001). Serum concentration of protein, glucose, and Ca were also decreased by AFB1 (P≤0.0015), while pancreatic activities of amylase and lipase were increased by AFB1 (P<0.005). Apparent N digestibility was not affected by dietary treatment, whereas apparent ileal digestible energy was reduced 7.6% by AFB1 (P=0.0003). Higher dietary CP improved BW gain, gain:feed ratio, and breast muscle weight (P≤0.021), and tended to improve feed intake (P=0.094), but did not improve serum measures, digestive enzyme activity, or nutrient digestibility. No statistical interaction of AFB1 by CP was observed for any measures. Results from the current study suggest that AFB1 at low concentration can significantly impair performance of Pekin ducklings primarily through inhibited feed intake, as well as influence nutrient digestion processes (jejunum morphology, digestive enzyme activity, and apparent energy digestibility). Higher dietary CP can improve growth performance of ducklings regardless of AF exposure, but did not interact with dietary AFB1 on performance, serum biochemistry, or nutrient digestion in Pekin ducklings from hatch to 14 d. PMID:26740138

  12. Effects of prebiotic, protein level, and stocking density on performance, immunity, and stress indicators of broilers.

    PubMed

    Houshmand, M; Azhar, K; Zulkifli, I; Bejo, M H; Kamyab, A

    2012-02-01

    An experiment was conducted to determine the effects of period on the performance, immunity, and some stress indicators of broilers fed 2 levels of protein and stocked at a normal or high stocking density. Experimental treatments consisted of a 2 × 2 × 2 factorial arrangement with 2 levels of prebiotic (with or without prebiotic), 2 levels of dietary CP [NRC-recommended or low CP level (85% of NRC-recommended level)], and 2 levels of stocking density (10 birds/m(2) as the normal density or 16 birds/m(2) as the high density), for a total of 8 treatments. Each treatment had 5 replicates (cages). Birds were reared in 3-tiered battery cages with wire floors in an open-sided housing system under natural tropical conditions. Housing and general management practices were similar for all treatment groups. Starter and finisher diets in mash form were fed from 1 to 21 d and 22 to 42 d of age, respectively. Supplementation with a prebiotic had no significant effect on performance, immunity, and stress indicators (blood glucose, cholesterol, corticosterone, and heterophil:lymphocyte ratio). Protein level significantly influenced broiler performance but did not affect immunity or stress indicators (except for cholesterol level). The normal stocking density resulted in better FCR and also higher antibody titer against Newcastle disease compared with the high stocking density. However, density had no significant effect on blood levels of glucose, cholesterol, corticosterone, and the heterophil:lymphocyte ratio. Significant interactions between protein level and stocking density were observed for BW gain and final BW. The results indicated that, under the conditions of this experiment, dietary addition of a prebiotic had no significant effect on the performance, immunity, and stress indicators of broilers. PMID:22252353

  13. Protein crystal growth and the International Space Station

    NASA Technical Reports Server (NTRS)

    DeLucas, L. J.; Moore, K. M.; Long, M. M.

    1999-01-01

    Protein structural information plays a key role in understanding biological structure-function relationships and in the development of new pharmaceuticals for both chronic and infectious diseases. The Center for Macromolecular Crystallography (CMC) has devoted considerable effort studying the fundamental processes involved in macromolecular crystal growth both in a 1-g and microgravity environment. Results from experiments performed on more than 35 U.S. space shuttle flights have clearly indicated that microgravity can provide a beneficial environment for macromolecular crystal growth. This research has led to the development of a new generation of pharmaceuticals that are currently in preclinical or clinical trials for diseases such as cutaneous T-cell lymphoma, psoriasis, rheumatoid arthritis, AIDS, influenza, stroke and other cardiovascular complications. The International Space Station (ISS) provides an opportunity to have complete crystallographic capability on orbit, which was previously not possible with the space shuttle orbiter. As envisioned, the x-ray Crystallography Facility (XCF) will be a complete facility for growing protein crystals; selecting, harvesting, and mounting sample crystals for x-ray diffraction; cryo-freezing mounted crystals if necessary; performing x-ray diffraction studies; and downlinking the data for use by crystallographers on the ground. Other advantages of such a facility include crystal characterization so that iterations in the crystal growth conditions can be made, thereby optimizing the final crystals produced in a three month interval on the ISS.

  14. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    DOE PAGESBeta

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; et al

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of themore » intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.« less

  15. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    SciTech Connect

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; Drennan, Catherine L.; Baker, David; Ting, Alice Y.

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.

  16. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    PubMed Central

    Liu, Daniel S.; Nivón, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; Drennan, Catherine L.; Baker, David; Ting, Alice Y.

    2014-01-01

    Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies. PMID:25313043

  17. Ribosomal crystallography: from crystal growth to initial phasing

    NASA Astrophysics Data System (ADS)

    Thygesen, J.; Krumbholz, S.; Levin, I.; Zaytzev-Bashan, A.; Harms, J.; Bartels, H.; Schlünzen, F.; Hansen, H. A. S.; Bennett, W. S.; Volkmann, N.; Agmon, I.; Eisenstein, M.; Dribin, A.; Maltz, E.; Sagi, I.; Morlang, S.; Fua, M.; Franceschi, F.; Weinstein, S.; Böddeker, N.; Sharon, R.; Anagnostopoulos, K.; Peretz, M.; Geva, M.; Berkovitch-Yellin, Z.; Yonath, A.

    1996-10-01

    Preliminary phases were determined by the application of the isomorphous replacement method at low and intermediate resolution for structure factor amplitudes collected from crystals of large and small ribosomal subunits from halophilic and thermophilic bacteria. Derivatization was performed with dense heavy atom clusters, either by soaking or by specific covalent binding prior to the crystallization. The resulting initial electron density maps contain features comparable in size to those expected for the corresponding particles. The packing arrangements of these maps have been compared with motifs observed by electron microscopy in positively stained thin sections of embedded three-dimensional crystals, as well as with phase sets obtained by ab-initio computations. Aimed at higher resolution phasing, procedures are being developed for multi-site binding of relatively small dense metal clusters at selected locations. Potential sites are being inserted either by mutagenesis or by chemical modifications to facilitate cluster binding to the large halophilic and the small thermophilic ribosomal subunits which yield crystals diffracting to the highest resolution obtained so far for ribosomes, 2.9 and 7.3 Å, respectively. For this purpose the surfaces of these ribosomal particles have been characterized and conditions for quantitative reversible detachment of selected ribosomal proteins have been found. The corresponding genes are being cloned, sequenced, mutated to introduce the reactive side-groups (mainly cysteines) and overexpressed. To assist the interpretation of the anticipated electron density maps, sub-ribosomal stable complexes were isolated from H50S. One of these complexes is composed of two proteins and the other is made of a stretch of the rRNA and a protein. For exploiting the exposed parts of the surface of these complexes for heavy atom binding and for attempting the determination of their three-dimensional structure, their components are being produced

