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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. Protein crystallography with spallation neutrons

    SciTech Connect

    Langan, P.; Schoenborn, Benno P.

    2003-01-01

    proteins and oriented molecular complexes. With spallation neutrons and their time dependent wavelength structure, one can select data with an optimal wavelength bandwidth and cover the whole Laue spectrum as time (wavelength) resolved diffraction data. This optimizes data quality with best peak to background ratios and provides spatial and energy resolution to eliminate peak overlaps. Such a Protein Crystallography Station (PCS) has been built and tested at Los Alamos Neutron Science Center. A partially coupled moderator is used to increase flux and data are collected by a Cylindrical He3 detector covering 120' with 200mm height. The PCS is described along with examples of data collected from a number of proteins.

  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. Phase retrieval in protein crystallography.

    PubMed

    Liu, Zhong Chuan; Xu, Rui; Dong, Yu Hui

    2012-03-01

    Solution of the phase problem is central to crystallographic structure determination. An oversampling method is proposed, based on the hybrid input-output algorithm (HIO) [Fienup (1982). Appl. Opt. 21, 2758-2769], to retrieve the phases of reflections in crystallography. This method can extend low-resolution structures to higher resolution for structure determination of proteins without additional sample preparation. The method requires an envelope of the protein which divides a unit cell into the density region where the proteins are located and the non-density region occupied by solvents. After a few hundred to a few thousand iterations, the correct phases and density maps are recovered. The method has been used successfully in several cases to retrieve the phases from the experimental X-ray diffraction data and the envelopes of proteins constructed from structure files downloaded from the Protein Data Bank. It is hoped that this method will greatly facilitate the ab initio structure determination of proteins.

  8. 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

  9. History of protein crystallography in China.

    PubMed

    Rao, Zihe

    2007-06-29

    China has a strong background in X-ray crystallography dating back to the 1920s. Protein crystallography research in China was first developed following the successful synthesis of insulin in China in 1966. The subsequent determination of the three-dimensional structure of porcine insulin made China one of the few countries which could determine macromolecular structures by X-ray diffraction methods in the late 1960s and early 1970s. After a slow period during the 1970s and 1980s, protein crystallography in China has reached a new climax with a number of outstanding accomplishments. Here, I review the history and progress of protein crystallography in China and detail some of the recent research highlights, including the crystal structures of two membrane proteins as well as the structural genomics initiative in China.

  10. Direct methods in protein crystallography.

    PubMed

    Karle, J

    1989-11-01

    It is pointed out that the 'direct methods' of phase determination for small-structure crystallography do not have immediate applicability to macromolecular structures. The term 'direct methods in macromolecular crystallography' is suggested to categorize a spectrum of approaches to macromolecular structure determination in which the analyses are characterized by the use of two-phase and higher-order-phase invariants. The evaluation of the invariants is generally obtained by the use of heavy-atom techniques. The results of a number of the more recent algebraic and probabilistic studies involving isomorphous replacement and anomalous dispersion thus become valid subjects for discussion here. These studies are described and suggestions are also presented concerning future applicability. Additional discussion concerns the special techniques of filtering, the use of non-crystallographic symmetry, some features of maximum entropy and attempts to apply phase-determining formulas to the refinement of macromolecular structure. It is noted that, in addition to the continuing remarkable progress in macromolecular crystallography based on the traditional applications of isomorphous replacement and anomalous dispersion, recent valuable advances have been made in the application of non-crystallographic symmetry, in particular, to virus structures and in applications of filtering. Good progress has also been reported in the application of exact linear algebra to multiple-wavelength anomalous-dispersion investigations of structures containing anomalous scatterers of only moderate scattering power.

  11. Ab-initio phasing in protein crystallography

    NASA Astrophysics Data System (ADS)

    van der Plas, J. L.; Millane, Rick P.

    2000-11-01

    The central problem in the determination of protein structures form x-ray diffraction dada (x-ray crystallography) corresponds to a phase retrieval problem with undersampled amplitude data. Algorithms for this problem that have an increased radius of convergence have the potential for reducing the amount of experimental work, and cost, involved in determining protein structures. We describe such an algorithm. Application of the algorithm to a simulated crystallographic problem shows that it converges to the correct solution, with no initial phase information, where currently used algorithms fail. The results lend support to the possibility of ab initio phasing in protein crystallography.

  12. 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

  13. CCD(charge-coupled device)-based synchrotron x-ray detector for protein crystallography: Performance projected from an experiment

    SciTech Connect

    Strauss, M.G.; Naday, I.; Sherman, I.S.; Kraimer, M.R.; Westbrook, E.M.

    1986-01-01

    The intense x radiation from a synchrotron source could, with a suitable detector, provide a complete set of diffraction images from a protein crystal before the crystal is damaged by radiation (2 to 3 min). An area detector consisting of a 40 mm dia. x-ray fluorescing phosphor, coupled with an image intensifier and lens to a CCD image sensor, was developed to determine the effectiveness of such a detector in protein crystallography. The detector was used in an experiment with a rotating anode x-ray generator. Diffraction patterns from a lysozyme crystal obtained with this detector are compared to those obtained with film. The two images appear to be virtually identical. The flux of 10/sup 4/ x-ray photons/s was observed on the detector at the rotating anode generator. At the 6-GeV synchrotron being designed at Argonne, the flux on an 80 x 80 mm/sup 2/ detector is expected to be >10/sup 9/ photons/s. The projected design of such a synchrotron detector shows that a diffraction-peak count >10/sup 6/ could be obtained in approx.0.5 s. With an additional approx.0.5 s readout time of a 512 x 512 pixel CCD, the data acquisition time per frame would be approx.1 s so that ninety 1/sup 0/ diffraction images could be obtained, with approximately 1% precision, in less than 3 min.

  14. Kinetic protein crystallography: a tool to watch proteins in action

    SciTech Connect

    Bourgeois, D.; Weik, M.

    2009-04-14

    Many proteins function in the crystalline state, making crystallography a tool that, beside structure, can address mechanism. By initiating biological turnover in the crystal, transient structural species form, which may be filmed 'on the fly' by Laue diffraction or captured by trapping methods. These strategies are jointly referred to as 'kinetic crystallography'. In this article, we review the general concepts of kinetic crystallography in the context of the conformational energy landscape of a protein. Whereas Laue diffraction is best suited to the investigation of cyclic, ultra-fast and light-triggered reactions, trapping approaches, on the other hand, are applicable to a wider range of biological systems but require care to avoid artefacts. Complementary methods - mainly UV/visible single-crystal spectroscopy - have proven essential to design, interpret and validate kinetic crystallography experiments. Achievements in the field as well as remaining puzzling questions are considered through the examination of recently published work: real-time-resolved crystallography of dimeric haemoglobin based on pump-probe Laue diffraction, temperature-trapping crystallography of acetylcholinesterase based on photo- and radio-induced ligand cleavage, and lattice-trapping crystallography of superoxide reductase based on product soaking and the combined use of X-ray diffraction and Raman spectroscopy.

  15. 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

  16. Fifteen years of the Protein Crystallography Station: The coming of age of macromolecular neutron crystallography

    SciTech Connect

    Chen, Julian C.-H.; Unkefer, Clifford Jay

    2017-01-01

    The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002–2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technical outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallography in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. As a result, this review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s.

  17. Fifteen years of the Protein Crystallography Station: the coming of age of macromolecular neutron crystallography.

    PubMed

    Chen, Julian C-H; Unkefer, Clifford J

    2017-01-01

    The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002-2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technical outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallo-graphy in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. This review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s.

  18. Fifteen years of the Protein Crystallography Station: The coming of age of macromolecular neutron crystallography

    DOE PAGES

    Chen, Julian C.-H.; Unkefer, Clifford Jay

    2017-01-01

    The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002–2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technicalmore » outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallography in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. As a result, this review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s.« less

  19. Fifteen years of the Protein Crystallography Station: the coming of age of macromolecular neutron crystallography

    PubMed Central

    Chen, Julian C.-H.

    2017-01-01

    The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002–2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technical outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallo­graphy in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. This review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s. PMID:28250943

  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.

    2012-09-05

    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.

  1. 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

  2. Proline: Mother Nature's cryoprotectant applied to protein crystallography.

    PubMed

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

    2012-08-01

    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.

  3. 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

  4. 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.

  5. Quantification of detergent using colorimetric methods in membrane protein crystallography.

    PubMed

    Prince, Chelsy; Jia, Zongchao

    2015-01-01

    Membrane protein crystallography has the potential to greatly aid our understanding of membrane protein biology. Yet, membrane protein crystals remain challenging to produce. Although robust methods for the expression and purification of membrane proteins continue to be developed, the detergent component of membrane protein samples is equally important to crystallization efforts. This chapter describes the development of three colorimetric assays for the quantitation of detergent in membrane protein samples and provides detailed protocols. All of these techniques use small sample volumes and have potential applications in crystallography. The application of these techniques in crystallization prescreening, detergent concentration modification, and detergent exchange experiments is demonstrated. It has been observed that the concentration of detergent in a membrane protein sample can be just as important as the protein concentration when attempting to reproduce crystallization lead conditions. © 2015 Elsevier Inc. All rights reserved.

  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.

  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.

  9. 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

  10. High-resolution protein structure determination by serial femtosecond crystallography.

    PubMed

    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

    2012-07-20

    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.

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

    DOE PAGES

    Fromme, Raimund; Ishchenko, Andrii; Metz, Markus; ...

    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. A prototype direct-detection CCD for protein crystallography.

    PubMed

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

    2013-08-01

    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.

  14. 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.

  15. 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.

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

    DOE PAGES

    Ng, Joseph D.; Baird, James K.; Coates, Leighton; ...

    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

  17. 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.

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

    PubMed Central

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

    2015-01-01

    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

  19. 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.

  20. 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.

  1. SIBYLS - A SAXS and protein crystallography beamline at the ALS

    SciTech Connect

    Trame, Christine; MacDowell, Alastair A.; Celestre, Richard S.; Padmore, Howard A.; Cambie, Daniella; Domning, Edward E.; Duarte, Robert M.; Kelez, Nicholas; Plate, David W.; Holton, James M.; Frankel, Kenneth; Tsutakawa, Susan; Tsuruta, Hiro; Tainer, John A.; Cooper, Priscilla K.

    2003-08-22

    The new Structurally Integrated BiologY for Life Sciences (SIBYLS) beamline at the Advanced Light Source will be dedicated to Macromolecular Crystallography (PX) and Small Angle X-ray Scattering (SAXS). SAXS will provide structural information of macromolecules in solutions and will complement high resolution PX studies on the same systems but in a crystalline state. The x-ray source is one of the 5 Tesla superbend dipoles recently installed at the ALS that allows for a hard x-ray program to be developed on the relatively low energy Advanced Light Source (ALS) ring (1.9 GeV). The beamline is equipped with fast interchangeable monochromator elements, consisting of either a pair of single Si(111) crystals for crystallography, or a pair of multilayers for the SAXS mode data collection (E/{Delta}E {approx} 1/110). Flux rates with Si(111) crystals for PX are measured as 2 x 10{sup 11} hv/sec/400 mA through a 100 {micro}m pinhole at 12.4 KeV. For SAXS the flux is up to 3 x 10{sup 13} photons/sec at 10 KeV with all apertures open when using the multilayer monochromator elements. The performance characteristics of this unique beamline will be described.

  2. 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.

  3. 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

  4. 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.

  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. 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

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

    PubMed

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

    2014-07-17

    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. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  8. 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

  9. Microbeam MAD Beamline for Challenging Protein Crystallography in TPS

    NASA Astrophysics Data System (ADS)

    Liu, D. G.; Chao, C. H.; Chang, C. H.; Juang, J. M.; Liu, C. Y.; Chang, S. H.; Chang, C. F.; Chou, C. K.; Tseng, C. C.; Chiang, C. H.; Jean, Y. C.; Tang, M. T.; Chung, S. C.; Chang, S. L.

    2013-03-01

    The TPS-05A beamline is the first X-ray beamline at NSRRC built for micro protein crystallography experiment as well as one of the seven ID beamlines in phase I at the TPS synchrotron facility. A 2-meter in-vacuum undulator (IU22) serves as the photon source from which the harmonics #3 to #9 will provide brilliance of 1018-1020 photons s-1 mrad-2 mm-2 (0.1% bandwidth)-1 and photon flux of 1013-1014 photons s-1 (0.1% bandwidth)-1 in the required energy range of 5.7-20 keV (2.175-0.620 Å) to cover MAD phasing experiments at 1 Å and SAD phasing experiments at 2 Å. The beamline optics consists of a cryo-cooled double crystal monochromator (DCM) and a pair of focusing K-B mirrors. Requirements from the user group include a target focus size of 50 μm × 50 μm (H × V) at the sample position, photon flux greater than 2 × 1012 photons s-1 at Se K-edge (0.9795 Å), pinholes for adjusting the beam size down to 5 μm. Calculation of heat load for the first optical element, i.e. the first crystal of DCM, is included in this paper.

  10. Protein crystallography beamline BL2S1 at the Aichi synchrotron

    PubMed Central

    Watanabe, Nobuhisa; Nagae, Takayuki; Yamada, Yusuke; Tomita, Ayana; Matsugaki, Naohiro; Tabuchi, Masao

    2017-01-01

    The protein crystallography beamline BL2S1, constructed at one of the 5 T superconducting bending-magnet ports of the Aichi synchrotron, is available to users associated with academic and industrial organizations. The beamline is mainly intended for use in X-ray diffraction measurements of single-crystals of macromolecules such as proteins and nucleic acids. Diffraction measurements for crystals of other materials are also possible, such as inorganic and organic compounds. BL2S1 covers the energy range 7–17 keV (1.8–0.7 Å) with an asymmetric-cut curved single-crystal monochromator [Ge(111) or Ge(220)], and a platinum-coated Si mirror is used for vertical focusing and as a higher-order cutoff filter. The beamline is equipped with a single-axis goniometer, a CCD detector, and an open-flow cryogenic sample cooler. High-pressure protein crystallography with a diamond anvil cell can also be performed using this beamline. PMID:28009576

  11. 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.

  12. Protein crystallography beamline BL2S1 at the Aichi synchrotron

    SciTech Connect

    Watanabe, Nobuhisa; Nagae, Takayuki; Yamada, Yusuke; Tomita, Ayana; Matsugaki, Naohiro; Tabuchi, Masao

    2017-01-01

    The protein crystallography beamline BL2S1, constructed at one of the 5 T superconducting bending-magnet ports of the Aichi synchrotron, is available to users associated with academic and industrial organizations. The beamline is mainly intended for use in X-ray diffraction measurements of single-crystals of macromolecules such as proteins and nucleic acids. Diffraction measurements for crystals of other materials are also possible, such as inorganic and organic compounds. BL2S1 covers the energy range 7–17 keV (1.8–0.7 Å) with an asymmetric-cut curved single-crystal monochromator [Ge(111) or Ge(220)], and a platinum-coated Si mirror is used for vertical focusing and as a higher-order cutoff filter. The beamline is equipped with a single-axis goniometer, a CCD detector, and an open-flow cryogenic sample cooler. Lastly, high-pressure protein crystallography with a diamond anvil cell can also be performed using this beamline.

  13. Design and performance of U7B beamline and X-ray diffraction and scattering station at NSRL and its preliminary experiments in protein crystallography

    NASA Astrophysics Data System (ADS)

    Pan, Guoqiang; Xu, Chaoyin; Fan, Rong; Gao, Chen; Lou, Xiaohua; Teng, Maikun; Huang, Qingqiu; Niu, Liwen

    2005-03-01

    This publication describes the design and performance of the U7B beamline and X-ray diffraction and diffuse scattering station at National Synchrotron Radiation Laboratory (NSRL). The beamline optics comprise a Pt-coated toroidal focusing mirror and a double-crystal Si(1 1 1) monochromator. A preliminary experiment of diffraction data collection and processing was carried out using a commercial imaging plate detector system (Mar345). The data collected from one single crystal of acutohaemolysin, a Lys49-type PLA2 from Agkistrodon acutus venom, are of high quality.

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. 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.