  18. Goniometer-based femtosecond crystallography with X-ray free electron lasers

    DOE PAGESBeta

    Cohen, Aina E.; Soltis, S. Michael; González, Ana; Aguila, Laura; Alonso-Mori, Roberto; Barnes, Christopher O.; Baxter, Elizabeth L.; Brehmer, Winnie; Brewster, Aaron S.; Brunger, Axel T.; et al

    2014-10-31

    The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. With smaller crystals, high-density grids were usedmore » to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.« less

  19. Goniometer-based femtosecond crystallography with X-ray free electron lasers

    SciTech Connect

    Cohen, Aina E.; Soltis, S. Michael; González, Ana; Aguila, Laura; Alonso-Mori, Roberto; Barnes, Christopher O.; Baxter, Elizabeth L.; Brehmer, Winnie; Brewster, Aaron S.; Brunger, Axel T.; Calero, Guillermo; Chang, Joseph F.; Chollet, Matthieu; Ehrensberger, Paul; Eriksson, Thomas L.; Feng, Yiping; Hattne, Johan; Hedman, Britt; Hollenbeck, Michael; Holton, James M.; Keable, Stephen; Kobilka, Brian K.; Kovaleva, Elena G.; Kruse, Andrew C.; Lemke, Henrik T.; Lin, Guowu; Lyubimov, Artem Y.; Manglik, Aashish; Mathews, Irimpan I.; McPhillips, Scott E.; Nelson, Silke; Peters, John W.; Sauter, Nicholas K.; Smith, Clyde A.; Song, Jinhu; Stevenson, Hilary P.; Tsai, Yingssu; Uervirojnangkoorn, Monarin; Vinetsky, Vladimir; Wakatsuki, Soichi; Weis, William I.; Zadvornyy, Oleg A.; Zeldin, Oliver B.; Zhu, Diling; Hodgson, Keith O.

    2014-10-31

    The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. With smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.

  20. Impact of blocking and detection chemistries on antibody performance for reverse phase protein arrays.

    PubMed

    Ambroz, Kristi

    2011-01-01

    Careful selection of well-qualified antibodies is critical for accurate data collection from reverse phase protein arrays (RPPA). The most common way to qualify antibodies for RPPA analysis is by Western blotting because the detection mechanism is based on the same immunodetection principles. Western blots of tissue or cell lysates that result in single bands and low cross-reactivity indicate appropriate antibodies for RPPA detection. Western blot conditions used to validate antibodies for RPPA experiments, including blocking and detection reagents, have significant effects on aspects of antibody performance such as cross-reactivity against other proteins in the sample. We have found that there can be a dramatic impact on antibody behavior with changes in blocking reagent and detection method, and offer an alternative method that allows detection reagents and conditions to be held constant in both antibody validation and RPPA experiments. PMID:21901590

  1. Exploring the speed and performance of molecular replacement with AMPLE using QUARK ab initio protein models

    SciTech Connect

    Keegan, Ronan M.; Bibby, Jaclyn; Thomas, Jens; Xu, Dong; Zhang, Yang; Mayans, Olga; Winn, Martyn D.; Rigden, Daniel J.

    2015-02-01

    Two ab initio modelling programs solve complementary sets of targets, enhancing the success of AMPLE with small proteins. AMPLE clusters and truncates ab initio protein structure predictions, producing search models for molecular replacement. Here, an interesting degree of complementarity is shown between targets solved using the different ab initio modelling programs QUARK and ROSETTA. Search models derived from either program collectively solve almost all of the all-helical targets in the test set. Initial solutions produced by Phaser after only 5 min perform surprisingly well, improving the prospects for in situ structure solution by AMPLE during synchrotron visits. Taken together, the results show the potential for AMPLE to run more quickly and successfully solve more targets than previously suspected.

  2. Scientist prepare Lysozyme Protein Crystal

    NASA Technical Reports Server (NTRS)

    1996-01-01

    Dan Carter and Charles Sisk center a Lysozyme Protein crystal grown aboard the USML-2 shuttle mission. Protein isolated from hen egg-white and functions as a bacteriostatic enzyme by degrading bacterial cell walls. First enzyme ever characterized by protein crystallography. It is used as an excellent model system for better understanding parameters involved in microgravity crystal growth experiments. The goal is to compare kinetic data from microgravity experiments with data from laboratory experiments to study the equilibrium.

  3. Determination of plasma protein binding of positron emission tomography radioligands by high-performance frontal analysis.

    PubMed

    Amini, Nahid; Nakao, Ryuji; Schou, Magnus; Halldin, Christer

    2014-09-01

    Positron emission tomography (PET) is an imaging technique based on the use of radioligands labeled with short lived radionuclides, such as (11)C (t½=20.4min) and (18)F (t½=109.8min), which as a consequence often requires rapid plasma protein binding analysis methods. In addition, PET radioligands can suffer from non-specific binding to the membrane when ultrafiltraion, which is the most commonly used method for measuring protein binding in PET, is employed. In this study a high-performance frontal analysis (HPFA) method based on incorporation of a gel filtration column (discovery(®) BIO GFC 100, 50mm×4.6mm, 5μm, 100Å) into a radio-LC system with phosphate buffered saline (PBS, pH 7.4) at a flow rate of 3ml/min as mobile phase was developed and investigated for four PET radioligands. The minimum injection volume (MIV) of plasma, which is a crucial factor in HPFA, was determined to be 200μl (human), 500μl (monkey), 700μl (human) and 1000μl (monkey) for these four radioligands. The MIV values increased as a higher fraction of the radioligand was present in the protein-free form. The protein binding results obtained were in good agreement with ultrafiltration and the method did not suffer from non-specific binding. The short analysis time (<12min) allowed multiple protein binding measurements during time course of a human [(11)C]PBR28 PET study. PMID:24922085

  4. Use of a combined cryo-EM and X-ray crystallography approach to reveal molecular details of bacterial pilus assembly by the chaperone/usher pathway

    SciTech Connect

    Li, H.; Thanassi, D. G.

    2009-06-01

    Many bacteria assemble hair-like fibers termed pili or fimbriae on their cell surface. These fibers mediate adhesion to various surfaces, including host cells, and play crucial roles in pathogenesis. Pili are polymers composed of thousands of individual subunit proteins. Understanding how these subunit proteins cross the bacterial envelope and correctly assemble at the cell surface is important not only for basic biology but also for the development of novel antimicrobial agents. The chaperone/usher pilus biogenesis pathway is one of the best-understood protein secretion systems, thanks largely to innovative efforts in biophysical techniques such as X-ray crystallography and cryo-electron microscopy. Such a combined approach holds promise for further elucidating remaining questions regarding the multi-step and highly dynamic pilus assembly process, as well as for studying other protein secretion and organelle biogenesis systems.

  5. Automated High Throughput Protein Crystallization Screening at Nanoliter Scale and Protein Structural Study on Lactate Dehydrogenase

    SciTech Connect

    Fenglei Li

    2006-08-09

    The purposes of our research were: (1) To develop an economical, easy to use, automated, high throughput system for large scale protein crystallization screening. (2) To develop a new protein crystallization method with high screening efficiency, low protein consumption and complete compatibility with high throughput screening system. (3) To determine the structure of lactate dehydrogenase complexed with NADH by x-ray protein crystallography to study its inherent structural properties. Firstly, we demonstrated large scale protein crystallization screening can be performed in a high throughput manner with low cost, easy operation. The overall system integrates liquid dispensing, crystallization and detection and serves as a whole solution to protein crystallization screening. The system can dispense protein and multiple different precipitants in nanoliter scale and in parallel. A new detection scheme, native fluorescence, has been developed in this system to form a two-detector system with a visible light detector for detecting protein crystallization screening results. This detection scheme has capability of eliminating common false positives by distinguishing protein crystals from inorganic crystals in a high throughput and non-destructive manner. The entire system from liquid dispensing, crystallization to crystal detection is essentially parallel, high throughput and compatible with automation. The system was successfully demonstrated by lysozyme crystallization screening. Secondly, we developed a new crystallization method with high screening efficiency, low protein consumption and compatibility with automation and high throughput. In this crystallization method, a gas permeable membrane is employed to achieve the gentle evaporation required by protein crystallization. Protein consumption is significantly reduced to nanoliter scale for each condition and thus permits exploring more conditions in a phase diagram for given amount of protein. In addition

  6. Biomolecular membrane protein crystallization

    NASA Astrophysics Data System (ADS)

    Reddy Bolla, Jani; Su, Chih-Chia; Yu, Edward W.

    2012-07-01

    Integral membrane proteins comprise approximately 30% of the sequenced genomes, and there is an immediate need for their high-resolution structural information. Currently, the most reliable approach to obtain these structures is X-ray crystallography. However, obtaining crystals of membrane proteins that diffract to high resolution appears to be quite challenging, and remains a major obstacle in structural determination. This brief review summarizes a variety of methodologies for use in crystallizing these membrane proteins. Hopefully, by introducing the available methods, techniques, and providing a general understanding of membrane proteins, a rational decision can be made about now to crystallize these complex materials.

  7. MEASUREMENT OF DRUG-PROTEIN DISSOCIATION RATES BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND PEAK PROFILING

    PubMed Central

    Schiel, John E.; Ohnmacht, Corey M.; Hage, David S.

    2012-01-01

    The rate at which a drug or other small solute interacts with a protein is important in understanding the biological and pharmacokinetic behavior of these agents. One approach that has been developed for examining these rates involves the use of high-performance affinity chromatography (HPAC) and estimates of band-broadening through peak profiling. Previous work with this method has been based on a comparison of the statistical moments for a retained analyte versus non-retained species at a single, high flow rate to obtain information on stationary phase mass transfer. In this study an alternative approach was created that allows a broad range of flow rates to be used for examining solute-protein dissociation rates. Chromatographic theory was employed to derive equations that could be used with this approach on a single column, as well as with multiple columns to evaluate and correct for the impact of stagnant mobile phase mass transfer. The interaction of L-tryptophan with human serum albumin was used as a model system to test this method. A dissociation rate constant of 2.7 (± 0.2) s−1 was obtained by this approach at pH 7.4 and 37°C, which was in good agreement with previous values determined by other methods. The techniques described in this report can be applied to other biomolecular systems and should be valuable for the determination of drug-protein dissociation rates. PMID:19422253

  8. Performance of hybrid methods for large-scale unconstrained optimization as applied to models of proteins.

    PubMed

    Das, B; Meirovitch, H; Navon, I M

    2003-07-30

    Energy minimization plays an important role in structure determination and analysis of proteins, peptides, and other organic molecules; therefore, development of efficient minimization algorithms is important. Recently, Morales and Nocedal developed hybrid methods for large-scale unconstrained optimization that interlace iterations of the limited-memory BFGS method (L-BFGS) and the Hessian-free Newton method (Computat Opt Appl 2002, 21, 143-154). We test the performance of this approach as compared to those of the L-BFGS algorithm of Liu and Nocedal and the truncated Newton (TN) with automatic preconditioner of Nash, as applied to the protein bovine pancreatic trypsin inhibitor (BPTI) and a loop of the protein ribonuclease A. These systems are described by the all-atom AMBER force field with a dielectric constant epsilon = 1 and a distance-dependent dielectric function epsilon = 2r, where r is the distance between two atoms. It is shown that for the optimal parameters the hybrid approach is typically two times more efficient in terms of CPU time and function/gradient calculations than the two other methods. The advantage of the hybrid approach increases as the electrostatic interactions become stronger, that is, in going from epsilon = 2r to epsilon = 1, which leads to a more rugged and probably more nonlinear potential energy surface. However, no general rule that defines the optimal parameters has been found and their determination requires a relatively large number of trial-and-error calculations for each problem. PMID:12820130

  9. Evaluation of the performance of protein separation in figure-8 centrifugal counter-current chromatography

    PubMed Central

    Yang, Yi; Gu, Dongyu; Aisa, Haji Akber; Ito, Yoichiro

    2011-01-01

    The performance of protein separation using the figure-8 column configuration in centrifugal counter-current chromatography was investigated under various flow rates and revolution speeds. The separation was performed with a two-phase solvent system composed of polyethylene glycol 1000/potassium phosphate each at 12.5% (w/w) in water and with lysozyme and myoglobin as test samples. In order to improve tracing of the elution curve, a hollow fiber membrane dialyzer was inserted at the inlet of the UV detector. The results showed that the retention of stationary phase (Sf) and resolution (Rs) increased with decreased flow rate and increased revolution speed. The highest Rs of approximately 1 was obtained at a flow rate of 0.01 mL/min under a revolution speed of 1200 rpm with a 3.4 ml capacity column. PMID:22100551

  10. Fabrication and characterization of aligned macroporous monolith for high-performance protein chromatography.

    PubMed

    Du, Kaifeng; Zhang, Qi; Dan, Shunmin; Yang, Min; Zhang, Yongkui; Chai, Dezhi

    2016-04-22

    In the present study, a freeze casting method combined with particle accumulation was applied to fabricate the aligned macroporous monolith for high-performance protein chromatography. For the preparation, the reactive colloids were first prepared by using glycidyl methacrylate and ethylene glycol dimethacrylate as monomers. Subsequently, these colloids accumulated regularly and polymerized into the aligned macroporous monolith. The aligned porous structure of the monolith was characterized by SEM, mercury intrusion, and flow hydrodynamics. The results revealed that the generated monolith was possessed of aligned macropores in size of about 10 μm and high column permeability. Finally, after being modified with sulfonated groups, the monolith was evaluated for its chromatographic performance. It demonstrated that the aligned macropores endowed the monolith with excellent adsorption capacity and high column efficiency. PMID:27016114