  1. Present and future of membrane protein structure determination by electron crystallography

    PubMed Central

    Ubarretxena-Belandia, Iban; Stokes, David L.

    2011-01-01

    Membrane proteins are critical to cell physiology, playing roles in signaling, trafficking, transport, adhesion, and recognition. Despite their relative abundance in the proteome and their prevalence as targets of therapeutic drugs, structural information about membrane proteins is in short supply. This review describes the use of electron crystallography as a tool for determining membrane protein structures. Electron crystallography offers distinct advantages relative to the alternatives of X-ray crystallography and NMR spectroscopy. Namely, membrane proteins are placed in their native membranous environment, which is likely to favor a native conformation and allow changes in conformation in response to physiological ligands. Nevertheless, there are significant logistical challenges in finding appropriate conditions for inducing membrane proteins to form two-dimensional arrays within the membrane and in using electron cryo-microscopy to collect the data required for structure determination. A number of developments are described for high-throughput screening of crystallization trials and for automated imaging of crystals with the electron microscope. These tools are critical for exploring the necessary range of factors governing the crystallization process. There have also been recent software developments to facilitate the process of structure determination. However, further innovations in the algorithms used for processing images and electron diffraction are necessary to improve throughput and to make electron crystallography truly viable as a method for determining atomic structures of membrane proteins. PMID:21115172

  2. 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.

  3. 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.

  4. 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.

  5. 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

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

    PubMed

    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.

  7. 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

  8. 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.

  9. An exponential modeling algorithm for protein structure completion by X-ray crystallography.

    PubMed

    Shneerson, V L; Wild, D L; Saldin, D K

    2001-03-01

    An exponential modeling algorithm is developed for protein structure completion by X-ray crystallography and tested on experimental data from a 59-residue protein. An initial noisy difference Fourier map of missing residues of up to half of the protein is transformed by the algorithm into one that allows easy identification of the continuous tube of electron density associated with that polypeptide chain. The method incorporates the paradigm of phase hypothesis generation and cross validation within an automated scheme.

  10. 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

  11. 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)

  12. Charge-coupled device/fiber optic taper array x-ray detector for protein crystallography

    SciTech Connect

    Naday, I.; Ross, S.; Westbrook, E.M.; Zentai, G.

    1998-04-01

    A large area charge-coupled device (CCD) based fiber optic taper array detector (APS-1) is installed at the insertion-device beamline of the Structural Biology Center at the Argonne Advanced Photon Source x-ray synchrotron. The detector is used in protein crystallography diffraction experiments, where the objective is to measure the position and intensity of x-ray Bragg peaks in diffraction images. Large imaging area, very high spatial resolution, high x-ray sensitivity, good detective quantum efficiency, low noise, wide dynamic range, excellent stability and short readout time are all fundamental requirements in this application. The APS-1 detector converts the 2-D x-ray patterns to visible light images by a thin layer of x-ray sensitive phosphor. The phosphor coating is directly deposited on the large ends of nine fiber optic tapers arranged in a 3{times}3 array. Nine, thermoelectrically cooled 1024{times}1024pixel CCDs image the patterns, demagnified by the tapers. After geometrical and uniformity corrections, the nine areas give a continuous image of the detector face with virtually no gaps between the individual tapers. The 18 parallel analog signal-processing channels and analog-to-digital converters ensure short readout time and low readout noise. We discuss the design and measured performance of the detector. {copyright} {ital 1998 Society of Photo-Optical Instrumentation Engineers.}{ital Key words:} charge-coupled device; fiber optic taper; x-ray diffraction; crystallography; imaging detector. {copyright} {ital 1998} {ital Society of Photo-Optical Instrumentation Engineers}

  13. Personal remarks on the future of protein crystallography and structural biology.

    PubMed

    Jaskolski, Mariusz

    2010-01-01

    Protein crystallography, the main experimental method of structural biology, has undergone in the recent past three revolutionary changes leading to its unexpected renaissance. They were connected with (i) the introduction of synchrotron radiation sources for X-ray diffraction experiments, (ii) implementation of Se-Met multiwavelength anomalous diffraction (MAD) for phasing, and (iii) initiation of structural genomics (SG) programs. It can be foreseen that in the next 10-15 years protein crystallography will continue to be in this revolutionary phase. We can expect not only an avalanche of protein crystal structures from SG centers, but also attacking of more demanding projects, such as the structure of membrane proteins and of very large macromolecular complexes. On the technological front, the introduction of X-ray radiation from free-electron lasers will revolutionize the experimental possibilities, making feasible even the imaging of single molecules and of intact biological cells.

  14. Dynamically polarized samples for neutron protein crystallography at the Spallation Neutron Source

    NASA Astrophysics Data System (ADS)

    Zhao, Jinkui; Pierce, Josh; Myles, Dean; Robertson, J. L.; Herwig, Kenneth W.; Standaert, Bob; Cuneo, Matt; Li, Le; Meilleur, Flora

    2016-09-01

    To prepare for the next generation neutron scattering instruments for the planned second target station at the Spallation Neutron Source (SNS) and to broaden the scientific impact of neutron protein crystallography at the Oak Ridge National Laboratory, we have recently ramped up our efforts to develop a dynamically polarized target for neutron protein crystallography at the SNS. Proteins contain a large amount of hydrogen which contributes to incoherent diffraction background and limits the sensitivity of neutron protein crystallography. This incoherent background can be suppressed by using polarized neutron diffraction, which in the same time also improves the coherent diffraction signal. Our plan is to develop a custom Dynamic Nuclear Polarization (DNP) setup tailored to neutron protein diffraction instruments. Protein crystals will be polarized at a magnetic field of 5 T and temperatures of below 1 K. After the dynamic polarization process, the sample will be brought to a frozen-spin mode in a 0.5 T holding field and at temperatures below 100 mK. In a parallel effort, we are also investigating various ways of incorporating polarization agents needed for DNP, such as site specific spin labels, into protein crystals.

  15. 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.

  16. A monoclonal antibody for G protein-coupled receptor crystallography.

    PubMed

    Day, Peter W; Rasmussen, Søren G F; Parnot, Charles; Fung, Juan José; Masood, Asna; Kobilka, Tong Sun; Yao, Xiao-Jie; Choi, Hee-Jung; Weis, William I; Rohrer, Daniel K; Kobilka, Brian K

    2007-11-01

    G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human beta(2) adrenergic (beta(2)AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. Filling the missing cone in protein electron crystallography.

    PubMed

    Dorset, D L

    1999-07-15

    The hyper-resolution property of the Sayre equation is explored for extrapolating amplitudes and phases into the missing cone of data left after tilting a representative protein (rubredoxin) to restricted limits in the electron microscope. At 0.6 nm resolution, a reasonable prediction of crystallographic phases can be made to reconstruct the lost information. Best results are obtained if the goniometer tilt value is greater than approximately +/-60 degrees, but some missing information can be restored if the tilt is restricted to +/-45 degrees.

  3. 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

  4. 10 years of protein crystallography at AR-NW12A beamline

    NASA Astrophysics Data System (ADS)

    Chavas, L. M. G.; Yamada, Y.; Hiraki, M.; Igarashi, N.; Matsugaki, N.; Wakatsuki, S.

    2013-03-01

    The exponential growth of protein crystallography can be observed in the continuously increasing demand for synchrotron beam time, both from academic and industrial users. Nowadays, the screening of a profusion of sample crystals for more and more projects is being implemented by taking advantage of fully automated procedures at every level of the experiments. The insertion device AR-NW12A beamline is one of the five macromolecular crystallography (MX) beamlines at the Photon Factory (PF). Currently the oldest MX beamline operational at the High Energy Accelerator Research Organization (KEK), the end-station was launched in 2001 as part of an upgrade of the PF Advanced Ring. Since its commissioning, AR-NW12A has been operating as a high-throughput beamline, slowly evolving to a multipurpose end-station for MX experiments. The development of the beamline took place about a decade ago, in parallel with a drastic development of protein crystallography and more general synchrotron technology. To keep the beamline up-to-date and competitive with other MX stations in Japan and worldwide, new features have been constantly added, with the goal of user friendliness of the various beamline optics and other instruments. Here we describe the evolution of AR-NW12A for its tenth anniversary. We also discuss the plans for upgrades for AR-NW12A, the future objectives in terms of the beamline developments, and especially the strong desire to open the beamline to a larger user community.

  5. High-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography.

    PubMed

    Mizutani, Kimihiko

    2015-01-01

    Homologous recombination is a system for repairing the broken genomes of living organisms by connecting two DNA strands at their homologous sequences. Today, homologous recombination in yeast is used for plasmid construction as a substitute for traditional methods using restriction enzymes and ligases. This method has various advantages over the traditional method, including flexibility in the position of DNA insertion and ease of manipulation. Recently, the author of this review reported the construction of plasmids by homologous recombination in the methanol-utilizing yeast Pichia pastoris, which is known to be an excellent expression host for secretory proteins and membrane proteins. The method enabled high-throughput construction of expression systems of proteins using P. pastoris; the constructed expression systems were used to investigate the expression conditions of membrane proteins and to perform X-ray crystallography of secretory proteins. This review discusses the mechanisms and applications of homologous recombination, including the production of proteins for X-ray crystallography.

  6. Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

    PubMed Central

    Ishchenko, Andrii; Cherezov, Vadim; Liu, Wei

    2016-01-01

    Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 µm crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include

  7. 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.

  8. 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.

  9. Performance of detectors for x-ray crystallography

    NASA Astrophysics Data System (ADS)

    Stanton, Martin J.; Phillips, Walter C.

    1993-12-01

    The performance of a detector can be characterized by its efficiency for measuring individual x-rays or for measuring Bragg peak intensities. The performance for detecting individual x- rays or for measuring Bragg peak intensities. The performance for detecting individual x-rays is well modeled by the DQE. The performance for measuring Bragg peak intensities in the presence of an x-ray background can be modeled by an expanded definition of the DQE which allows inclusion of experimental constraints, the XDCE. These constraints include the observation that by increasing the crystal-to-detector distance and using a larger detector, Bragg peaks can be better resolved and the x-ray background reduced. Calculation of the XDCE for a detector consisting of a fiberoptic taper with a phosphor x-ray convertor deposited on the large end and a CCD bonded to the small end demonstrate the need to make the detector area relatively large, possibly at the expense of a decrease in the DQE.

  10. 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.

  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. 3D shape reconstruction of loop objects in X-ray protein crystallography.

    PubMed

    Strutz, Tilo

    2011-01-01

    Knowledge of the shape of crystals can benefit data collection in X-ray crystallography. A preliminary step is the determination of the loop object, i.e., the shape of the loop holding the crystal. Based on the standard set-up of experimental X-ray stations for protein crystallography, the paper reviews a reconstruction method merely requiring 2D object contours and presents a dedicated novel algorithm. Properties of the object surface (e.g., texture) and depth information do not have to be considered. The complexity of the reconstruction task is significantly reduced by slicing the 3D object into parallel 2D cross-sections. The shape of each cross-section is determined using support lines forming polygons. The slicing technique allows the reconstruction of concave surfaces perpendicular to the direction of projection. In spite of the low computational complexity, the reconstruction method is resilient to noisy object projections caused by imperfections in the image-processing system extracting the contours. The algorithm developed here has been successfully applied to the reconstruction of shapes of loop objects in X-ray crystallography.

  13. 3D Manipulation of Protein Microcrystals with Optical Tweezers for X-ray Crystallography

    NASA Astrophysics Data System (ADS)

    Hikima, T.; Hashimoto, K.; Murakami, H.; Ueno, G.; Kawano, Y.; Hirata, K.; Hasegawa, K.; Kumasaka, T.; Yamamoto, M.

    2013-03-01

    In some synchrotron facilities such as SPring-8, X-ray microbeams have been utilized for protein crystallography, allowing users to collect diffraction data from a protein microcrystal. Usually, a protein crystal is picked up manually from a crystallization droplet. However it is very difficult to manipulate the protein microcrystals which are very small and fragile against a shock and changes of temperature and solvent condition. We have been developing an automatic system applying the optical tweezers with two lensed fiber probes to manipulate the fragile protein microcrystal. The system succeeded in trapping a crystal and levitating it onto the cryoloop in the solvent. X-ray diffraction measurement for the manipulated protein microcrystals indicated that laser irradiation and trap with 1064nm wavelength hardly affected the result of X-ray structural analysis.

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

    DOE PAGES

    Bunker, Richard D.; Mandal, Kalyaneswar; Bashiri, Ghader; ...

    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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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

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

    SciTech Connect

    Tenboer, Jason; Basu, Shibom; Zatsepin, Nadia; Pande, Kanupriya; Milathianaki, Despina; Frank, Matthias; Hunter, Mark; Boutet, Sebastien; Williams, Garth J.; Koglin, Jason E.; Oberthuer, Dominik; Heymann, Michael; Kupitz, Christopher; Conrad, Chelsie; Coe, Jesse; Roy-Chowdhury, Shatabdi; Weierstall, Uwe; James, Daniel; Wang, Dingjie; Grant, Thomas; Barty, Anton; Yefanov, Oleksandr; Scales, Jennifer; Gati, Cornelius; Seuring, Carolin; Srajer, Vukica; Henning, Robert; Schwander, Peter; Fromme, Raimund; Ourmazd, Abbas; Moffat, Keith; Van Thor, Jasper J.; Spence, John C. H.; Fromme, Petra; Chapman, Henry N.; Schmidt, Marius

    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.

  3. Time-Resolved Serial Crystallography Captures High Resolution Intermediates of Photoactive Yellow Protein

    PubMed Central

    Tenboer, Jason; Basu, Shibom; Zatsepin, Nadia; Pande, Kanupria; Milathianaki, Despina; Frank, Matthias; Hunter, Mark; Boutet, Sébastien; Williams, Garth J.; Koglin, Jason E.; Oberthuer, Dominik; Heymann, Michael; Kupitz, Christopher; Conrad, Chelsie; Coe, Jesse; Roy-Chowdhury, Shatabdi; Weierstall, Uwe; James, Daniel; Wang, Dingjie; Grant, Thomas; Barty, Anton; Yefanov, Oleksandr; Scales, Jennifer; Gati, Cornelius; Seuring, Carolin; Srajer, Vukica; Henning, Robert; Schwander, Peter; Fromme, Raimund; Ourmazd, Abbas; Moffat, Keith; Van Thor, Jasper; Spence, John H. C.; Fromme, Petra; Chapman, Henry N.; Schmidt, Marius

    2015-01-01

    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. PMID:25477465

  4. New micro-beam beamline at SPring-8, targeting at protein micro-crystallography

    SciTech Connect

    Hirata, Kunio; Ueno, Go; Nisawa, Atsushi; Kawano, Yoshiaki; Hikima, Takaaki; Tanaka, Takashi; Kitamura, Hideo; Yamamoto, Masaki; Shimizu, Nobutaka; Kumasaka, Takashi; Takeshita, Kunikazu; Ohashi, Haruhiko; Goto, Shunji

    2010-06-23

    A new protein micro-crystallography beamline BL32XU at SPring-8 is under construction and scheduled to start operation in 2010. The beamline is designed to provide the stabilized and brilliant micro-beam to collect high-quality data from micro-crystals. The beamline consists of a hybrid in-vacuum undulator, a liquid-nitrogen cooled double crystal monochromator, and K-B focusing mirrors with large magnification factor. Development of data acquisition system and end station consists of high-precision diffractometer, high-efficiency area detector, sample auto-changer etc. are also in progress.

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

    DOE PAGES

    Tenboer, Jason; Basu, Shibom; Zatsepin, Nadia; ...

    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.

  6. Watching proteins function with time-resolved x-ray crystallography

    NASA Astrophysics Data System (ADS)

    Šrajer, Vukica; Schmidt, Marius

    2017-09-01

    Macromolecular crystallography was immensely successful in the last two decades. To a large degree this success resulted from use of powerful third generation synchrotron x-ray sources. An expansive database of more than 100 000 protein structures, of which many were determined at resolution better than 2 Å, is available today. With this achievement, the spotlight in structural biology is shifting from determination of static structures to elucidating dynamic aspects of protein function. A powerful tool for addressing these aspects is time-resolved crystallography, where a genuine biological function is triggered in the crystal with a goal of capturing molecules in action and determining protein kinetics and structures of intermediates (Schmidt et al 2005a Methods Mol. Biol. 305 115-54, Schmidt 2008 Ultrashort Laser Pulses in Biology and Medicine (Berlin: Springer) pp 201-41, Neutze and Moffat 2012 Curr. Opin. Struct. Biol. 22 651-9, Šrajer 2014 The Future of Dynamic Structural Science (Berlin: Springer) pp 237-51). In this approach, short and intense x-ray pulses are used to probe intermediates in real time and at room temperature, in an ongoing reaction that is initiated synchronously and rapidly in the crystal. Time-resolved macromolecular crystallography with 100 ps time resolution at synchrotron x-ray sources is in its mature phase today, particularly for studies of reversible, light-initiated reactions. The advent of the new free electron lasers for hard x-rays (XFELs; 5-20 keV), which provide exceptionally intense, femtosecond x-ray pulses, marks a new frontier for time-resolved crystallography. The exploration of ultra-fast events becomes possible in high-resolution structural detail, on sub-picosecond time scales (Tenboer et al 2014 Science 346 1242-6, Barends et al 2015 Science 350 445-50, Pande et al 2016 Science 352 725-9). We review here state-of-the-art time-resolved crystallographic experiments both at synchrotrons and XFELs. We also outline

  7. 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. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  8. 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

    2016-03-03

    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.

  9. Large aperture CCD x ray detector for protein crystallography using a fiberoptic taper

    NASA Astrophysics Data System (ADS)

    Strauss, M. G.; Westbrook, E. M.; Naday, I.; Coleman, T. A.; Westbrook, M. L.; Travis, D. J.; Sweet, R. M.; Pflugrath, J. W.

    A detector with a 114 mm aperture, based on a charge-coupled device (CCD), has been designed for x-ray diffraction studies in protein crystallography. The detector was tested on a beamline of the National Synchrotron Light Source at Brookhaven National Laboratory with a beam intensity greater than 10(exp 9) x-ray photons/s. A fiberoptic taper, an image intensifier and a lens demagnify, intensify, and focus the image onto a CCD having 512 x 512 pixels. A detective quantum efficiency (DOE) of 0.36 was obtained by evaluating the statistical uncertainty in the detector output. The dynamic range of a 4 x 4 pixel resolution element, comparable in size to a diffraction peak, was 10(exp 4). The point-spread function shows FWHM resolution of approximately 1 pixel, where a pixel on the detector face is 160 microns. A complete data set, consisting of forty-five 1 deg rotation frames, was obtained in just 36 s of x-ray exposure to a crystal of chicken egg-white lysozyme. In a separate experiment, a lysozyme data set consisting of 495 0.1 deg frames, was processed by the MADNES data reduction program, yielding symmetry R-factors for the data of 3.2 to 3.5 percent. Diffraction images from crystals of the myosin S1 head (a = 275 A) were also recorded. The Bragg spots, only 5 pixels apart, were resolved but were not sufficiently separated to process these data. Changes in the detector design which will improve the DOE and spatial resolution are outlined. The overall performance showed that this type of detector is well suited for x-ray scattering investigations with synchrotron sources.

  10. CCD-based detector for protein crystallography with synchrotron X-rays

    NASA Astrophysics Data System (ADS)

    Strauss, M. G.; Westbrook, E. M.; Naday, I.; Coleman, T. A.; Westbrook, M. L.; Travis, D. J.; Sweet, R. M.; Pflugrath, J. W.; Stanton, M.

    1990-11-01

    A detector with a 114 mm aperture, based on a charge-coupled device (CCD), has been designed for X-ray diffraction studies in protein crystallography. The detector was tested at the National Synchrotron Light Source with a beam intensity, through a 0.3 mm collimator, of greater than 109 X-ray photons/s. A fiberoptic taper, an image intensifier, and a lens demagnify, intensify, and focus the image onto a CCD having 512×512 pixels. The statistical uncertainty in the detector output was evaluated as a function of conversion gain. From this, a detective quantum efficiency (DQE) of 0.36 was derived. The dynamic range of a 4×4 pixel resolution element, comparable in size to a diffraction peak, was 104. The point-spread function shows FWHM resolution of approximately 1 pixel, where a pixel is 160 μm on the detector face. A data set collected from a chicken egg-white lysozyme crystal, consisting of 495 0.1° frames, was processed by the MADNES data reduction program. The symmetry R-factors for the data were 3.2-3.5%. In a separate experiment a complete lysozyme data set consisting of 45 1° frames was obtained in just 36 s of X-ray exposure. Diffraction images from crystals of the myosin S1 head (a = 275 Å) were also recorded; the Bragg spots, only 5 pixels apart, were separated but not fully resolved. Changes in the detector design that will improve the DQE and spatial resolution are outlined. The overall performance showed that this type of detector is well suited for X-ray scattering investigations with synchrotron sources.

  11. Cell-free protein synthesis for structure determination by X-ray crystallography.

    PubMed

    Watanabe, Miki; Miyazono, Ken-ichi; Tanokura, Masaru; Sawasaki, Tatsuya; Endo, Yaeta; Kobayashi, Ichizo

    2010-01-01

    Structure determination has been difficult for those proteins that are toxic to the cells and cannot be prepared in a large amount in vivo. These proteins, even when biologically very interesting, tend to be left uncharacterized in the structural genomics projects. Their cell-free synthesis can bypass the toxicity problem. Among the various cell-free systems, the wheat-germ-based system is of special interest due to the following points: (1) Because the gene is placed under a plant translational signal, its toxic expression in a bacterial host is reduced. (2) It has only little codon preference and, especially, little discrimination between methionine and selenomethionine (SeMet), which allows easy preparation of selenomethionylated proteins for crystal structure determination by SAD and MAD methods. (3) Translation is uncoupled from transcription, so that the toxicity of the translation product on DNA and its transcription, if any, can be bypassed. We have shown that the wheat-germ-based cell-free protein synthesis is useful for X-ray crystallography of one of the 4-bp cutter restriction enzymes, which are expected to be very toxic to all forms of cells retaining the genome. Our report on its structure represents the first report of structure determination by X-ray crystallography using protein overexpressed with the wheat-germ-based cell-free protein expression system. This will be a method of choice for cytotoxic proteins when its cost is not a problem. Its use will become popular when the crystal structure determination technology has evolved to require only a tiny amount of protein.

  12. 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.

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

    DOE PAGES

    Coquelle, Nicolas; Brewster, Aaron S.; Kapp, Ulrike; ...

    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

  14. 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

  15. 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

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

    PubMed

    Tsujino, Soichiro; Tomizaki, Takashi

    2016-05-06

    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.

  17. 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.

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

    NASA Astrophysics Data System (ADS)

    Tsujino, Soichiro; Tomizaki, Takashi

    2016-05-01

    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.

  19. 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.

  20. Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation

    PubMed Central

    Conrad, Chelsie E.; Nelson, Garrett; Stander, Natasha; Zatsepin, Nadia A.; Zook, James; Zhu, Lan; Geiger, James; Chun, Eugene; Kissick, David; Hilgart, Mark C.; Ogata, Craig; Ishchenko, Andrii; Nagaratnam, Nirupa; Roy-Chowdhury, Shatabdi; Coe, Jesse; Subramanian, Ganesh; Schaffer, Alexander; Ketwala, Gihan; Venugopalan, Nagarajan; Xu, Shenglan; Corcoran, Stephen; Ferguson, Dale; Weierstall, Uwe; Spence, John C. H.; Cherezov, Vadim; Fromme, Petra; Fischetti, Robert F.; Liu, Wei

    2017-01-01

    Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5–20 µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A2A adenosine receptor (A2AAR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8 000 000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suites Cheetah and CrystFEL enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A2AAR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A2AAR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5–20 µm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS

  1. Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation

    DOE PAGES

    Martin-Garcia, Jose M.; Conrad, Chelsie E.; Nelson, Garrett; ...

    2017-05-24

    Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advancedmore » Photon Source (APS), are reported. Microcrystals (5–20 µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A2A adenosine receptor (A2AAR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8 000 000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suites Cheetah and CrystFEL enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A2AAR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A2AAR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5–20 µm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS

  2. Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent

    PubMed Central

    Nakane, Takanori; Hanashima, Shinya; Suzuki, Mamoru; Saiki, Haruka; Hayashi, Taichi; Kakinouchi, Keisuke; Sugiyama, Shigeru; Kawatake, Satoshi; Matsuoka, Shigeru; Matsumori, Nobuaki; Nango, Eriko; Kobayashi, Jun; Shimamura, Tatsuro; Kimura, Kanako; Mori, Chihiro; Kunishima, Naoki; Sugahara, Michihiro; Takakyu, Yoko; Inoue, Shigeyuki; Masuda, Tetsuya; Hosaka, Toshiaki; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Inoue, Tsuyoshi; Nureki, Osamu; Iwata, So; Murata, Michio; Mizohata, Eiichi

    2016-01-01

    The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams. PMID:27799539

  3. Two-dimensional crystallization of integral membrane proteins for electron crystallography.

    PubMed

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

    2010-01-01

    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 microscopy.

  4. 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)

  5. Continuous sample rotation data collection for protein crystallography with the PILATUS detector

    NASA Astrophysics Data System (ADS)

    Brönnimann, Ch.; Eikenberry, E. F.; Horisberger, R.; Hülsen, G.; Schmitt, B.; Schulze-Briese, C.; Tomizaki, T.

    2003-09-01

    The PILATUS detector (pixel apparatus for the SLS) is a large all silicon quantum-limited area X-ray detector for protein crystallography. A three-module array with 1120×157 pixels covering an active area of 24.3×3.6 cm 2 is in operation. Its main features are an excellent point-spread function, a very high dynamic range and a readout time of <7 ms. X-rays with energy above 6 keV can be detected in single photon counting mode. To demonstrate the potential of the detector, fine φ-sliced protein crystal data were collected in continuous sample rotation mode at beamline X06SA of the SLS.

  6. 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

  7. Some Aspects of Crystal Centering During X-ray High-throughput Protein Crystallography Experiment

    NASA Astrophysics Data System (ADS)

    Gaponov, Yu. A.; Matsugaki, N.; Sasajima, K.; Igarashi, N.; Wakatsuki, S.

    A set of algorithms and procedures of a crystal loop centering during X-ray high-throughput protein crystallography experiment has been designed and developed. A simple algorithm of the crystal loop detection and preliminary recognition has been designed and developed. The crystal loop detection algorithm is based on finding out the crystal loop ending point (opposite to the crystal loop pin) using image cross section (digital image column) profile analysis. The crystal loop preliminary recognition procedure is based on finding out the crystal loop sizes and position using image cross section profile analysis. The crystal loop fine recognition procedure based on Hooke-Jeeves pattern search method with an ellipse as a fitting pattern has been designed and developed. The procedure of restoring missing coordinate of the crystal loop is described. Based on developed algorithms and procedures the optimal auto-centering procedure has been designed and developed. A procedure of optimal manual crystal centering (Two Clicks Procedure) has been designed and developed. Developed procedures have been integrated into control software system PCCS installed at crystallography beamlines Photon Factory BL5A and PF-AR NW12, KEK.

  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. 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.

  10. 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.

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

    DOE PAGES

    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

  12. 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

  13. Cooperative water filling of a nonpolar protein cavity observed by high-pressure crystallography and simulation.

    PubMed

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

    2005-11-15

    Formation of a water-expelling nonpolar core is the paradigm of protein folding and stability. Although experiment largely confirms this picture, water buried in "hydrophobic" cavities is required for the function of some proteins. Hydration of the protein core has also been suggested as the mechanism of pressure-induced unfolding. We therefore are led to ask whether even the most nonpolar protein core is truly hydrophobic (i.e., water-repelling). To answer this question we probed the hydration of an approximately 160-A(3), highly hydrophobic cavity created by mutation in T4 lysozyme by using high-pressure crystallography and molecular dynamics simulation. We show that application of modest pressure causes approximately four water molecules to enter the cavity while the protein itself remains essentially unchanged. The highly cooperative filling is primarily due to a small change in bulk water activity, which implies that changing solvent conditions or, equivalently, cavity polarity can dramatically affect interior hydration of proteins and thereby influence both protein activity and folding.

  14. 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

  15. 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.

  16. 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

  17. Data processing in neutron protein crystallography using position-sensitive detectors

    SciTech Connect

    Schoenborn, B.P.

    1982-01-01

    Neutrons provide a unique probe for localizing hydrogen atoms and for distinguishing hydrogen from deuterons. Hydrogen atoms largely determine the three-dimensional structure of proteins and are responsible for many catalytic reactions. The study of hydrogen bonding and hydrogen exchange will therefore give insight into reaction mechanisms and conformational fluctuations. In addition, neutrons provide the ability to distinguish N from C and O and to allow correct orientation of groups such as histidine and glutamine. To take advantage of these unique features of neutron crystallography, one needs accurate Fourier maps depicting atomic structure to a high precision. In this paper, techniques are described for minimizing error in the observed structure factors by optimizing data collection and analysis procedures. Special attention is given to subtraction of the high background associated with hydrogen-containing molecules, which produces a disproportionately large statistical error.

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

    PubMed

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

    2015-06-01

    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, a surface acoustic wave-based method for manipulating and patterning crystals is developed. This method, which does not damage the fragile protein crystals, can precisely manipulate and pattern micrometer and submicrometer-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 will also make it possible to collect data on samples that were previously intractable.

  19. Combined use of XAFS and crystallography for studying protein-ligand interactions in metalloproteins.

    PubMed

    Strange, Richard W; Hasnain, S Samar

    2005-01-01

    This chapter describes the method of X-ray absorption spectroscopy when applied to the study of metal sites in proteins. The method requires the intense X-rays found only at synchrotron radiation sources, and is equally applicable to metalloproteins in dilute solutions, in fibers, films, and in crystalline states. In each case, structural changes occurring at metal sites during catalysis or ligand-binding are revealed with an accuracy and precision equivalent to atomic resolution crystallography. When combined with crystallographic data, of any resolution, X-ray absorption spectroscopy can yield atomic resolution three-dimensional structural models of the metal sites, thus providing the level of structural detail necessary for understanding the chemical mechanisms involved in the active states of metalloproteins.

  20. 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.

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

    DOE PAGES

    Das, Amit; Gerlits, Oksana O.; Parks, Jerry M.; ...

    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

  2. Disordered water within a hydrophobic protein cavity visualized by x-ray crystallography.

    PubMed

    Yu, B; Blaber, M; Gronenborn, A M; Clore, G M; Caspar, D L

    1999-01-05

    Water in the hydrophobic cavity of human interleukin 1beta, which was detected by NMR spectroscopy but was invisible by high resolution x-ray crystallography, has been mapped quantitatively by measurement and phasing of all of the low resolution x-ray diffraction data from a single crystal. Phases for the low resolution data were refined by iterative density modification of an initial flat solvent model outside the envelope of the atomic model. The refinement was restrained by the condition that the map of the difference between the electron density distribution in the full unit cell and that of the atomic model be flat within the envelope of the well ordered protein structure. Care was taken to avoid overfitting the diffraction data by maintaining phases for the high resolution data from the atomic model and by a resolution-dependent damping of the structure factor differences between data and model. The cavity region in the protein could accommodate up to four water molecules. The refined solvent difference map indicates that there are about two water molecules in the cavity region. This map is compatible with an atomic model of the water distribution refined by using XPLOR. About 70% of the time, there appears to be a water dimer in the central hydrophobic cavity, which is connected to the outside by two constricted channels occupied by single water molecules approximately 40% of the time on one side and approximately 10% on the other.