  11. Protein carbonyl levels correlate with performance in elite field hockey players.

    PubMed

    Rosa-Lima, Frederico Luis; Lannes, Luiz; Viana-Gomes, Diego; Pierucci, Anna Paola T R; Salerno, Verônica P

    2015-07-01

    Excess and incorrectly selected exercise can degrade athletic performance from an imbalance in redox homeostasis and oxidative stress, but well-planned training and nutrition can improve antioxidant capacity. The aim of the study was to investigate how nutrient intake could influence oxidative stress and cell lesion biomarkers after 5 days of training followed by a game. Blood was collected from 10 athletes at the start of training (basal), after training (pre-game), and postgame. Their acceleration capacity also was measured pre- and postgame. Blood analysis showed an increase in lactate concentration postgame (13%) and total antioxidant capacity increased both pre-game (13.1%) and postgame (12.7%), all in comparison with basal levels. An oxidative stress marker, protein carbonyl (PC), increased 3-fold over the course of the game, which correlated with a decreased acceleration (r = 0.749). For biomarkers of tissue damage, creatine kinase and aspartate transaminase (AST) increased postgame by 150% and 75%, respectively. The AST variation had a high negative correlation with energy and carbohydrate consumption and a moderate correlation with lipid and vitamin C intake. Protein intake had a positive but moderate correlation with reduced glutathione. The observed correlations suggest that nutritional monitoring can improve exercise physiological homeostasis and that PC serves as a good biomarker for oxidative stress and performance loss. PMID:25962716

  12. Surfactant-Bound Monolithic Columns for Separation of Proteins in Capillary High Performance Liquid Chromatography

    PubMed Central

    Gu, Congying; He, Jun; Jia, Jinping; Fang, Nenghu; Simmons, Robert; Shamsi, Shahab A.

    2011-01-01

    A surfactant bound monolithic stationary phase based on the co-polymerization of 11-acrylamino-undecanoic acid (AAUA) is designed for capillary high performance liquid chromatography (HPLC). Using D-optimal design, the effect of the polymerization mixture (concentrations of monomer, crosslinker and porogens) on the chromatographic performance (resolution and analysis time) of the AAUA-EDMA monolithic column was evaluated. The polymerization mixture was optimized using three proteins as model test solutes. The D-optimal design indicates a strong dependence of chromatographic parameters on the concentration of porogens (1,4-butanediol and water) in the polymerization mixture. Optimized solutions for fast separation and high resolution separation, respectively, were obtained using the proposed multivariate optimization. Differences less than 6.8% between the predicted and the experimental values in terms of resolution and retention time indeed confirmed that the proposed approach is practical. Using the optimized column, fast separation of proteins could be obtained in 2.5 min, and a tryptic digest of myoglobin was successfully separated on the high resolution column. The physical properties (i.e. morphology, porosity and permeability) of the optimized monolithic column were thoroughly investigated. It appears that this surfactant-bound monolith may have a great potential as a new generation of capillary HPLC stationary phase. PMID:20031139

  13. Protein-Pacing from Food or Supplementation Improves Physical Performance in Overweight Men and Women: The PRISE 2 Study

    PubMed Central

    Arciero, Paul J.; Edmonds, Rohan C.; Bunsawat, Kanokwan; Gentile, Christopher L.; Ketcham, Caitlin; Darin, Christopher; Renna, Mariale; Zheng, Qian; Zhang, Jun Zhu; Ormsbee, Michael J.

    2016-01-01

    We recently reported that protein-pacing (P; six meals/day @ 1.4 g/kg body weight (BW), three of which included whey protein (WP) supplementation) combined with a multi-mode fitness program consisting of resistance, interval sprint, stretching, and endurance exercise training (RISE) improves body composition in overweight individuals. The purpose of this study was to extend these findings and determine whether protein-pacing with only food protein (FP) is comparable to WP supplementation during RISE training on physical performance outcomes in overweight/obese individuals. Thirty weight-matched volunteers were prescribed RISE training and a P diet derived from either whey protein supplementation (WP, n = 15) or food protein sources (FP, n = 15) for 16 weeks. Twenty-one participants completed the intervention (WP, n = 9; FP, n = 12). Measures of body composition and physical performance were significantly improved in both groups (p < 0.05), with no effect of protein source. Likewise, markers of cardiometabolic disease risk (e.g., LDL (low-density lipoprotein) cholesterol, glucose, insulin, adiponectin, systolic blood pressure) were significantly improved (p < 0.05) to a similar extent in both groups. These results demonstrate that both whey protein and food protein sources combined with multimodal RISE training are equally effective at improving physical performance and cardiometabolic health in obese individuals. PMID:27187451

  14. Protein-Pacing from Food or Supplementation Improves Physical Performance in Overweight Men and Women: The PRISE 2 Study.

    PubMed

    Arciero, Paul J; Edmonds, Rohan C; Bunsawat, Kanokwan; Gentile, Christopher L; Ketcham, Caitlin; Darin, Christopher; Renna, Mariale; Zheng, Qian; Zhang, Jun Zhu; Ormsbee, Michael J

    2016-01-01

    We recently reported that protein-pacing (P; six meals/day @ 1.4 g/kg body weight (BW), three of which included whey protein (WP) supplementation) combined with a multi-mode fitness program consisting of resistance, interval sprint, stretching, and endurance exercise training (RISE) improves body composition in overweight individuals. The purpose of this study was to extend these findings and determine whether protein-pacing with only food protein (FP) is comparable to WP supplementation during RISE training on physical performance outcomes in overweight/obese individuals. Thirty weight-matched volunteers were prescribed RISE training and a P diet derived from either whey protein supplementation (WP, n = 15) or food protein sources (FP, n = 15) for 16 weeks. Twenty-one participants completed the intervention (WP, n = 9; FP, n = 12). Measures of body composition and physical performance were significantly improved in both groups (p < 0.05), with no effect of protein source. Likewise, markers of cardiometabolic disease risk (e.g., LDL (low-density lipoprotein) cholesterol, glucose, insulin, adiponectin, systolic blood pressure) were significantly improved (p < 0.05) to a similar extent in both groups. These results demonstrate that both whey protein and food protein sources combined with multimodal RISE training are equally effective at improving physical performance and cardiometabolic health in obese individuals. PMID:27187451

  15. Remote Access to the PXRR Macromolecular Crystallography Facilities at the NSLS

    SciTech Connect

    Soares, A.S.; Schneider, D. K.; Skinner, J. M.; Cowan, M.; Buono, R.; Robinson, H. H.; Heroux, A.; Carlucci-Dayton, M.; Saxena, A.; Sweet, R. M.

    2008-09-01

    The most recent surge of innovations that have simplified and streamlined the process of determining macromolecular structures by crystallography owes much to the efforts of the structural genomics community. However, this was only the last step in a long evolution that saw the metamorphosis of crystallography from an heroic effort that involved years of dedication and skill into a straightforward measurement that is occasionally almost trivial. Many of the steps in this remarkable odyssey involved reducing the physical labor that is demanded of experimenters in the field. Other steps reduced the technical expertise required for conducting those experiments.

  16. Remote Access to the PXRR Macromolecular Crystallography Facilities at the NSLS

    SciTech Connect

    A Soares; D Schneider; J Skinner; M Cowan; R Buono; H Robinson; A Heroux; M Carlucci-Dayton; A Saxena; R Sweet

    2011-12-31

    The most recent surge of innovations that have simplified and streamlined the process of determining macromolecular structures by crystallography owes much to the efforts of the structural genomics community. However, this was only the last step in a long evolution that saw the metamorphosis of crystallography from an heroic effort that involved years of dedication and skill into a straightforward measurement that is occasionally almost trivial. Many of the steps in this remarkable odyssey involved reducing the physical labor that is demanded of experimenters in the field. Other steps reduced the technical expertise required for conducting those experiments.

  17. Engineering fluorescent proteins towards ultimate performances: lessons from the newly developed cyan variants

    NASA Astrophysics Data System (ADS)

    Mérola, Fabienne; Erard, Marie; Fredj, Asma; Pasquier, Hélène

    2016-03-01

    New fluorescent proteins (FPs) are constantly discovered from natural sources, and submitted to intensive engineering based on random mutagenesis and directed evolution. However, most of these newly developed FPs fail to achieve all the performances required for their bioimaging applications. The design of highly optimised FP-based reporters, simultaneously displaying appropriate colour, multimeric state, chromophore maturation, brightness, photostability and environmental sensitivity will require a better understanding of the structural and dynamic determinants of FP photophysics. The recent development of cyan fluorescent proteins (CFPs) like mCerulean3, mTurquoise2 and Aquamarine brings a different view on these questions, as in this particular case, a step by step evaluation of critical mutations has been performed within a family of spectrally identical and evolutionary close variants. These efforts have led to CFPs with quantum yields close to unity, near single exponential emission decays, high photostability and complete insensitivity to pH, making them ideal choices as energy transfer donors in FRET and FLIM imaging applications. During this process, it was found that a proper amino-acid choice at only two positions (148 and 65) is sufficient to transform the performances of CFPs: with the help of structural and theoretical investigations, we rationalise here how these two positions critically control the CFP photophysics, in the context of FPs derived from the Aequorea victoria species. Today, these results provide a useful toolbox for upgrading the different CFP donors carried by FRET biosensors. They also trace the route towards the de novo design of FP-based optogenetic devices that will be perfectly tailored to dedicated imaging and sensing applications.

  18. Performance, immunity, and physiological responses of broilers to dietary energy and protein sequential variations.

    PubMed

    Ale Saheb Fosoul, S S; Toghyani, M; Gheisari, A; Tabeidiyan, S A; Mohammadrezaei, M; Azarfar, A

    2016-09-01

    The current trial was designed to examine effects of sequential feeding of diets varying in energy and protein contents on performance, gut development, welfare indices, and immune responses of broiler chicks. A total of 240 one-day-old broiler chicks (Ross 308) were randomly allotted to 4 treatments with 5 replicates in a completely randomized design. Sequential feeding was evaluated in 48-h cycles during 8 to 28 d of age. Diets varied in energy (E+ = 3,210 kcal/kg and E- = 2,790 kcal/kg) and protein (P+ = 25.14% CP and P- = 16.76% CP) contents. The 4 feeding programs were: control (ME: 3,000 kcal/kg, CP: 20.95%); P+/P- (P+ followed by P- containing 3,000 kcal/kg energy); E-/E+ (E- followed by E+ containing 20.95% CP), and E-P+ / E+P- (E-P+ followed by E+P-). The experiment lasted 42 d and birds were fed by a standard finishing diet from d 28 to 42. Broilers fed on E-/E+ and E-P+/E+P- had lower daily feed intake than control ones during 8 to 28 d of age (P < 0.05), while daily weight gain and feed conversion ratio were not affected significantly. Footpad dermatitis was lower in birds sequentially fed E-/E+ and E-P+/E+P- at 35 d of age (P < 0.05). Sequential feeding of diets varying in either energy or protein or both increased duration of tonic immobility at d 39 of age (P < 0.05). Feeding with E-/E+ regime decreased jejunal crypt depth, while feeding P+/P- regime increased villus height and crypt depth in the duodenum and ileum (P < 0.05). However, no relationship was found between intestinal morphology and growth performance of broiler chickens. Antibody production against sheep red blood cells as well as Newcastle and influenza disease viruses was not affected by sequential feeding. In conclusion, digestive organs and intestinal morphology might be adjusted to energy and protein variations. Sequential feeding increased the fear level in chickens. PMID:26994199

  19. Serial crystallographic analysis of protein isomorphous replacement data from a mixture of native and derivative microcrystals.

    PubMed

    Zhang, Tao; Yao, Deqiang; Wang, Jiawei; Gu, Yuanxin; Fan, Haifu

    2015-12-01

    A post-experimental identification/purification procedure similar to that described in Zhang et al. [(2015), IUCrJ, 2, 322-326] has been proposed for use in the treatment of multiphase protein serial crystallography (SX) diffraction snapshots. As a proof of concept, the procedure was tested using theoretical serial femtosecond crystallography (SFX) data from a mixture containing native and derivatized crystals of a protein. Two known proteins were taken as examples. Multiphase diffraction snapshots were subjected to two rounds of indexing using the program CrystFEL [White et al. (2012). J. Appl. Cryst. 45, 335-341]. In the first round, an ab initio indexing was performed to derive a set of approximate primitive unit-cell parameters, which are roughly the average of those from the native protein and the derivative. These parameters were then used in a second round of indexing as input to CrystFEL. The results were then used to separate the diffraction snapshots into two subsets corresponding to the native and the derivative. For each test sample, integration of the two subsets of snapshots separately led to two sets of three-dimensional diffraction intensities, one belonging to the native and the other to the derivative. Based on these two sets of intensities, a conventional single isomorphous replacement (SIR) procedure solved the structure easily. PMID:26627658

  20. The Amicon Pro system--a centrifugal device capable of performing all steps in the protein purification workflow.

    PubMed

    Cappione, Amedeo; Mabuchi, Masaharu; Suhrawardy, Saosan; Briggs, David; Nadler, Timothy

    2013-01-01

    raditional protein purification is a long process with many steps utilizing multiple devices, often resulting in protein degradation and loss. The Amicon Pro device streamlines the affinity purification process by providing a single adaptable centrifugation unit capable of performing all steps in the affinity purification process. The device combines affinity-based spin column purification with downstream sample concentration and buffer exchange, eliminating the need for multiple sample transfers, thereby minimizing protein loss. The results presented in this work indicate that purification of His-tagged protein using the Amicon Pro device is faster, easier, and provides better yields than other traditional methods (eg. spin-column and slurry method). PMID:24364216

  1. ADEQUATE DIETARY PROTEIN IS ASSOCIATED WITH BETTER PHYSICAL PERFORMANCE AMONG POST-MENOPAUSAL WOMEN 60–90 YEARS

    PubMed Central

    Gregorio, L.; Brindisi, J.; Kleppinger, A.; Sullivan, R.; Mangano, K.M; Bihuniak, J.D.; Kenny, A.M.; Kerstetter, J.E.; Insogna, K.L.