  3. Protein crystallography and drug discovery: recollections of knowledge exchange between academia and industry

    PubMed Central

    2017-01-01

    The development of structure-guided drug discovery is a story of knowledge exchange where new ideas originate from all parts of the research ecosystem. Dorothy Crowfoot Hodgkin obtained insulin from Boots Pure Drug Company in the 1930s and insulin crystallization was optimized in the company Novo in the 1950s, allowing the structure to be determined at Oxford University. The structure of renin was developed in academia, on this occasion in London, in response to a need to develop antihypertensives in pharma. The idea of a dimeric aspartic protease came from an international academic team and was discovered in HIV; it eventually led to new HIV antivirals being developed in industry. Structure-guided fragment-based discovery was developed in large pharma and biotechs, but has been exploited in academia for the development of new inhibitors targeting protein–protein interactions and also antimicrobials to combat mycobacterial infections such as tuberculosis. These observations provide a strong argument against the so-called ‘linear model’, where ideas flow only in one direction from academic institutions to industry. Structure-guided drug discovery is a story of applications of protein crystallography and knowledge exhange between academia and industry that has led to new drug approvals for cancer and other common medical conditions by the Food and Drug Administration in the USA, as well as hope for the treatment of rare genetic diseases and infectious diseases that are a particular challenge in the developing world. PMID:28875019

  4. 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.

  5. Charge-coupled-device/fiberoptic taper array x-ray detector for protein crystallography

    SciTech Connect

    Naday, I.; Ross, S.; Westbrook, E.M.; Zentai, G.

    1997-12-31

    A large area, charge-couple-device (CCD) based fiberoptic taper array detector (APS-1) has been installed at the insertion-device beamline of the Structural Biology Center at the ANL Advanced Photon Source. The detector is used in protein crystallography diffraction experiments, where the objective is to measure the position and intensity of X-ray Bragg peaks in diffraction images. Large imaging area, very high spatial resolution, high X-ray sensitivity, good detective quantum efficiency, low noise, wide dynamic range, excellent stability and short readout time are all fundamental requirements in this application. The APS-1 detector converts the two-dimensional X-ray patterns to a visible light images by a thin layer of X-ray sensitive phosphor. The phosphor coating is directly deposited on the large ends of nine fiberoptic tapers arranged in a 3 x 3 array. Nine, thermoelectrically cooled 1,024 x 1,024 pixel CCD`s image the patterns, demagnified by the tapers. After geometrical and uniformity corrections, the nine areas give a continuous image of the detector face with virtually no gaps between the individual tapers. The 18 parallel analog signal-processing channels and analog-to-digital converters assure short readout time and low readout noise.

  6. Charge-coupled-device/fiberoptic taper array X-ray detector for protein crystallography

    SciTech Connect

    Naday, I.; Ross, S.; Westbrook, E.M.; Zentai, G.

    1997-03-01

    A large area, charge-couple-device (CCD) based fiberoptic taper array detector (APS-1) has been installed at the insertion-device beamline of the Structural Biology Center at the ANL Advanced Photon Source. The detector is used in protein crystallography diffraction experiments, where the objective is to measure the position and intensity of X-ray Bragg peaks in diffraction images. Large imaging area, very high spatial resolution, high X-ray sensitivity, good detective quantum efficiency, low noise, wide dynamic range, excellent stability and short readout time are all fundamental requirements in this application. The APS-1 detector converts the two-dimensional X-ray patterns to a visible light images by a thin layer of X-ray sensitive phosphor. The phosphor coating is directly deposited on the large ends of nine fiberoptic tapers arranged in a 3x3 array. Nine, thermoelectrically cooled 1024 x 1024 pixel CCD`s image the patterns, demagnified by the tapers. After geometrical and uniformity corrections, the nine areas give a continuous image of the detector face with virtually no gaps between the individual tapers. The 18 parallel analog signal-processing channels and analog-to-digital converters assure short readout time and low readout noise.

  7. 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? © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  8. Purification of Transmembrane Proteins from Saccharomyces cerevisiae for X-ray Crystallography

    PubMed Central

    Clark, Kathleen M.; Fedoriw, Nadia; Robinson, Katrina; Connelly, Sara M.; Randles, Joan; Malkowski, Michael G.; DeTitta, George T.; Dumont, Mark E.

    2010-01-01

    To enhance the quantity and quality of eukaryotic transmembrane proteins (TMPs) available for structure determination by x-ray crystallography, we have optimized protocols for purification of TMPs expressed in the yeast Saccharomyces cerevisiae. We focused on a set of the highest-expressing endogenous yeast TMPs for which there are established biochemical assays. Genes encoding the target TMPs are transferred via ligation-independent cloning to a series of vectors that allow expression of reading frames fused to C-terminal His10 and ZZ (IgG-binding) domains that are separated from the reading frame by a cleavage site for rhinovirus 3C protease. Several TMP targets expressed from these vectors have been purified via affinity chromatography and gel filtration chromatography at levels and purities sufficient for ongoing crystallization trials. Initial purifications were based on expression of the genes under control of a galactose-inducible promoter, but higher cell densities and improved expression have been obtained through use of the yeast ADH2 promoter. Wide variations have been observed in the behavior of different TMP targets during purification-some can be readily purified, while others do not bind efficiently to affinity matrices, are not efficiently cleaved from the matrices, or remain tightly associated with the matrices even after cleavage of the affinity tags. The size, oligomeric state, and composition of purified protein-detergent complexes purified under different conditions were analyzed using a colorimetric assay of detergent concentration and by analytical size exclusion chromatography using static light scattering, refractive index detection, and UV absorption to monitor the elution profiles. Effective procedures were developed for obtaining high concentrations of purified TMPs without excessively concentrating detergents. PMID:20045057

  9. Signal to noise considerations for single crystal femtosecond time resolved crystallography of the Photoactive Yellow Protein.

    PubMed

    van Thor, Jasper J; Warren, Mark M; Lincoln, Craig N; Chollet, Matthieu; Lemke, Henrik Till; Fritz, David M; Schmidt, Marius; Tenboer, Jason; Ren, Zhong; Srajer, Vukica; Moffat, Keith; Graber, Tim

    2014-01-01

    Femtosecond time resolved pump-probe protein X-ray crystallography requires highly accurate measurements of the photoinduced structure factor amplitude differences. In the case of femtosecond photolysis of single P63 crystals of the Photoactive Yellow Protein, it is shown that photochemical dynamics place a considerable restraint on the achievable time resolution due to the requirement to stretch and add second order dispersion in order to generate threshold concentration levels in the interaction region. Here, we report on using a 'quasi-cw' approach to use the rotation method with monochromatic radiation and 2 eV bandwidth at 9.465 keV at the Linac Coherent Light Source operated in SASE mode. A source of significant Bragg reflection intensity noise is identified from the combination of mode structure and jitter with very small mosaic spread of the crystals and very low convergence of the XFEL source. The accuracy with which the three dimensional reflection is approximated by the 'quasi-cw' rotation method with the pulsed source is modelled from the experimentally collected X-ray pulse intensities together with the measured rocking curves. This model is extended to predict merging statistics for recently demonstrated self seeded mode generated pulse train with improved stability, in addition to extrapolating to single crystal experiments with increased mosaic spread. The results show that the noise level can be adequately modelled in this manner, indicating that the large intensity fluctuations dominate the merged signal-to-noise (I/σI) value. Furthermore, these results predict that using the self seeded mode together with more mosaic crystals, sufficient accuracy may be obtained in order to resolve typical photoinduced structure factor amplitude differences, as taken from representative synchrotron results.

  10. 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

  11. 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.

  12. 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.

  13. Development of an X-ray HARP–FEA detector system for high-throughput protein crystallography

    PubMed Central

    Miyoshi, Toshinobu; Igarashi, Noriyuki; Matsugaki, Naohiro; Yamada, Yusuke; Hirano, Keiichi; Hyodo, Kazuyuki; Tanioka, Kenkichi; Egami, Norifumi; Namba, Masakazu; Kubota, Misao; Kawai, Teruo; Wakatsuki, Soichi

    2008-01-01

    A new detector system for protein crystallography is now being developed based on an X-ray HARP–FEA (high-gain avalanche rushing amorphous photoconductor–field emitter array), which consists of an amorphous selenium membrane and a matrix field emitter array. The combination of the membrane avalanche effect with a single driven FEA has several advantages over currently available area detectors, including higher sensitivity, higher spatial resolution and a higher frame rate. Preliminary evaluation of the detector has been carried out and its effectiveness has been confirmed. Next, diffraction images were measured with continuous rotation of a protein crystal, and the images were compared with those measured by the existing CCD detector; the system successfully obtained high-spatial-resolution images. Using shutterless measurement, the total measurement time can be reduced significantly, making the method appropriate for high-throughput protein crystallography. The X-ray HARP–FEA detector is an attractive candidate for the next generation of X-ray area detectors. PMID:18421159

  14. Large-aperture CCD x-ray detector for protein crystallography using a fiber-optic taper

    NASA Astrophysics Data System (ADS)

    Strauss, Michael G.; Westbrook, Edwin M.; Naday, Istvan; Coleman, T. A.; Westbrook, Mary L.; Travis, D. J.; Sweet, Robert M.; Pflugrath, J. W.; Stanton, Martin J.

    1991-07-01

    A detector with a 114 mm aperture, based on a charge-coupled device (CCD), has been designed for x-ray diffraction studies in protein crystallography. The detector was tested on a beamline of the National Synchrotron Light Source at Brookhaven National Laboratory with a beam intensity greater than 10(superscript 9) x-ray photons/s. A fiber-optic taper, an image intensifier, and a lens demagnify, intensify, and focus the image onto a CCD having 512 X 512 pixels. A detective quantum efficiency (DQE) of 0.36 was obtained by evaluating the statistical uncertainty in the detector output. The dynamic range of a 4 X 4 pixel resolution element, comparable in size to a diffraction peak, was 10 (superscript 4). The point-spread function shows FWHM resolution of approximately 1 pixel, where a pixel on the detector face is 160 micrometers . A complete data set, consisting of forty-five 1 degree(s) rotation frames, was obtained in just 36 s of x-ray exposure to a crystal of chicken egg-white lysozyme. In a separate experiment, a lysozyme data set consisting of 495 0.1 degree(s) frames, was processed by the MADNES data reduction program, yielding symmetry R-factors for the data of 3.2- 3.5%. Diffraction images from crystals of the myosin S1 head (a equals 275 angstroms) were also recorded. The Bragg spots, only 5 pixels apart, were resolved but were not sufficiently separated to process these data. Changes in the detector design which will improve the DQE and spatial resolution are outlined. The overall performance showed that this type of detector is well suited for x-ray scattering investigations with synchrotron sources.

  15. 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

  16. Bragg coherent diffraction imaging and metrics for radiation damage in protein micro-crystallography

    PubMed Central

    Coughlan, H. D.; Darmanin, C.; Kirkwood, H. J.; Phillips, N. W.; Hoxley, D.; Clark, J. N.; Vine, D. J.; Hofmann, F.; Harder, R. J.; Maxey, E.; Abbey, B.

    2017-01-01

    The proliferation of extremely intense synchrotron sources has enabled ever higher-resolution structures to be obtained using data collected from smaller and often more imperfect biological crystals (Helliwell, 1984 ▸). Synchrotron beamlines now exist that are capable of measuring data from single crystals that are just a few micrometres in size. This provides renewed motivation to study and understand the radiation damage behaviour of small protein crystals. Reciprocal-space mapping and Bragg coherent diffractive imaging experiments have been performed on cryo-cooled microcrystals of hen egg-white lysozyme as they undergo radiation damage. Several well established metrics, such as intensity-loss and lattice expansion, are applied to the diffraction data and the results are compared with several new metrics that can be extracted from the coherent imaging experiments. Individually some of these metrics are inconclusive. However, combining metrics, the results suggest that radiation damage behaviour in protein micro-crystals differs from that of larger protein crystals and may allow them to continue to diffract for longer. A possible mechanism to account for these observations is proposed. PMID:28009549

  17. Bragg coherent diffraction imaging and metrics for radiation damage in protein micro-crystallography

    DOE PAGES

    Coughlan, H. D.; Darmanin, C.; Kirkwood, H. J.; ...

    2017-01-01

    The proliferation of extremely intense synchrotron sources has enabled ever higher-resolution structures to be obtained using data collected from smaller and often more imperfect biological crystals. Synchrotron beamlines now exist that are capable of measuring data from single crystals that are just a few micrometres in size. This provides renewed motivation to study and understand the radiation damage behaviour of small protein crystals. Reciprocal-space mapping and Bragg coherent diffractive imaging experiments have been performed on cryo-cooled microcrystals of hen egg-white lysozyme as they undergo radiation damage. Several well established metrics, such as intensity-loss and lattice expansion, are applied to themore » diffraction data and the results are compared with several new metrics that can be extracted from the coherent imaging experiments. Individually some of these metrics are inconclusive. However, combining metrics, the results suggest that radiation damage behaviour in protein micro-crystals differs from that of larger protein crystals and may allow them to continue to diffract for longer. As a result, a possible mechanism to account for these observations is proposed.« less

  18. Bragg coherent diffraction imaging and metrics for radiation damage in protein micro-crystallography

    SciTech Connect

    Coughlan, H. D.; Darmanin, C.; Kirkwood, H. J.; Phillips, N. W.; Hoxley, D.; Clark, J. N.; Vine, D. J.; Hofmann, F.; Harder, R. J.; Maxey, E.; Abbey, B.

    2017-01-01

    The proliferation of extremely intense synchrotron sources has enabled ever higher-resolution structures to be obtained using data collected from smaller and often more imperfect biological crystals. Synchrotron beamlines now exist that are capable of measuring data from single crystals that are just a few micrometres in size. This provides renewed motivation to study and understand the radiation damage behaviour of small protein crystals. Reciprocal-space mapping and Bragg coherent diffractive imaging experiments have been performed on cryo-cooled microcrystals of hen egg-white lysozyme as they undergo radiation damage. Several well established metrics, such as intensity-loss and lattice expansion, are applied to the diffraction data and the results are compared with several new metrics that can be extracted from the coherent imaging experiments. Individually some of these metrics are inconclusive. However, combining metrics, the results suggest that radiation damage behaviour in protein micro-crystals differs from that of larger protein crystals and may allow them to continue to diffract for longer. As a result, a possible mechanism to account for these observations is proposed.

  19. Bragg coherent diffraction imaging and metrics for radiation damage in protein micro-crystallography.

    PubMed

    Coughlan, H D; Darmanin, C; Kirkwood, H J; Phillips, N W; Hoxley, D; Clark, J N; Vine, D J; Hofmann, F; Harder, R J; Maxey, E; Abbey, B

    2017-01-01

    The proliferation of extremely intense synchrotron sources has enabled ever higher-resolution structures to be obtained using data collected from smaller and often more imperfect biological crystals (Helliwell, 1984). Synchrotron beamlines now exist that are capable of measuring data from single crystals that are just a few micrometres in size. This provides renewed motivation to study and understand the radiation damage behaviour of small protein crystals. Reciprocal-space mapping and Bragg coherent diffractive imaging experiments have been performed on cryo-cooled microcrystals of hen egg-white lysozyme as they undergo radiation damage. Several well established metrics, such as intensity-loss and lattice expansion, are applied to the diffraction data and the results are compared with several new metrics that can be extracted from the coherent imaging experiments. Individually some of these metrics are inconclusive. However, combining metrics, the results suggest that radiation damage behaviour in protein micro-crystals differs from that of larger protein crystals and may allow them to continue to diffract for longer. A possible mechanism to account for these observations is proposed.

  20. Current status and future prospects of an automated sample exchange system PAM for protein crystallography

    NASA Astrophysics Data System (ADS)

    Hiraki, M.; Yamada, Y.; Chavas, L. M. G.; Matsugaki, N.; Igarashi, N.; Wakatsuki, S.

    2013-03-01

    To achieve fully-automated and/or remote data collection in high-throughput X-ray experiments, the Structural Biology Research Centre at the Photon Factory (PF) has installed PF automated mounting system (PAM) for sample exchange robots at PF macromolecular crystallography beamlines BL-1A, BL-5A, BL-17A, AR-NW12A and AR-NE3A. We are upgrading the experimental systems, including the PAM for stable and efficient operation. To prevent human error in automated data collection, we installed a two-dimensional barcode reader for identification of the cassettes and sample pins. Because no liquid nitrogen pipeline in the PF experimental hutch is installed, the users commonly add liquid nitrogen using a small Dewar. To address this issue, an automated liquid nitrogen filling system that links a 100-liter tank to the robot Dewar has been installed on the PF macromolecular beamline. Here we describe this new implementation, as well as future prospects.