    2015-01-01

    Objectives Sarcopenia, the involuntary loss of skeletal muscle with age, affects up to one-quarter of older adults. Evidence indicates a positive association between dietary protein intake and lean muscle mass and strength among older persons, but information on dietary protein’s effect on physical performance in older adults has received less attention. Design Cross-sectional observational analysis of the relationship of dietary protein on body composition and physical performance. Setting Clinical research center. Participants 387 healthy women aged 60 – 90 years (mean 72.7 ± 7.0 y). Measurements Measures included body composition (fat-free mass, appendicular skeletal mass and fat mass) via dual x-ray absorptiometry (DXA), physical performance (Physical Performance Test [PPT] and Short Physical Performance Battery [SPPB]), handgrip strength, Physical Activity Scale in the Elderly (PASE), quality of life measure (SF-8), falls, fractures, nutrient and macromolecule intake (four-day food record). Independent samples t-tests determined mean differences between the above or below RDA protein groups. Statistical Analysis Analysis of covariance was used to control for body mass index (BMI) between groups when assessing physical performance, physical activity and health-related quality of life. Results The subjects consumed an average of 72.2 g protein/day representing 1.1 g protein/kg body weight/day. Subjects were categorized as below the recommended daily allowance (RDA) for protein (defined as less than 0.8 g protein/kg) or at or above the RDA (equal to or higher than 0.8 g protein/kg). Ninety-seven subjects (25%) were in the low protein group, and 290 (75%) were in the higher protein group. Women in the higher protein group had lower body mass, including fat and lean mass, and fat-to-lean ratio than those in the lower-protein group (p <0.001). Composite scores of upper and lower extremity strength were impaired in the group with low protein intake; SPPB score

  2. Effects of Forage Sources on Rumen Fermentation Characteristics, Performance, and Microbial Protein Synthesis in Midlactation Cows

    PubMed Central

    Xu, Jun; Hou, Yujie; Yang, Hongbo; Shi, Renhuang; Wu, Caixia; Huo, Yongjiu; Zhao, Guoqi

    2014-01-01

    Eight multiparous Holstein cows (632±12 kg BW; 135±16 DIM) were used in a replicated 4×4 Latin square design to evaluate the effects of forage sources on rumen fermentation characteristics, performance, and microbial protein (MCP) synthesis. The forage portion of the diets contained alfalfa hay (AH), oat hay (OH), Leymus chinensis (LC), or rice straw (RS) as the primary source of fiber. Diets were isonitrogenous and isocaloric, and cows were fed four corn silages based total mixed rations with equivalent nonfiber carbohydrate (NFC) and forage neutral detergent fiber (NDF). Dry matter intake was not affected by the source of dietary forages, ranging from 18.83 to 19.20 kg/d, consequently, milk yield was similar among diets. Because of the numerical differences in milk fat and milk protein concentrations, 4% FCM and ECM yields were unchanged (p>0.05). Mean rumen pH, NH3-N content, and concentrations of volatile fatty acids in the rumen fluid were not affected by the treatments (p>0.05). Dietary treatments did not affect the total tract apparent digestibility of dry matter, organic matter, and crude protein (p>0.05); however, digestibility of NDF and acid detergent fiber in RS diet was higher compared with AH, OH, and LC diets (p<0.05). Total purine derivative excretion was higher in cows fed AH, OH, and LC diets compared with those fed RS diet (p<0.05), consequently, estimated MCP synthesis was 124.35 g/d higher in cows fed AH diet compared with those fed RS diet (p<0.05). The results indicated that cows fed AH, OH, LC, and RS diets with an equivalent forage NDF and NFC have no unfavourable effect on the ruminal fermentation and productive parameters. PMID:25050001

  3. Effects of forage sources on rumen fermentation characteristics, performance, and microbial protein synthesis in midlactation cows.

    PubMed

    Xu, Jun; Hou, Yujie; Yang, Hongbo; Shi, Renhuang; Wu, Caixia; Huo, Yongjiu; Zhao, Guoqi

    2014-05-01

    Eight multiparous Holstein cows (632±12 kg BW; 135±16 DIM) were used in a replicated 4×4 Latin square design to evaluate the effects of forage sources on rumen fermentation characteristics, performance, and microbial protein (MCP) synthesis. The forage portion of the diets contained alfalfa hay (AH), oat hay (OH), Leymus chinensis (LC), or rice straw (RS) as the primary source of fiber. Diets were isonitrogenous and isocaloric, and cows were fed four corn silages based total mixed rations with equivalent nonfiber carbohydrate (NFC) and forage neutral detergent fiber (NDF). Dry matter intake was not affected by the source of dietary forages, ranging from 18.83 to 19.20 kg/d, consequently, milk yield was similar among diets. Because of the numerical differences in milk fat and milk protein concentrations, 4% FCM and ECM yields were unchanged (p>0.05). Mean rumen pH, NH3-N content, and concentrations of volatile fatty acids in the rumen fluid were not affected by the treatments (p>0.05). Dietary treatments did not affect the total tract apparent digestibility of dry matter, organic matter, and crude protein (p>0.05); however, digestibility of NDF and acid detergent fiber in RS diet was higher compared with AH, OH, and LC diets (p<0.05). Total purine derivative excretion was higher in cows fed AH, OH, and LC diets compared with those fed RS diet (p<0.05), consequently, estimated MCP synthesis was 124.35 g/d higher in cows fed AH diet compared with those fed RS diet (p<0.05). The results indicated that cows fed AH, OH, LC, and RS diets with an equivalent forage NDF and NFC have no unfavourable effect on the ruminal fermentation and productive parameters. PMID:25050001

  4. Effects of surface microstructure of hydroxyapatite on protein adsorption and biological performance of osteoblasts

    NASA Astrophysics Data System (ADS)

    He, H. W.; Li, G. D.; Li, B.; Chen, Z. Q.

    2008-11-01

    The aim of this study was to investigate the effect of surface microstructure on the serum protein adsorption and the biological performance of osteoblasts cultured in vitro, when seeded onto the surface of ceramics with different grain size: conventional HA, micron-sized HA and nano-sized HA. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to comparatively analyze the protein adsorption solution. The content of alkaline phosphatase (ALP) was determined, and then by using wash way method, the adhesion ability was tested. XPS tests indicated that the content of N on the surface was significant different between the three groups ( P < 0.05). SDS-PAGE analysis indicated that all the materials in these three groups could adsorb a large amount albumin, while the material in the nHA group adsorbed more albumin than the other groups. There were significant differences among them on the levels of osteoblast proliferation and adhesion in vitro. The biocompatibility of nHA is the best and of conventional HA is the worst.

  5. Visible vs near-infrared optical fiber plasmonics: performance comparison for protein sensing

    NASA Astrophysics Data System (ADS)

    Caucheteur, Christophe; Ribaut, Clotilde; Wattiez, Ruddy

    2016-04-01

    In this work, two plasmonic optical fiber sensor configurations are used for protein sensing and their relative performances in terms of limit of detection and sensitivity are compared. The first configuration consists in unclad 200 μm optical fibers that produce a broadband resonance in the visible wavelength range around 650 nm while the second configuration makes use of multiple narrowband resonances produced in the C+L bands with weakly tilted fiber Bragg gratings photo-inscribed in telecommunication-grade single-mode optical fibers. In both cases, the sensitive regions are surrounded by a ~50 nm gold layer so that the evanescent wave can excite a surface plasmon polariton at the metalsurrounding medium interface. Both configurations are used to sense green fluorescent proteins. Our experimental results demonstrate that the two sensor configurations present a complementary measurement dynamics as a function of the investigated concentration in the range 10-12 - 10-7 g/ml. We attribute this difference of sensitivity to the difference of penetration depth of the evanescent wave in the surrounding medium, which is proportional to the light wavelength.

  6. [Determination of hydroxyproline in collagenous proteins by high performance liquid chromatography-mass spectrometry].

    PubMed

    Xia, Jingen; Chen, Bo; Yao, Shouzhuo

    2008-09-01

    A simple, rapid and accurate method for the determination of hydroxyproline in collagenous proteins by high performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI/MS) was developed. After hydrolysis of collagenous proteins with hydrochloric acid, the hydroxyproline was separated on a Spherigel C8 column using acetonitrile-0.05% trifluoroacetic acid aqueous solution (5:95, v/v) at a flow rate of 1.0 mL/min. Theanine was used as the internal standard for the quantification. Hydroxyproline was identified and quantified by electrospray ionization mass spectrometry (ESI-MS), which was operated in positive ion mode while the m/z 132 and m/z 175 ions were recorded in the selective ionization monitoring (SIM) mode. The peak area ratio of hydroxyproline to theanine versus the hydroxyproline concentration was linear over a concentration range of 11.7-117 mg/L for hydroxyproline. The correlation coefficient was 0.999 3. The reproducibility and average recovery of the method were 1.87% and 98.85% respectively. The method has the advantages of easy operation, without derivatization, less analysis time, good precision and accuracy. PMID:19160759

  7. Analysis of Lidocaine Interactions with Serum Proteins Using High-Performance Affinity Chromatography

    PubMed Central

    Soman, Sony; Yoo, Michelle J.; Jang, Yoon Jeong; Hage, David S.

    2010-01-01

    High-performance affinity chromatography was used to study binding by the drug lidocaine to human serum albumin (HSA) and α1–acid glycoprotein (AGP). AGP had strong binding to lidocaine, with an association equilibrium constant (Ka) of 1.1-1.7 × 105 M-1 at 37 °C and pH 7.4. Lidocaine had weak-to-moderate binding to HSA, with a Ka in the range of 103 to 104 M-1. Competitive experiments with site selective probes showed that lidocaine was interacting with Sudlow site II of HSA and the propranolol site of AGP. These results agree with previous observations in the literature and provide a better quantitative understanding of how lidocaine binds to these serum proteins and is transported in the circulation. This study also demonstrates how HPAC can be used to examine the binding of a drug with multiple serum proteins and provide detailed information on the interaction sites and equilibrium constants that are involved in such processes. PMID:20138813

  8. Research Results Ultra-fast Energy Transfer from Monomer to Dimer within a Trimeric Molecule New Progress in Heterogeneous Catalysis Research Key Progress in Research on Terrestrial Carbon Cycle in China A New Progress in Research on the Mechanism of Bio-Invasion New Findings in Anti-viral infection and Control of Inflammation Major Headway in Avian Origin Research New Progress in Gold-Nanoparticle-Based Biochips Topological Insulator Research Made Important Progress Major Progress in Biodiversity Achieved New Developments of Direct Methods in Protein Crystallography Major Progress in China-UK Collaboration on the Causal Relationship between Volcanic Activity and Biological Distinction News in Brief: NSFC set up "Research Fund for Young Foreign Scholars" How Often Does Human DNA Mutate? Research Progress on Colossal Anisotropic Magneto Resistive Effect

    NASA Astrophysics Data System (ADS)

    2009-01-01

    Ultra-fast Energy Transfer from Monomer to Dimer within a Trimeric Molecule New Progress in Heterogeneous Catalysis Research Key Progress in Research on Terrestrial Carbon Cycle in China A New Progress in Research on the Mechanism of Bio-Invasion New Findings in Anti-viral infection and Control of Inflammation Major Headway in Avian Origin Research New Progress in Gold-Nanoparticle-Based Biochips Topological Insulator Research Made Important Progress Major Progress in Biodiversity Achieved New Developments of Direct Methods in Protein Crystallography Major Progress in China-UK Collaboration on the Causal Relationship between Volcanic Activity and Biological Distinction News in Brief: NSFC set up "Research Fund for Young Foreign Scholars" How Often Does Human DNA Mutate? Research Progress on Colossal Anisotropic Magneto Resistive Effect

  9. Lactational performance of dairy cows fed raw soybeans, with or without animal by-product proteins, or roasted soybeans.

    PubMed

    Grummer, R R; Luck, M L; Barmore, J A

    1994-05-01

    Twelve multiparous Holstein cows averaged 10 wk postpartum and were used in a replicated 3 x 3 Latin square design to compare two feeding strategies for increasing the ratio of dietary undegradable to degradable protein. Treatments were raw soybeans, with or without meat and bone meal plus blood meal, and roasted soybeans as the primary protein supplements. Meat and bone meal and blood meal were fed at 4.0 and .9% of dietary DM, respectively. Basal diets were 30% alfalfa silage, 18% corn silage, and 52% corn-based concentrate mix. Diets were formulated to be isonitrogenous and isocaloric. Estimated undegradable protein contents, as a percentage of total CP, were 32.2, 36.2, and 34.3 for diets containing raw soybeans, raw soybeans plus animal by-product proteins, and roasted soybeans, respectively. The undegradable protein estimates were calculated from NRC values for basal feeds and from results of in vitro analysis of test protein supplements. Yields of milk and 3.5% FCM of cows receiving raw soybeans plus animal by-product proteins (45.5 and 43.4 kg/d) and roasted soybeans (44.7 and 42.7 kg/d) were greater than those of cows receiving raw soybeans alone (43.2 and 41.3 kg/d). Increasing the ratio of undegradable to degradable dietary protein also increased yields of milk protein and fat. No differences occurred in lactation performance among cows fed the two diets containing higher undegradable protein. The DMI was not influenced by treatment. Increasing the ratio of undegradable to degradable dietary protein by feeding animal by-product proteins or heated soybeans enhanced lactation performance. PMID:8046075