  1. 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

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

    SciTech Connect

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

    2015-02-20

    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. We also show 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.

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

    DOE PAGES

    Dziedzic, Pawel; Cisneros, José A.; Robertson, Michael J.; ...

    2015-02-20

    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. Activitymore » 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. We also show 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.« less

  4. Neutron Crystallography for Macromolecular Structure Analysis

    NASA Astrophysics Data System (ADS)

    Kuroki, Ryota

    Hydrogen atoms in proteins as well as protein-bound water molecules play a significant role in many chemical reaction processes in living systems, such as catalytic reaction and molecular recognition. Neutron crystallography is a powerful tool to identify locations of light atoms like hydrogen. In the field of neutron crystallography, the development of diffractometers and techniques for preparation and crystallization of target samples has been developed to complement the low flux of neutron sources. In Japan, single-crystal diffractometers named BIX-3 and BIX-4 have been developed, and contribute to the effective collection of neutron diffraction data. Recent developments on the complementary use of neutron and X-ray diffraction data have begun solving previously undetermined problems of protein function. Further efforts to acquire higher measurement performance are now in progress to increase the application of neutron crystallographic studies.

  5. 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.

  6. An evaluation of automated model-building procedures for protein crystallography.

    PubMed

    Badger, John

    2003-05-01

    The computer programs ARP/wARP, MAID and RESOLVE are designed to build protein structures into experimentally phased electron-density maps without any user intervention, requiring only diffraction data and sequence information. However, the MAID and RESOLVE systems, which seek to extend the range of automated model-building to approximately 3 A resolution, have yet to receive significant testing outside the small numbers of data sets used in their development. Since these two systems employ a large number of scoring functions and decision-making heuristics, additional tests are required to establish their usefulness to the crystallographic community. To independently evaluate these programs, their performance was tested using a database containing 41 experimentally phased maps between 1.3 and 2.9 A resolution from a diverse set of protein structures. At resolutions higher than 2.3 A the most successful program was ARP/wARP 6.0, which accurately built an average of 90% of the main chain. This system builds somewhat larger fractions of the model than the previous version ARP/wARP 5.1, which accurately built an average of 87% of the main chain. Although not specifically designed for model building into high-resolution maps, MAID and RESOLVE were also quite successful in this resolution regime, typically building approximately 80% of the main chain. At 2.3-2.7 A resolution the MAID and RESOLVE programs automatically built approximately 75% of the main-chain atoms in the protein structures used in these tests, which would significantly accelerate the model-building process. Data sets at lower resolution proved more problematic for these programs, although many of the secondary-structure elements were correctly identified and fitted.

  7. Accurate protein crystallography at ultra-high resolution: Valence electron distribution in crambin

    PubMed Central

    Jelsch, Christian; Teeter, Martha M.; Lamzin, Victor; Pichon-Pesme, Virginie; Blessing, Robert H.; Lecomte, Claude

    2000-01-01

    The charge density distribution of a protein has been refined experimentally. Diffraction data for a crambin crystal were measured to ultra-high resolution (0.54 Å) at low temperature by using short-wavelength synchrotron radiation. The crystal structure was refined with a model for charged, nonspherical, multipolar atoms to accurately describe the molecular electron density distribution. The refined parameters agree within 25% with our transferable electron density library derived from accurate single crystal diffraction analyses of several amino acids and small peptides. The resulting electron density maps of redistributed valence electrons (deformation maps) compare quantitatively well with a high-level quantum mechanical calculation performed on a monopeptide. This study provides validation for experimentally derived parameters and a window into charge density analysis of biological macromolecules. PMID:10737790

  8. Accurate protein crystallography at ultra-high resolution: valence electron distribution in crambin.

    PubMed

    Jelsch, C; Teeter, M M; Lamzin, V; Pichon-Pesme, V; Blessing, R H; Lecomte, C

    2000-03-28

    The charge density distribution of a protein has been refined experimentally. Diffraction data for a crambin crystal were measured to ultra-high resolution (0.54 A) at low temperature by using short-wavelength synchrotron radiation. The crystal structure was refined with a model for charged, nonspherical, multipolar atoms to accurately describe the molecular electron density distribution. The refined parameters agree within 25% with our transferable electron density library derived from accurate single crystal diffraction analyses of several amino acids and small peptides. The resulting electron density maps of redistributed valence electrons (deformation maps) compare quantitatively well with a high-level quantum mechanical calculation performed on a monopeptide. This study provides validation for experimentally derived parameters and a window into charge density analysis of biological macromolecules.

  9. 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.

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

    PubMed Central

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

    2013-01-01

    Trans-to-cis isomerization, the key reaction in photoactive proteins, cannot usually occur through the standard one-bond-flip mechanism. Due to spatial constraints imposed by a protein environment, isomerization is likely to proceed via a “volume-conserving” mechanism in which highly-choreographed atomic motions are expected, the details of which have not yet been directly observed. Here we employ time-resolved X-ray crystallography to structurally visualize isomerization of the p-coumaric acid chromophore in photoactive yellow protein with 100 picosecond time resolution and 1.6 Å spatial resolution. 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 via E46Q mutation, which switches off the bicycle-pedal pathway. PMID:23422563

  11. Fully Convergent Chemical Synthesis of Ester Insulin: Determination of the High Resolution X-ray Structure by Racemic Protein Crystallography

    PubMed Central

    Avital-Shmilovici, Michal; Mandal, Kalyaneswar; Gates, Zachary P.; Phillips, Nelson B.; Weiss, Michael A.; Kent, Stephen B.H.

    2013-01-01

    Efficient total synthesis of insulin is important to enable the application of medicinal chemistry to the optimization of the properties of this important protein molecule. Recently we described ‘ester insulin’ – a novel form of insulin in which the function of the 35 residue C-peptide of proinsulin is replaced by a single covalent bond – as a key intermediate for the efficient total synthesis of insulin. Here we describe a fully convergent synthetic route to the ester insulin molecule from three unprotected peptide segments of approximately equal size. The synthetic ester insulin polypeptide chain folded much more rapidly than proinsulin, and at physiological pH. Both the D-protein and L-protein enantiomers of monomeric DKP ester insulin (i.e. [AspB10, LysB28, ProB29]ester insulin) were prepared by total chemical synthesis. The atomic structure of the synthetic ester insulin molecule was determined by racemic protein X-ray crystallography to a resolution of 1.6 Å. Diffraction quality crystals were readily obtained from the racemic mixture of {D-DKP ester insulin + L-DKP ester insulin}, whereas crystals were not obtained from the L-ester insulin alone even after extensive trials. Both the D-protein and L-protein enantiomers of monomeric DKP ester insulin were assayed for receptor binding and in diabetic rats, before and after conversion by saponification to the corresponding DKP insulin enantiomers. L-DKP ester insulin bound weakly to the insulin receptor, while synthetic L-DKP insulin derived from the L-DKP ester insulin intermediate was fully active in binding to the insulin receptor. The D- and L-DKP ester insulins and D-DKP insulin were inactive in lowering blood glucose in diabetic rats, while synthetic L-DKP insulin was fully active in this biological assay. The structural basis of the lack of biological activity of ester insulin is discussed. PMID:23343390

  12. 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

  13. 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.

  14. Bent Diamond Crystals and Multilayer Based Optics at the new 5-Station Protein Crystallography Beamline 'Cassiopeia' at MAX-lab

    SciTech Connect

    Mammen, Christian B.; Als-Nielsen, Jens; Ursby, Thomas; Thunnissen, Marjolein

    2004-05-12

    A new 5-station beamline for protein crystallography is being commissioned at the Swedish synchrotron light source MAX-II at Lund University. Of the 2K/{gamma} = 14 mrad horizontal wiggler fan, the central 2 mrad are used and split in three parts. The central 1 mrad will be used for a station optimized for MAD experiments and on each side of the central fan, from 0.5 mrad to 1 mrad, there are two fixed energy stations using different energies of the same part of the beam. These, in total five stations, can be used simultaneously and independently for diffraction data collection. The two upstream monochromators for the side stations are meridionally bent asymmetric diamond(111) crystals in Laue transmission geometry. The monochromators for the downstream side stations are bent Ge(111) crystals in asymmetric Bragg reflection geometry. Curved multilayer mirrors inserted in the monochromatic beams provide focusing in the vertical plane. The first side station is under commissioning, and a preliminary test protein data set has been collected.

  15. 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.

  16. Watching a protein as it functions with 150-ps time-resolved x-ray crystallography.

    PubMed

    Schotte, Friedrich; Lim, Manho; Jackson, Timothy A; Smirnov, Aleksandr V; Soman, Jayashree; Olson, John S; Phillips, George N; Wulff, Michael; Anfinrud, Philip A

    2003-06-20

    We report picosecond time-resolved x-ray diffraction from the myoglobin (Mb) mutant in which Leu29 is replaced by Phe (L29Fmutant). The frame-by-frame structural evolution, resolved to 1.8 angstroms, allows one to literally "watch" the protein as it executes its function. Time-resolved mid-infrared spectroscopy of flash-photolyzed L29F MbCO revealed a short-lived CO intermediate whose 140-ps lifetime is shorter than that found in wild-type protein by a factor of 1000. The electron density maps of the protein unveil transient conformational changes far more dramatic than the structural differences between the carboxy and deoxy states and depict the correlated side-chain motion responsible for rapidly sweeping CO away from its primary docking site.

  17. 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.

  18. Harvesting and cryo-cooling crystals of membrane proteins grown in lipidic mesophases for structure determination by macromolecular crystallography.

    PubMed

    Li, Dianfan; Boland, Coilín; Aragao, David; Walsh, Kilian; Caffrey, Martin

    2012-09-02

    An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases(1-5), has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field(6-21) (www.mpdb.tcd.ie). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting(22,23). Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)(24,25) are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection. The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies(4,26). The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been

  19. Total chemical synthesis and X-ray structure of kaliotoxin by racemic protein crystallography

    SciTech Connect

    Pentelute, Brad L.; Mandal, Kalyaneswar; Gates, Zachary P.; Sawaya, Michael R.; Yeates, Todd O.; Kent, Stephen B.H.

    2010-11-05

    Here we report the total synthesis of kaliotoxin by 'one pot' native chemical ligation of three synthetic peptides. A racemic mixture of D- and L-kaliotoxin synthetic protein molecules gave crystals in the centrosymmetric space groupP that diffracted to atomic-resolution (0.95 {angstrom}), enabling the X-ray structure of kaliotoxin to be determined by direct methods.

  20. 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.

  1. 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

  2. Direct observation of hydrogen atom dynamics and interactions by ultrahigh resolution neutron protein crystallography.

    PubMed

    Chen, Julian C-H; Hanson, B Leif; Fisher, S Zoë; Langan, Paul; Kovalevsky, Andrey Y

    2012-09-18

    The 1.1 Å, ultrahigh resolution neutron structure of hydrogen/deuterium (H/D) exchanged crambin is reported. Two hundred ninety-nine out of 315, or 94.9%, of the hydrogen atom positions in the protein have been experimentally derived and resolved through nuclear density maps. A number of unconventional interactions are clearly defined, including a potential O─H…π interaction between a water molecule and the aromatic ring of residue Y44, as well as a number of potential C─H…O hydrogen bonds. Hydrogen bonding networks that are ambiguous in the 0.85 Å ultrahigh resolution X-ray structure can be resolved by accurate orientation of water molecules. Furthermore, the high resolution of the reported structure has allowed for the anisotropic description of 36 deuterium atoms in the protein. The visibility of hydrogen and deuterium atoms in the nuclear density maps is discussed in relation to the resolution of the neutron data.

  3. 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.

  4. Anisotropic atomic motions in high-resolution protein crystallography molecular dynamics simulations.

    PubMed

    Burden, Conrad J; Oakley, Aaron J

    2007-06-11

    Molecular dynamics (MD) simulations using empirical force fields are popular for the study of proteins. In this work, we compare anisotropic atomic fluctuations in nanosecond-timescale MD simulations with those observed in an ultra-high-resolution crystal structure of crambin. In order to make our comparisons, we have developed a compact graphical technique for assessing agreement between spatial atomic distributions determined by MD simulations and observed anisotropic temperature factors.

  5. 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

  6. Neutron crystallography of photoactive yellow protein reveals unusual protonation state of Arg52 in the crystal.

    PubMed

    Yonezawa, Kento; Shimizu, Nobutaka; Kurihara, Kazuo; Yamazaki, Yoichi; Kamikubo, Hironari; Kataoka, Mikio

    2017-08-24

    Because of its high pKa, arginine (Arg) is believed to be protonated even in the hydrophobic environment of the protein interior. However, our neutron crystallographic structure of photoactive yellow protein, a light sensor, demonstrated that Arg52 adopts an electrically neutral form. We also showed that the hydrogen bond between the chromophore and Glu46 is a so-called low barrier hydrogen bond (LBHB). Because both the neutral Arg and LBHB are unusual in proteins, these observations remain controversial. To validate our findings, we carried out neutron crystallographic analysis of the E46Q mutant of PYP. The resultant structure revealed that the proportion of the cationic form is higher in E46Q than in WT, although the cationic and neutral forms of Arg52 coexist in E46Q. These observations were confirmed by the occupancy of the deuterium atom bound to the N η1 atom combined with an alternative conformation of the N(η2)D2 group comprising sp(2) hybridisation. Based on these results, we propose that the formation of the LBHB decreases the proton affinity of Arg52, stabilizing the neutral form in the crystal.

  7. 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.

  8. Direct observation of hydrogen atom dynamics and interactions by ultrahigh resolution neutron protein crystallography

    PubMed Central

    Chen, Julian C.-H.; Hanson, B. Leif; Fisher, S. Zoë; Langan, Paul; Kovalevsky, Andrey Y.

    2012-01-01

    The 1.1 Å, ultrahigh resolution neutron structure of hydrogen/deuterium (H/D) exchanged crambin is reported. Two hundred ninety-nine out of 315, or 94.9%, of the hydrogen atom positions in the protein have been experimentally derived and resolved through nuclear density maps. A number of unconventional interactions are clearly defined, including a potential O─H…π interaction between a water molecule and the aromatic ring of residue Y44, as well as a number of potential C─H…O hydrogen bonds. Hydrogen bonding networks that are ambiguous in the 0.85 Å ultrahigh resolution X-ray structure can be resolved by accurate orientation of water molecules. Furthermore, the high resolution of the reported structure has allowed for the anisotropic description of 36 deuterium atoms in the protein. The visibility of hydrogen and deuterium atoms in the nuclear density maps is discussed in relation to the resolution of the neutron data. PMID:22949690

  9. Inhibition of Retinoblastoma Protein Inactivation

    DTIC Science & Technology

    2016-09-01

    Retinoblastoma protein, E2F transcription factor, high throughput screen, drug discovery, x - ray crystallography 16. SECURITY CLASSIFICATION OF: 17...developed a method to perform fragment based screening by x - ray crystallography. 2.0 KEYWORDS Retinoblastoma (Rb) pathway, E2F transcription factor...cancer, cell-cycle inhibition, activation, modulation, inhibition, high throughput screening, fragment-based screening, x - ray crystallography

  10. 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.

  11. Contaminant inclusion into protein crystals analyzed by electrospray mass spectrometry and X-ray crystallography.

    PubMed Central

    Hirschler, J.; Halgand, F.; Forest, E.; Fontecilla-Camps, J. C.

    1998-01-01

    The inclusion of protein contaminants into crystals of turkey egg white lysozyme (TEWL) was investigated by electrospray mass spectrometry of the dissolved crystals. The results show that significant amounts of the structurally related contaminant hen egg white lysozyme (HEWL) are included in the crystals of TEWL. The structurally unrelated contaminant RNAse A, on the other hand, is not included. The X-ray diffraction data statistics of a hybrid TEWL/HEWL crystal and an uncontaminated TEWL crystal were of similar quality. This indicates that, even though the crystals contain much higher levels of the contaminant than one would have expected after a recrystallization experiment, they are still suitable for X-ray diffraction experiments. However, attempts to detect the presence of the contaminant in the crystal by crystallographic structure refinement did not yield conclusive results. PMID:9514273

  12. 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.