  10. Profiling of soluble proteins in wine by nano-high-performance liquid chromatography/tandem mass spectrometry.

    PubMed

    Kwon, Sung Won

    2004-12-01

    Wine proteins play an important role in a wine's quality as they affect taste, clarity, and stability. To enhance our understanding of the proteins in wine, nano-high-performance liquid chromatography (HPLC)/tandem mass spectrometry was used to profile soluble proteins in wine. Twenty proteins were identified from a Sauvignon Blanc wine including five proteins derived from the grape, 12 from yeast, two from bacteria, and one from fungi. The findings are somewhat peculiar at first glance, but reasonable explanations can account for the results. The grape proteins identified are less in number, which may be due to the availability of an incomplete database and possibly bentonite fining. The relatively large number of identified yeast proteins may be due to their complete protein database. The identified bacterial and fungal proteins could possibly be attributed to sources in the vineyard including natural infections and improper handling during harvest. The use of nano-HPLC/tandem mass spectrometry is an important tool for identifying wine proteins and understanding how they affect its characteristics. PMID:15563204

  11. Contributions of NIST/NBS Researchers to the Crystallography of Construction Materials

    PubMed Central

    Stutzman, Paul

    2001-01-01

    For more than 100 years, the primary theme underlying the NBS/NIST staff contribution to the crystallography of building materials has been the development of an improved understanding of concrete materials performance. Over that time period, portland cement concrete has become one of the most important of our construction materials for roads, buildings, and other large municipal structures. At the beginning of the 20th century our understanding of portland cement composition, performance, use in concrete, and how the concrete performs in harsh environments was lacking. The efforts of NIST have served to advance construction materials science and technology through the combined efforts of experimental, field study, and theoretical computational materials science. One major achievement in the late 1920s, derived from studies on phase equilibria in cement clinker, allows calculation of potential cement clinker composition. Known as the Bogue calculation, this continues to be an essential tool in cement plant process control to this day. Additionally, contributions of NIST scientists to our knowledge of the chemistry and nature of cement hydration products have been crucial in our understanding of cement hydration and concrete durability. Today, computational materials science is a rapidly developing discipline, and NIST is developing tools incorporating predictive models aided by empirical studies. Examples include a computer-integrated knowledge system for prediction and optimization of performance and life-cycle cost of high performance concrete and the Virtual Cement and Concrete Testing Laboratory. Understanding the relationships between material and performance properties has not been confined only to portland cements. One of the longest running experiments at NIST, the stone test wall, has stood for over 50 years as one of the world’s largest single collections of building stone, and is invaluable for studying weathering effects associated with stone

  12. Frontiers of Crystallography: A Project-Based Research-Led Learning Exercise

    ERIC Educational Resources Information Center

    Wilson, Chick C.; Parkin, Andrew; Thomas, Lynne H.

    2012-01-01

    A highly interactive research-led learning session for chemistry undergraduates is described, which aims to lead students to an awareness of the applications of crystallography technique through a mentored hands-on crystal structure solution and refinement session. The research-based environment is inherent throughout the 4.5 h program and is…

  13. Using the Plan View to Teach Basic Crystallography in General Chemistry

    ERIC Educational Resources Information Center

    Cushman, Cody V.; Linford, Matthew R.

    2015-01-01

    The plan view is used in crystallography and materials science to show the positions of atoms in crystal structures. However, it is not widely used in teaching general chemistry. In this contribution, we introduce the plan view, and show these views for the simple cubic, body-centered cubic, face-centered cubic, hexagonal close packed, CsCl, NaCl,…

  14. Celebrating the International Year of Crystallography with a Wisconsin High School Crystal Growing Competition

    ERIC Educational Resources Information Center

    Guzei, Ilia A.

    2014-01-01

    In honor of the 2014 International Year of Crystallography, the first Wisconsin Crystal Growing Competition was successfully organized and conducted. High school students from 26 schools across the state competed for prizes by growing large crystals of CuSO[subscript4]·5(H[subscript2]O). This paper describes how the event was planned and carried…

  15. 100 Years Later: Celebrating the Contributions of X-ray Crystallography to Allergy and Clinical Immunology

    PubMed Central

    Pomés, Anna; Chruszcz, Maksymilian; Gustchina, Alla; Minor, Wladek; Mueller, Geoffrey A.; Pedersen, Lars C.; Wlodawer, Alexander; Chapman, Martin D.

    2015-01-01

    Current knowledge of molecules involved in immunology and allergic disease results from significant contributions of X-ray crystallography, a discipline that just celebrated its 100th anniversary. The histories of allergens and X-ray crystallography are intimately intertwined. The first enzyme structure to be determined was lysozyme, also known as the chicken food allergen Gal d 4. Crystallography determines the exact three-dimensional positions of atoms in molecules. Structures of molecular complexes in the disciplines of immunology and allergy have revealed the atoms involved in molecular interactions and in mechanisms of disease. These complexes include peptides presented by MHC class II molecules, cytokines bound to their receptors, allergen-antibody complexes, and innate immune receptors with their ligands. The information derived from crystallographic studies provides insights into the function of molecules. Allergen function is one of the determinants of environmental exposure, which is essential for IgE sensitization. Proteolytic activity of allergens or their capacity to bind lipopolysaccharides may also contribute to allergenicity. The atomic positions define the molecular surface that is accessible to antibodies. This surface in turn determines antibody specificity and cross-reactivity that are important factors for the selection of allergen panels used for molecular diagnosis and for the interpretation of clinical symptoms. This review celebrates the contributions of X-ray crystallography to clinical immunology and allergy, focusing on new molecular perspectives that influence the diagnosis and treatment of allergic diseases. PMID:26145985

  16. Textural performance of crosslinked or reduced-calcium milk protein ingredients in model high-protein nutrition bars.

    PubMed

    Banach, J C; Clark, S; Metzger, L E; Lamsal, B P

    2016-08-01

    Transglutaminase (Tgase) crosslinking and calcium reduction were investigated as ways to improve the texture and storage stability of high-protein nutrition (HPN) bars formulated with milk protein concentrate (MPC) and micellar casein concentrate (MCC). The MPC and MCC crosslinked at none, low, and high levels, and a reduced-calcium MPC (RCMPC) were each formulated into model HPN bars. Hardness, crumbliness, moisture content, pH, color, and water activity of the HPN bars were measured during accelerated storage. The HPN bars prepared with MPC were harder and more cohesive than those prepared with MCC. Higher levels of Tgase crosslinking improved HPN bar cohesiveness and decreased hardening during storage. The RCMPC produced softer, yet crumblier HPN bars. Small textural differences were observed for the HPN bars formulated with the transglutaminase crosslinked proteins or RCMPC when compared with their respective controls. However, modification only slightly improved protein ingredient ability to slow hardening while balancing cohesion and likely requires further improvement for increased applicability in soft-texture HPN bars. PMID:27236767

  17. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  18. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are