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

    SciTech Connect

    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 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. 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.

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

    DOE PAGES

    Van Benschoten, Andrew H.; Liu, Lin; Gonzalez, Ana; ...

    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

  15. 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.

  16. 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.

  17. Performance of PILATUS detector technology for long-wavelength macromolecular crystallography

    NASA Astrophysics Data System (ADS)

    Marchal, J.; Wagner, A.

    2011-05-01

    The long-wavelength MX beamline I23 currently under design at Diamond Light Source will be optimized in the X-ray energy range between 3 and 5 keV. At the moment no commercial off-the-shelf detector with high quantum efficiency and dynamic range is available to cover the large area required for diffraction experiments in this energy range. The hybrid pixel detector technology used in PILATUS detectors could overcome these limitations as the modular design could allow a large coverage in reciprocal space and high detection efficiency. Experiments were carried out on the Microfocus Spectroscopy beamline I18 at Diamond Light Source to test the performance of a 100K PILATUS module in the low-energy range from 2.3 to 3.7 keV.

  18. Serial Crystallography

    NASA Astrophysics Data System (ADS)

    Spence, John

    2007-03-01

    To solve proteins which cannot be crystallized we have devised an aerodynamic focussing, monodispered droplet beam, which runs in single file across a synchrotron X-ray beam (LBNL Advanced Light Source) as it freezes in vacuum. The aim is to obtain a charge-density map of the protein at 0.7nm resolution , sufficient to locate alpha-helices. Water is removed before the proteins, coated by a thin ice-jacket, are aligned by the dipole moment induced by a 100 W NIR polarized fiber laser. All three orthogonal beams (proteins, X-rays, laser) intersect in a 10 micron diameter volume, and run continuously without sychronization. Elliptical polarization aligns molecular axes in direction but not sense. Data is collected continuously until adequate statistics are achieved before rotating the polarization to a new orientation. Details of the adiabatic laser-alignment and damping processes will be given. Misalignment is shown to be proportional to temperature and inversely to laser power and molecular volume. Polarizability tensor calculations for proteins will be discussed. Preliminary X-ray results (without laser) will be shown. Iterative methods for solving the phase problem will be demonstrated using experimental soft X-ray diffraction patterns from non-crystalline particles. Detailed simulations of the continuous diffraction patterns from a moving stream of partially aligned large hydrated molecules will be shown, and inverted to density maps using the Fienup-Gerchberg-Saxton algorithm. From this the exposure time and resolution may be estimated for tomographic reconstruction. Experimental comparisons of Rayleigh, electrospray and aerodynamic focussing droplet beam sources will also be described. The research team includes B.Doak, U. Weierstall, D. Shapiro (LBNL), D. Deponte, P. Fromme, D. Starodub, G. Hembree and H. Chapman (LLNL).

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

    PubMed

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

    2014-06-01

    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.

  20. 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

  1. 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

  2. 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).

  3. Room-temperature serial crystallography using a kinetically optimized microfluidic device for protein crystallization and on-chip X-ray diffraction

    PubMed Central

    Heymann, Michael; Opthalage, Achini; Wierman, Jennifer L.; Akella, Sathish; Szebenyi, Doletha M. E.; Gruner, Sol M.; Fraden, Seth

    2014-01-01

    An emulsion-based serial crystallographic technology has been developed, in which nanolitre-sized droplets of protein solution are encapsulated in oil and stabilized by surfactant. Once the first crystal in a drop is nucleated, the small volume generates a negative feedback mechanism that lowers the supersaturation. This mechanism is exploited to produce one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room-temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different unoriented crystals. As proof of concept, the structure of glucose isomerase was solved to 2.1 Å, demonstrating the feasibility of high-throughput serial X-ray crystallography using synchrotron radiation. PMID:25295176

  4. X-ray crystallography of viruses.

    PubMed

    Verdaguer, Nuria; Garriga, Damià; Fita, Ignacio

    2013-01-01

    For about 30 years X-ray crystallography has been by far the most powerful approach for determining virus structures at close to atomic resolutions. Information provided by these studies has deeply and extensively enriched and shaped our vision of the virus world. In turn, the ever increasing complexity and size of the virus structures being investigated have constituted a major driving force for methodological and conceptual developments in X-ray macromolecular crystallography. Landmarks of new virus structures determinations, such as the ones from the first animal viruses or from the first membrane-containing viruses, have often been associated to methodological breakthroughs in X-ray crystallography. In this chapter we present the common ground of proteins and virus crystallography with an emphasis in the peculiarities of virus studies. For example, the solution of the phase problem, a central issue in X-ray diffraction, has benefited enormously from the presence of non-crystallographic symmetry in virus crystals.

  5. Determination of the GH3.12 protein conformation through HPLC-integrated SAXS measurements combined with X-ray crystallography.

    PubMed

    Round, Adam; Brown, Elizabeth; Marcellin, Romain; Kapp, Ulrike; Westfall, Corey S; Jez, Joseph M; Zubieta, Chloe

    2013-10-01

    The combination of protein crystallography and small-angle X-ray scattering (SAXS) provides a powerful method to investigate changes in protein conformation. These complementary structural techniques were used to probe the solution structure of the apo and the ligand-bound forms of the Arabidopsis thaliana acyl acid-amido synthetase GH3.12. This enzyme is part of the extensive GH3 family and plays a critical role in the regulation of plant hormones through the formation of amino-acid-conjugated hormone products via an ATP-dependent reaction mechanism. The enzyme adopts two distinct C-terminal domain orientations with `open' and `closed' active sites. Previous studies suggested that ATP only binds in the open orientation. Here, the X-ray crystal structure of GH3.12 is presented in the closed conformation in complex with the nonhydrolysable ATP analogue AMPCPP and the substrate salicylate. Using on-line HPLC purification combined with SAXS measurements, the most likely apo and ATP-bound protein conformations in solution were determined. These studies demonstrate that the C-terminal domain is flexible in the apo form and favours the closed conformation upon ATP binding. In addition, these data illustrate the efficacy of on-line HPLC purification integrated into the SAXS sample-handling environment to reliably monitor small changes in protein conformation through the collection of aggregate-free and highly redundant data.

  6. 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.

  7. 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

  8. 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

  9. Synchrotron X-Radiation Protein Crystallography and its Use in the Crystal Structure Determination of Purine Nucleoside Phosphorylase.

    NASA Astrophysics Data System (ADS)

    Rule, Stephen Anthony

    Available from UMI in association with The British Library. The availability of intense, tunable Synchrotron X-radiation (SR) has, in recent years, revolutionised certain aspects of macromolecular crystallography. Data collection times have been dramatically reduced, the range of suitable samples has been widened, and techniques such as optimised anomalous scattering and Laue diffraction have become feasible. An understanding of the characteristics of the SR beam and its interaction with single crystal monochromator systems leads to the possibility of a polychromatic diffraction experiment in which each ray's energy is correlated with its direction within the beam. Furthermore, this correlation is preserved in the reflections from a crystal sample, thereby allowing the wavelength dependent effects of an anomalous scatterer to be recorded simultaneously and yet remain resolved spatially. The potential of this approach is demonstrated by an experiment in which a small Re complex was exposed to a polychromatic beam centred on the Re L _{rm III} absorption edge wavelength. Purine nucleoside phosphorylase (PNP) is an important enzyme in purine catabolism and salvage. PNP deficiency is associated with selective T-cell immunodeficiency and, consequently, an effective PNP inhibitor might prove to be useful in treating various conditions including T-cell leukemias. With a view towards rational drug design, the crystal structure of PNP was initially determined at 6.0 A resolution using diffractometer data. However, the crystals diffract weakly and data to high resolution from native and derivative crystals were therefore collected at the Daresbury SRS. Phases were calculated by the method of isomorphous replacement and the final electron density map at 3.16 A resolution is of high quality, enabling the entire polypeptide chain to be followed. The structure consists principally of a six stranded beta sheet flanked by several alpha-helices. The region of the active centre is

  10. 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.

  11. Surface Proteins of Gram-Positive Pathogens: Using Crystallography to Uncover Novel Features in Drug and Vaccine Candidates

    NASA Astrophysics Data System (ADS)

    Baker, Edward N.; Proft, Thomas; Kang, Haejoo

    Proteins displayed on the cell surfaces of pathogenic organisms are the front-line troops of bacterial attack, playing critical roles in colonization, infection and virulence. Although such proteins can often be recognized from genome sequence data, through characteristic sequence motifs, their functions are often unknown. One such group of surface proteins is attached to the cell surface of Gram-positive pathogens through the action of sortase enzymes. Some of these proteins are now known to form pili: long filamentous structures that mediate attachment to human cells. Crystallographic analyses of these and other cell surface proteins have uncovered novel features in their structure, assembly and stability, including the presence of inter- and intramolecular isopeptide crosslinks. This improved understanding of structures on the bacterial cell surface offers opportunities for the development of some new drug targets and for novel approaches to vaccine design.

  12. Crystallography: Sources of inspiration

    NASA Astrophysics Data System (ADS)

    McSweeney, Sean; Fromme, Petra

    2014-01-01

    Synchrotrons have long been the preferred X-ray sources for crystallography, but competition has arrived with the advent of X-ray free-electron lasers. A synchrotron expert and an advocate of free-electron lasers discuss the prospects of the respective source types for applications in structural biology.

  13. 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.

  14. Asymmetry in serial femtosecond crystallography data

    PubMed Central

    Sharma, Amit; Johansson, Linda; Dunevall, Elin; Wahlgren, Weixiao Y.; Neutze, Richard

    2017-01-01

    Serial crystallography is an increasingly important approach to protein crystallography that exploits both X-ray free-electron laser (XFEL) and synchrotron radiation. Serial crystallography recovers complete X-ray diffraction data by processing and merging diffraction images from thousands of randomly oriented non-uniform microcrystals, of which all observations are partial Bragg reflections. Random fluctuations in the XFEL pulse energy spectrum, variations in the size and shape of microcrystals, integrating over millions of weak partial observations and instabilities in the XFEL beam position lead to new types of experimental errors. The quality of Bragg intensity estimates deriving from serial crystallography is therefore contingent upon assumptions made while modeling these data. Here it is observed that serial femtosecond crystallography (SFX) Bragg reflections do not follow a unimodal Gaussian distribution and it is recommended that an idealized assumption of single Gaussian peak profiles be relaxed to incorporate apparent asymmetries when processing SFX data. The phenomenon is illustrated by re-analyzing data collected from microcrystals of the Blastochloris viridis photosynthetic reaction center and comparing these intensity observations with conventional synchrotron data. The results show that skewness in the SFX observations captures the essence of the Wilson plot and an empirical treatment is suggested that can help to separate the diffraction Bragg intensity from the background. PMID:28248658

  15. High-throughput Crystallography for Structural Genomics

    PubMed Central

    Joachimiak, Andrzej

    2009-01-01

    Protein X-ray crystallography recently celebrated its 50th anniversary. The structures of myoglobin and hemoglobin determined by Kendrew and Perutz provided the first glimpses into the complex protein architecture and chemistry. Since then, the field of structural molecular biology has experienced extraordinary progress and now over 53,000 proteins structures have been deposited into the Protein Data Bank. In the past decade many advances in macromolecular crystallography have been driven by world-wide structural genomics efforts. This was made possible because of third-generation synchrotron sources, structure phasing approaches using anomalous signal and cryo-crystallography. Complementary progress in molecular biology, proteomics, hardware and software for crystallographic data collection, structure determination and refinement, computer science, databases, robotics and automation improved and accelerated many processes. These advancements provide the robust foundation for structural molecular biology and assure strong contribution to science in the future. In this report we focus mainly on reviewing structural genomics high-throughput X-ray crystallography technologies and their impact. PMID:19765976

  16. 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

  17. Development of a dose-limiting data collection strategy for serial synchrotron rotation crystallography

    PubMed Central

    Hasegawa, Kazuya; Yamashita, Keitaro; Murai, Tomohiro; Nuemket, Nipawan; Hirata, Kunio; Ueno, Go; Ago, Hideo; Nakatsu, Toru; Kumasaka, Takashi; Yamamoto, Masaki

    2017-01-01

    Serial crystallography, in which single-shot diffraction images are collected, has great potential for protein microcrystallography. Although serial femtosecond crystallography (SFX) has been successfully demonstrated, limited beam time prevents its routine use. Inspired by SFX, serial synchrotron crystallography (SSX) has been investigated at synchrotron macromolecular crystallography beamlines. Unlike SFX, the longer exposure time of milliseconds to seconds commonly used in SSX causes radiation damage. However, in SSX, crystals can be rotated during the exposure, which can achieve efficient coverage of the reciprocal space. In this study, mercury single-wavelength anomalous diffraction (Hg-SAD) phasing of the luciferin regenerating enzyme (LRE) was performed using serial synchrotron rotation crystallography. The advantages of rotation and influence of dose on the data collected were evaluated. The results showed that sample rotation was effective for accurate data collection, and the optimum helical rotation step depended on multiple factors such as multiplicity and partiality of reflections, exposure time per rotation angle and the contribution from background scattering. For the LRE microcrystals, 0.25° was the best rotation step for the achievable resolution limit, whereas a rotation step larger than or equal to 1° was favorable for Hg-SAD phasing. Although an accumulated dose beyond 1.1 MGy caused specific damage at the Hg site, increases in resolution and anomalous signal were observed up to 3.4 MGy because of a higher signal-to-noise ratio. PMID:28009544

  18. 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.

  19. Crystallography and chemistry should always go together: a cautionary tale of protein complexes with cisplatin and carboplatin

    PubMed Central

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

    2015-01-01

    The anticancer activity of platinum-containing drugs such as cisplatin and carboplatin is considered to primarily arise from their interactions with nucleic acids; nevertheless, these drugs, or the products of their hydrolysis, also bind to proteins, potentially leading to the known side effects of the treatments. Here, over 40 crystal structures deposited in the Protein Data Bank (PDB) of cisplatin and carboplatin complexes of several proteins were analysed. Significant problems of either a crystallographic or a chemical nature were found in most of the presented atomic models and they could be traced to less or more serious deficiencies in the data-collection and refinement procedures. The re-evaluation of these data and models was possible thanks to their mandatory or voluntary deposition in publicly available databases, emphasizing the point that the availability of such data is critical for making structural science reproducible. Based on this analysis of a selected group of macromolecular structures, the importance of deposition of raw diffraction data is stressed and a procedure for depositing, tracking and using re-refined crystallographic models is suggested. PMID:26327386

  20. Three-dimensional structure of the surface layer protein of Aquaspirillum serpens VHA determined by electron crystallography.

    PubMed Central

    Dickson, M R; Downing, K H; Wu, W H; Glaeser, R M

    1986-01-01

    The three-dimensional structure of the protein which forms the S layer of Aquaspirillum serpens strain VHA has been determined by electron microscopy. Structures have been reconstructed to a resolution of about 1.6 nm for single-layered specimens and about 4 nm for two-layered specimens. The structure, which has hexagonal symmetry, consists of a core in the shape of a cup, with six projections arising from the rim of the cup to join adjacent subunits at the threefold symmetry axes. The model is consistent with edge views of the S layer which have been obtained in this and other work. It is now clear from this work and from three-dimensional reconstructions of other bacterial S layers that a wide diversity exists in the morphology of surface layers. Images PMID:3745114

  1. Following DNA chain extension and protein conformational changes in crystals of a Y-family DNA polymerase by Raman crystallography

    PubMed Central

    Espinoza-Herrera, Shirly J.; Gaur, Vineet; Suo, Zucai; Carey, Paul R.

    2013-01-01

    Y-family DNA polymerases are known to bypass DNA lesions in vitro and in vivo. Sulfolobus solfataricus DNA polymerase (Dpo4) was chosen as a model Y-family enzyme for investigating the mechanism of DNA synthesis in single crystals. Crystals of Dpo4 in complexes with DNA (the binary complex) in the presence or absence of an incoming nucleotide were analyzed by Raman microscopy. 13C, 15N labeled d*CTP, or unlabeled dCTP, were soaked into the binary crystals with G as the templating base. In the presence of the catalytic metal ions, Mg2+ or Mn2+, nucleotide incorporation was detected by the disappearance of the triphosphate band of dCTP and the retention of C* modes in the crystal following soaking out of noncovalently bound C(or *C)TP. The addition of the second coded base, thymine, was observed by adding cognate dTTP to the crystal following single d*CTP addition. Adding these two bases caused visible damage to the crystal possibly caused by protein and/or DNA conformational change within the crystal. When d*CTP is soaked into the Dpo4 crystal in the absence of Mn2+ or Mg2+, the primer extension reaction did not occur; instead a ternary protein/template/d*CTP complex was formed. In the Raman difference spectra of both binary and ternary complexes, in addition to the modes of d(*C)CTP, features appear due to ring modes from the template/primer bases being perturbed and from the DNA backbone, as well as from perturbed peptide and amino acid side chain modes. These effects are more pronounced in the ternary than in the binary complex. Using standardized Raman intensities followed as a function of time C(*C)TP population in the crystal maximized at about 20 min. These remained unchanged in the ternary complex but declined in the binary complexes as chain incorporation occurred. PMID:23855392

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

    NASA Astrophysics Data System (ADS)

    McDdonald, William T.; Lewis, Johanna L.; Smith, Craig D.; Delucas, Lawrence J.

    1997-01-01

    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. 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.

  4. Radiation damage to protein specimens from electron beam imaging and diffraction: a mini-review of anti-damage approaches, with special reference to synchrotron X-ray crystallography.

    PubMed

    Massover, William H

    2007-01-01

    Recent research progress using X-ray cryo-crystallography with the photon beams from third-generation synchrotron sources has resulted in recognition that this intense radiation commonly damages protein samples even when they are held at 100 K. Other structural biologists examining thin protein crystals or single particle specimens encounter similar radiation damage problems during electron diffraction and imaging, but have developed some effective countermeasures. The aim of this concise review is to examine whether analogous approaches can be utilized to alleviate the X-ray radiation damage problem in synchrotron macromolecular crystallography. The critical discussion of this question is preceded by presentation of background material on modern technical procedures with electron beam instruments using 300-400 kV accelerating voltage, low-dose exposures for data recording, and protection of protein specimens by cryogenic cooling; these practical approaches to dealing with electron radiation damage currently permit best resolution levels of 6 A (0.6 nm) for single particle specimens, and of 1.9 A for two-dimensional membrane protein crystals. Final determination of the potential effectiveness and practical value of using such new or unconventional ideas will necessitate showing, by experimental testing, that these produce significantly improved protection of three-dimensional protein crystals during synchrotron X-ray diffraction.

  5. 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

  6. 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

  7. 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.

  8. Shoot-and-Trap: Use of specific x-ray damage to study structural protein dynamics by temperature-controlled cryo-crystallography

    PubMed Central

    Colletier, Jacques-Philippe; Bourgeois, Dominique; Sanson, Benoît; Fournier, Didier; Sussman, Joel L.; Silman, Israel; Weik, Martin

    2008-01-01

    Although x-ray crystallography is the most widely used method for macromolecular structure determination, it does not provide dynamical information, and either experimental tricks or complementary experiments must be used to overcome the inherently static nature of crystallographic structures. Here we used specific x-ray damage during temperature-controlled crystallographic experiments at a third-generation synchrotron source to trigger and monitor (Shoot-and-Trap) structural changes putatively involved in an enzymatic reaction. In particular, a nonhydrolyzable substrate analogue of acetylcholinesterase, the “off-switch” at cholinergic synapses, was radiocleaved within the buried enzymatic active site. Subsequent product clearance, observed at 150 K but not at 100 K, indicated exit from the active site possibly via a “backdoor.” The simple strategy described here is, in principle, applicable to any enzyme whose structure in complex with a substrate analogue is available and, therefore, could serve as a standard procedure in kinetic crystallography studies. PMID:18701720

  9. 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

  10. Resolution of structural heterogeneity in dynamic crystallography.

    PubMed

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

    2013-06-01

    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.

  11. 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

  12. 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

  13. In situ macromolecular crystallography using microbeams.

    PubMed

    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

    2012-05-01

    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.

  14. Benzimidazole inhibitors of the protein kinase CHK2: clarification of the binding mode by flexible side chain docking and protein-ligand crystallography.

    PubMed

    Matijssen, Cornelis; Silva-Santisteban, M Cris; Westwood, Isaac M; Siddique, Samerene; Choi, Vanessa; Sheldrake, Peter; van Montfort, Rob L M; Blagg, Julian

    2012-11-15

    Two closely related binding modes have previously been proposed for the ATP-competitive benzimidazole class of checkpoint kinase 2 (CHK2) inhibitors; however, neither binding mode is entirely consistent with the reported SAR. Unconstrained rigid docking of benzimidazole ligands into representative CHK2 protein crystal structures reveals an alternative binding mode involving a water-mediated interaction with the hinge region; docking which incorporates protein side chain flexibility for selected residues in the ATP binding site resulted in a refinement of the water-mediated hinge binding mode that is consistent with observed SAR. The flexible docking results are in good agreement with the crystal structures of four exemplar benzimidazole ligands bound to CHK2 which unambiguously confirmed the binding mode of these inhibitors, including the water-mediated interaction with the hinge region, and which is significantly different from binding modes previously postulated in the literature.

  15. 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

  16. 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

  17. 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

  18. O-Alkylated heavy atom carbohydrate probes for protein X-ray crystallography: Studies towards the synthesis of methyl 2-O-methyl-L-selenofucopyranoside

    PubMed Central

    Sommer, Roman; Hauck, Dirk; Varrot, Annabelle; Imberty, Anne; Künzler, Markus

    2016-01-01

    Selenoglycosides are used as reactive glycosyl donors in the syntheses of oligosaccharides. In addition, such heavy atom analogs of natural glycosides are useful tools for structure determination of their lectin receptors using X-ray crystallography. Some lectins, e.g., members of the tectonin family, only bind to carbohydrate epitopes with O-alkylated ring hydroxy groups. In this context, we report the first synthesis of an O-methylated selenoglycoside, specifically methyl 2-O-methyl-L-selenofucopyranoside, a ligand of the lectin tectonin-2 from the mushroom Laccaria bicolor. The synthetic route required a strategic revision and further optimization due to the intrinsic lability of alkyl selenoglycosides, in particular for the labile fucose. Here, we describe a successful synthetic access to methyl 2-O-methyl-L-selenofucopyranoside in 9 linear steps and 26% overall yield starting from allyl L-fucopyranoside. PMID:28144356

  19. O-Alkylated heavy atom carbohydrate probes for protein X-ray crystallography: Studies towards the synthesis of methyl 2-O-methyl-L-selenofucopyranoside.

    PubMed

    Sommer, Roman; Hauck, Dirk; Varrot, Annabelle; Imberty, Anne; Künzler, Markus; Titz, Alexander

    2016-01-01

    Selenoglycosides are used as reactive glycosyl donors in the syntheses of oligosaccharides. In addition, such heavy atom analogs of natural glycosides are useful tools for structure determination of their lectin receptors using X-ray crystallography. Some lectins, e.g., members of the tectonin family, only bind to carbohydrate epitopes with O-alkylated ring hydroxy groups. In this context, we report the first synthesis of an O-methylated selenoglycoside, specifically methyl 2-O-methyl-L-selenofucopyranoside, a ligand of the lectin tectonin-2 from the mushroom Laccaria bicolor. The synthetic route required a strategic revision and further optimization due to the intrinsic lability of alkyl selenoglycosides, in particular for the labile fucose. Here, we describe a successful synthetic access to methyl 2-O-methyl-L-selenofucopyranoside in 9 linear steps and 26% overall yield starting from allyl L-fucopyranoside.

  20. 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.

  1. Ultrafast X-Ray Crystallography and Liquidography

    NASA Astrophysics Data System (ADS)

    Ki, Hosung; Oang, Key Young; Kim, Jeongho; Ihee, Hyotcherl

    2017-05-01

    Time-resolved X-ray diffraction provides direct information on three-dimensional structures of reacting molecules and thus can be used to elucidate structural dynamics of chemical and biological reactions. In this review, we discuss time-resolved X-ray diffraction on small molecules and proteins with particular emphasis on its application to crystalline (crystallography) and liquid-solution (liquidography) samples. Time-resolved X-ray diffraction has been used to study picosecond and slower dynamics at synchrotrons and can now access even femtosecond dynamics with the recent arrival of X-ray free-electron lasers.

  2. Ultrafast X-Ray Crystallography and Liquidography.

    PubMed

    Ki, Hosung; Oang, Key Young; Kim, Jeongho; Ihee, Hyotcherl

    2017-05-05

    Time-resolved X-ray diffraction provides direct information on three-dimensional structures of reacting molecules and thus can be used to elucidate structural dynamics of chemical and biological reactions. In this review, we discuss time-resolved X-ray diffraction on small molecules and proteins with particular emphasis on its application to crystalline (crystallography) and liquid-solution (liquidography) samples. Time-resolved X-ray diffraction has been used to study picosecond and slower dynamics at synchrotrons and can now access even femtosecond dynamics with the recent arrival of X-ray free-electron lasers.

  3. CLIMS: crystallography laboratory information management system.

    PubMed

    Fulton, Kate F; Ervine, Shaun; Faux, Noel; Forster, Richard; Jodun, Rachel A; Ly, Wayson; Robilliard, Lee; Sonsini, Jai; Whelan, Dan; Whisstock, James C; Buckle, Ashley M

    2004-09-01

    Macromolecular crystallography requires simple yet effective means of organizing and managing the large amounts of data generated by crystallization experiments. There are several freely available web-based Laboratory Information Management Systems (LIMS) that assist in these tasks. These, however, rely on the limited user interfaces allowed in HTML-based web pages. To address this limitation, a new LIMS for protein crystallization, which features a novel rich graphical user interface (GUI) to a relational database, has been developed. This application, which is called CLIMS (Crystallography LIMS), assists in all aspects of protein-crystallization projects: protein expression, handling, crystallization optimization, visualization of results and preliminary diffraction data. Extensive use of templates, particularly for commercial screens and common optimization grid screens, exploits the redundancy in experimental setups. The crystallization tray is the central focus of the graphical interface, thus facilitating rapid visualization and annotation of results. CLIMS was developed specifically to cater for the needs of individual laboratories requiring an intuitive and robust system for managing crystallization experiments and is freely available.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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

    2016-08-02

    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.

  10. The bacterial cell-division protein ZipA and its interaction with an FtsZ fragment revealed by X-ray crystallography

    PubMed Central

    Mosyak, Lidia; Zhang, Yan; Glasfeld, Elizabeth; Haney, Steve; Stahl, Mark; Seehra, Jasbir; Somers, William S.

    2000-01-01

    In Escherichia coli, FtsZ, a homologue of eukaryotic tubulins, and ZipA, a membrane-anchored protein that binds to FtsZ, are two essential components of the septal ring structure that mediates cell division. Recent data indicate that ZipA is involved in the assembly of the ring by linking FtsZ to the cytoplasmic membrane and that the ZipA–FtsZ interaction is mediated by their C-terminal domains. We present the X-ray crystal structures of the C-terminal FtsZ-binding domain of ZipA and a complex between this domain and a C-terminal fragment of FtsZ. The ZipA domain is a six-stranded β-sheet packed against three α-helices and contains the split β–α–β motif found in many RNA-binding proteins. The uncovered side of the sheet incorporates a shallow hydrophobic cavity exposed to solvent. In the complex, the 17-residue FtsZ fragment occupies this entire cavity of ZipA and binds as an extended β-strand followed by α-helix. An alanine-scanning mutagenesis analysis of the FtsZ fragment was also performed, which shows that only a small cluster of the buried FtsZ side chains is critical in binding to ZipA. PMID:10880432

  11. Intact Protein Analysis at 21 Tesla and X-Ray Crystallography Define Structural Differences in Single Amino Acid Variants of Human Mitochondrial Branched-Chain Amino Acid Aminotransferase 2 (BCAT2)

    NASA Astrophysics Data System (ADS)

    Anderson, Lissa C.; Håkansson, Maria; Walse, Björn; Nilsson, Carol L.

    2017-09-01

    Structural technologies are an essential component in the design of precision therapeutics. Precision medicine entails the development of therapeutics directed toward a designated target protein, with the goal to deliver the right drug to the right patient at the right time. In the field of oncology, protein structural variants are often associated with oncogenic potential. In a previous proteogenomic screen of patient-derived glioblastoma (GBM) tumor materials, we identified a sequence variant of human mitochondrial branched-chain amino acid aminotransferase 2 as a putative factor of resistance of GBM to standard-of-care-treatments. The enzyme generates glutamate, which is neurotoxic. To elucidate structural coordinates that may confer altered substrate binding or activity of the variant BCAT2 T186R, a 45 kDa protein, we applied combined ETD and CID top-down mass spectrometry in a LC-FT-ICR MS at 21 T, and X-Ray crystallography in the study of both the variant and non-variant intact proteins. The combined ETD/CID fragmentation pattern allowed for not only extensive sequence coverage but also confident localization of the amino acid variant to its position in the sequence. The crystallographic experiments confirmed the hypothesis generated by in silico structural homology modeling, that the Lys59 side-chain of BCAT2 may repulse the Arg186 in the variant protein (PDB code: 5MPR), leading to destabilization of the protein dimer and altered enzyme kinetics. Taken together, the MS and novel 3D structural data give us reason to further pursue BCAT2 T186R as a precision drug target in GBM. [Figure not available: see fulltext.

  12. Intact Protein Analysis at 21 Tesla and X-Ray Crystallography Define Structural Differences in Single Amino Acid Variants of Human Mitochondrial Branched-Chain Amino Acid Aminotransferase 2 (BCAT2)

    NASA Astrophysics Data System (ADS)

    Anderson, Lissa C.; Håkansson, Maria; Walse, Björn; Nilsson, Carol L.

    2017-07-01

    Structural technologies are an essential component in the design of precision therapeutics. Precision medicine entails the development of therapeutics directed toward a designated target protein, with the goal to deliver the right drug to the right patient at the right time. In the field of oncology, protein structural variants are often associated with oncogenic potential. In a previous proteogenomic screen of patient-derived glioblastoma (GBM) tumor materials, we identified a sequence variant of human mitochondrial branched-chain amino acid aminotransferase 2 as a putative factor of resistance of GBM to standard-of-care-treatments. The enzyme generates glutamate, which is neurotoxic. To elucidate structural coordinates that may confer altered substrate binding or activity of the variant BCAT2 T186R, a 45 kDa protein, we applied combined ETD and CID top-down mass spectrometry in a LC-FT-ICR MS at 21 T, and X-Ray crystallography in the study of both the variant and non-variant intact proteins. The combined ETD/CID fragmentation pattern allowed for not only extensive sequence coverage but also confident localization of the amino acid variant to its position in the sequence. The crystallographic experiments confirmed the hypothesis generated by in silico structural homology modeling, that the Lys59 side-chain of BCAT2 may repulse the Arg186 in the variant protein (PDB code: 5MPR), leading to destabilization of the protein dimer and altered enzyme kinetics. Taken together, the MS and novel 3D structural data give us reason to further pursue BCAT2 T186R as a precision drug target in GBM.

  13. 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.

  14. 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

  15. 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

  16. Viscous hydrophilic injection matrices for serial crystallography

    PubMed Central

    Kovácsová, Gabriela; Grünbein, Marie Luise; Kloos, Marco; Barends, Thomas R. M.; Heberle, Joachim; Kabsch, Wolfgang; Shoeman, Robert L.; Doak, R. Bruce; Schlichting, Ilme

    2017-01-01

    Serial (femtosecond) crystallography at synchrotron and X-ray free-electron laser (XFEL) sources distributes the absorbed radiation dose over all crystals used for data collection and therefore allows measurement of radiation damage prone systems, including the use of microcrystals for room-temperature measurements. Serial crystallography relies on fast and efficient exchange of crystals upon X-ray exposure, which can be achieved using a variety of methods, including various injection techniques. The latter vary significantly in their flow rates – gas dynamic virtual nozzle based injectors provide very thin fast-flowing jets, whereas high-viscosity extrusion injectors produce much thicker streams with flow rates two to three orders of magnitude lower. High-viscosity extrusion results in much lower sample consumption, as its sample delivery speed is commensurate both with typical XFEL repetition rates and with data acquisition rates at synchrotron sources. An obvious viscous injection medium is lipidic cubic phase (LCP) as it is used for in meso membrane protein crystallization. However, LCP has limited compatibility with many crystallization conditions. While a few other viscous media have been described in the literature, there is an ongoing need to identify additional injection media for crystal embedding. Critical attributes are reliable injection properties and a broad chemical compatibility to accommodate samples as heterogeneous and sensitive as protein crystals. Here, the use of two novel hydro­gels as viscous injection matrices is described, namely sodium carb­oxy­methyl cellulose and the thermo-reversible block polymer Pluronic F-127. Both are compatible with various crystallization conditions and yield acceptable X-ray background. The stability and velocity of the extruded stream were also analysed and the dependence of the stream velocity on the flow rate was measured. In contrast with previously characterized injection media, both new matrices

  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. Expression, purification, and crystallography of the conserved methionine-rich domain of human signal recognition particle 54 kDa protein.

    PubMed Central

    Gowda, K.; Clemons, W. M.; Zwieb, C.; Black, S. D.

    1999-01-01

    Protein SRP54 is an essential component of eukaryotic signal recognition particle (SRP). The methionine-rich M-domain (SRP54M or 54M) interacts with the SRP RNA and is also involved in the binding to signal peptides of secretory proteins during their targeting to cellular membranes. To gain insight into the molecular details of SRP-mediated protein targeting, we studied the human 54M polypeptide. The recombinant human protein was expressed successfully in Escherichia coli and was purified to homogeneity. Our studies determined the sites that were susceptible to limited proteolysis, with the goal to design smaller functional mutant derivatives that lacked nonessential amino acid residues from both termini. Of the four polypeptides produced by V8 protease or chymotrypsin, 54MM-2 was the shortest (120 residues; Mr = 13,584.8), but still contained the conserved amino acids suggested to associate with the signal peptide or the SRP RNA. 54MM-2 was cloned, expressed, purified to homogeneity, and was shown to bind human SRP RNA in the presence of protein SRP19, indicating that it was functional. Highly reproducible conditions for the crystallization of 54MM-2 were established. Examination of the crystals by X-ray diffraction showed an orthorhombic unit cell of dimensions a = 29.127 A, b = 63.693 A, and c = 129.601 A, in space group P2(1)2(1)2(1), with reflections extending to at least 2.0 A. PMID:10338025

  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. Fab Chaperone-Assisted RNA Crystallography (Fab CARC).

    PubMed

    Sherman, Eileen; Archer, Jennifer; Ye, Jing-Dong

    2016-01-01

    Recent discovery of structured RNAs such as ribozymes and riboswitches shows that there is still much to learn about the structure and function of RNAs. Knowledge learned can be employed in both biochemical research and clinical applications. X-ray crystallography gives unparalleled atomic-level structural detail from which functional inferences can be deduced. However, the difficulty in obtaining high-quality crystals and their phasing information make it a very challenging task. RNA crystallography is particularly arduous due to several factors such as RNA's paucity of surface chemical diversity, lability, repetitive anionic backbone, and flexibility, all of which are counterproductive to crystal packing. Here we describe Fab chaperone assisted RNA crystallography (CARC), a systematic technique to increase RNA crystallography success by facilitating crystal packing as well as expediting phase determination through molecular replacement of conserved Fab domains. Major steps described in this chapter include selection of a synthetic Fab library displayed on M13 phage against a structured RNA crystallization target, ELISA for initial choice of binding Fabs, Fab expression followed by protein A affinity then cation exchange chromatography purification, final choice of Fab by binding specificity and affinity as determined by a dot blot assay, and lastly gel filtration purification of a large quantity of chosen Fabs for crystallization.

  1. Benzimidazole inhibitors of the protein kinase CHK2: Clarification of the binding mode by flexible side chain docking and protein–ligand crystallography

    PubMed Central

    Matijssen, Cornelis; Silva-Santisteban, M. Cris; Westwood, Isaac M.; Siddique, Samerene; Choi, Vanessa; Sheldrake, Peter; van Montfort, Rob L.M.; Blagg, Julian

    2012-01-01

    Two closely related binding modes have previously been proposed for the ATP-competitive benzimidazole class of checkpoint kinase 2 (CHK2) inhibitors; however, neither binding mode is entirely consistent with the reported SAR. Unconstrained rigid docking of benzimidazole ligands into representative CHK2 protein crystal structures reveals an alternative binding mode involving a water-mediated interaction with the hinge region; docking which incorporates protein side chain flexibility for selected residues in the ATP binding site resulted in a refinement of the water-mediated hinge binding mode that is consistent with observed SAR. The flexible docking results are in good agreement with the crystal structures of four exemplar benzimidazole ligands bound to CHK2 which unambiguously confirmed the binding mode of these inhibitors, including the water-mediated interaction with the hinge region, and which is significantly different from binding modes previously postulated in the literature. PMID:23058106

  2. Does a fast nuclear magnetic resonance spectroscopy- and X-ray crystallography hybrid approach provide reliable structural information of ligand-protein complexes? A case study of metalloproteinases.

    PubMed

    Isaksson, Johan; Nyström, Susanne; Derbyshire, Dean; Wallberg, Hans; Agback, Tatiana; Kovacs, Helena; Bertini, Ivano; Giachetti, Andrea; Luchinat, Claudio

    2009-03-26

    A human matrix metalloproteinase (MMP) hydroxamic acid inhibitor (CGS27023A) was cross-docked into 15 MMP-12, MMP-13, MMP-9, and MMP-1 cocrystal structures. The aim was to validate a fast protocol for ligand binding conformation elucidation and to probe the feasibility of using inhibitor-protein NMR contacts to dock an inhibitor into related MMP crystal structures. Such an approach avoids full NMR structure elucidation, saving both spectrometer- and analysis time. We report here that for the studied MMPs, one can obtain docking results well within 1 A compared to the corresponding reference X-ray structure, using backbone amide contacts only. From the perspective of the pharmaceutical industry, these results are relevant for the binding studies of inhibitor series to a common target and have the potential advantage of obtaining information on protein-inhibitor complexes that are difficult to crystallize.

  3. Wrinkling crystallography on spherical surfaces.

    PubMed

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

    2015-01-06

    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.

  4. 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.

  5. Busting out of crystallography's Sisyphean prison

    NASA Astrophysics Data System (ADS)

    Cranswick, L. M. D.

    2008-01-01

    The history of crystallographic computing and use of crystallographic software is one which traces the escape from the drudgery of manual human calculations to a world where the user delegates most of the travail to electronic computers. This review traces the development of small-molecule single-crystal and powder diffraction hardware, starting with the use of Hollerith tabulators of the late 1930's through to today's use of high-performance personal computers. It also emphasizes that the main challenge for current and future crystallography computing is not that of hardware development, or even specific scientific challenges, but rather in maintaining a critical mass of human expertise with which these computational challenges can be undertaken.

  6. Neutron crystallography for the study of hydrogen bonds in macromolecules

    DOE PAGES

    Oksanen, Esko; Chen, Julian C.; Fisher, Zoe

    2017-04-07

    The hydrogen bond (H bond) is one of the most important interactions that form the foundation of secondary and tertiary protein structure. Beyond holding protein structures together, H bonds are also intimately involved in solvent coordination, ligand binding, and enzyme catalysis. The H bond by definition involves the light atom, H, and it is very difficult to study directly, especially with X-ray crystallographic techniques, due to the poor scattering power of H atoms. Neutron protein crystallography provides a powerful, complementary tool that can give unambiguous information to structural biologists on solvent organization and coordination, the electrostatics of ligand binding, themore » protonation states of amino acid side chains and catalytic water species. The method is complementary to X-ray crystallography and the dynamic data obtainable with NMR spectroscopy. Also, as it gives explicit H atom positions, it can be very valuable to computational chemistry where exact knowledge of protonation and solvent orientation can make a large difference in modeling. Finally, this article gives general information about neutron crystallography and shows specific examples of how the method has contributed to structural biology, structure-based drug design; and the understanding of fundamental questions of reaction mechanisms.« less

  7. Neutron Crystallography for the Study of Hydrogen Bonds in Macromolecules.

    PubMed

    Oksanen, Esko; Chen, Julian C-H; Fisher, Suzanne Zoë

    2017-04-07

    Abstract: The hydrogen bond (H bond) is one of the most important interactions that form the foundation of secondary and tertiary protein structure. Beyond holding protein structures together, H bonds are also intimately involved in solvent coordination, ligand binding, and enzyme catalysis. The H bond by definition involves the light atom, H, and it is very difficult to study directly, especially with X-ray crystallographic techniques, due to the poor scattering power of H atoms. Neutron protein crystallography provides a powerful, complementary tool that can give unambiguous information to structural biologists on solvent organization and coordination, the electrostatics of ligand binding, the protonation states of amino acid side chains and catalytic water species. The method is complementary to X-ray crystallography and the dynamic data obtainable with NMR spectroscopy. Also, as it gives explicit H atom positions, it can be very valuable to computational chemistry where exact knowledge of protonation and solvent orientation can make a large difference in modeling. This article gives general information about neutron crystallography and shows specific examples of how the method has contributed to structural biology, structure-based drug design; and the understanding of fundamental questions of reaction mechanisms.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. Crystallography, evolution, and the structure of viruses.

    PubMed

    Rossmann, Michael G

    2012-03-16

    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.

  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. 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.

  15. 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

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

    DOE PAGES

    Conrad, Chelsie E.; Basu, Shibom; James, Daniel; ...

    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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. Automated data collection for macromolecular crystallography.

    PubMed

    Winter, Graeme; McAuley, Katherine E

    2011-09-01

    An overview, together with some practical advice, is presented of the current status of the automation of macromolecular crystallography (MX) data collection, with a focus on MX beamlines at Diamond Light Source, UK.

  4. Statistical learning for chemical crystallography

    NASA Astrophysics Data System (ADS)

    Balachandran, Prasanna V.

    A novel computational approach is developed for the study of chemical crystallography in materials science using the tools of information theory and data science. By integrating the information derived from phase homologies, electronic structure calculations and known crystal structure data, a high-dimensional data space is created. From this data space, we seek to extract statistically robust and yet physically meaningful relationships linking structure with chemistry and property in the form of chemical design rules that identifies the exact role of key structure governing factors without any a priori assumptions. The powerful role of data dimensionality reduction, clustering analysis and data mining methods for both classification and prediction of structure-property relationships in materials is discussed. Using examples from two different crystal chemistry families, apatites and perovskite oxides, it is shown how high-dimensional data can be used to assess patterns of behavior as well as establish predictive Quantitative Structure-Activity Relationships (QSARs). A library of potentially new "virtual" stoichiometric and non-stoichiometric apatites and high TC piezoelectric perovskite materials chemistries were identified, thereby reinforcing the value of statistical learning methods in rational materials design.

  5. Structure validation in chemical crystallography

    PubMed Central

    Spek, Anthony L.

    2009-01-01

    Automated structure validation was introduced in chemical crystallography about 12 years ago as a tool to assist practitioners with the exponential growth in crystal structure analyses. Validation has since evolved into an easy-to-use checkCIF/PLATON web-based IUCr service. The result of a crystal structure determination has to be supplied as a CIF-formatted computer-readable file. The checking software tests the data in the CIF for completeness, quality and consistency. In addition, the reported structure is checked for incomplete analysis, errors in the analysis and relevant issues to be verified. A validation report is generated in the form of a list of ALERTS on the issues to be corrected, checked or commented on. Structure validation has largely eliminated obvious problems with structure reports published in IUCr journals, such as refinement in a space group of too low symmetry. This paper reports on the current status of structure validation and possible future extensions. PMID:19171970

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

    SciTech Connect

    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

    2013-05-01

    New software has been developed for automating the experimental and data-processing stages of fragment-based drug discovery at a macromolecular crystallography beamline. A new workflow-automation framework orchestrates beamline-control and data-analysis software while organizing results from multiple samples. 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

  7. 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. Copyright © 2011. Published by Elsevier Ltd.

  8. 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.

  9. Fragment screening using X-ray crystallography.

    PubMed

    Davies, Thomas G; Tickle, Ian J

    2012-01-01

    The fragment-based approach is now well established as an important component of modern drug discovery. A key part in establishing its position as a viable technique has been the development of a range of biophysical methodologies with sufficient sensitivity to detect the binding of very weakly binding molecules. X-ray crystallography was one of the first techniques demonstrated to be capable of detecting such weak binding, but historically its potential for screening was under-appreciated and impractical due to its relatively low throughput. In this chapter we discuss the various benefits associated with fragment-screening by X-ray crystallography, and describe the technical developments we have implemented to allow its routine use in drug discovery. We emphasize how this approach has allowed a much greater exploitation of crystallography than has traditionally been the case within the pharmaceutical industry, with the rapid and timely provision of structural information having maximum impact on project direction.

  10. MolProbity: all-atom structure validation for macromolecular crystallography.

    PubMed

    Chen, Vincent B; Arendall, W Bryan; Headd, Jeffrey J; Keedy, Daniel A; Immormino, Robert M; Kapral, Gary J; Murray, Laura W; Richardson, Jane S; Richardson, David C

    2010-01-01

    MolProbity is a structure-validation web service that provides broad-spectrum solidly based evaluation of model quality at both the global and local levels for both proteins and nucleic acids. It relies heavily on the power and sensitivity provided by optimized hydrogen placement and all-atom contact analysis, complemented by updated versions of covalent-geometry and torsion-angle criteria. Some of the local corrections can be performed automatically in MolProbity and all of the diagnostics are presented in chart and graphical forms that help guide manual rebuilding. X-ray crystallography provides a wealth of biologically important molecular data in the form of atomic three-dimensional structures of proteins, nucleic acids and increasingly large complexes in multiple forms and states. Advances in automation, in everything from crystallization to data collection to phasing to model building to refinement, have made solving a structure using crystallography easier than ever. However, despite these improvements, local errors that can affect biological interpretation are widespread at low resolution and even high-resolution structures nearly all contain at least a few local errors such as Ramachandran outliers, flipped branched protein side chains and incorrect sugar puckers. It is critical both for the crystallographer and for the end user that there are easy and reliable methods to diagnose and correct these sorts of errors in structures. MolProbity is the authors' contribution to helping solve this problem and this article reviews its general capabilities, reports on recent enhancements and usage, and presents evidence that the resulting improvements are now beneficially affecting the global database.

  11. MolProbity: all-atom structure validation for macromolecular crystallography

    SciTech Connect

    Chen, Vincent B.; Arendall, W. Bryan III; Headd, Jeffrey J.; Keedy, Daniel A.; Immormino, Robert M.; Kapral, Gary J.; Murray, Laura W.; Richardson, Jane S.; Richardson, David C.

    2010-01-01

    MolProbity structure validation will diagnose most local errors in macromolecular crystal structures and help to guide their correction. MolProbity is a structure-validation web service that provides broad-spectrum solidly based evaluation of model quality at both the global and local levels for both proteins and nucleic acids. It relies heavily on the power and sensitivity provided by optimized hydrogen placement and all-atom contact analysis, complemented by updated versions of covalent-geometry and torsion-angle criteria. Some of the local corrections can be performed automatically in MolProbity and all of the diagnostics are presented in chart and graphical forms that help guide manual rebuilding. X-ray crystallography provides a wealth of biologically important molecular data in the form of atomic three-dimensional structures of proteins, nucleic acids and increasingly large complexes in multiple forms and states. Advances in automation, in everything from crystallization to data collection to phasing to model building to refinement, have made solving a structure using crystallography easier than ever. However, despite these improvements, local errors that can affect biological interpretation are widespread at low resolution and even high-resolution structures nearly all contain at least a few local errors such as Ramachandran outliers, flipped branched protein side chains and incorrect sugar puckers. It is critical both for the crystallographer and for the end user that there are easy and reliable methods to diagnose and correct these sorts of errors in structures. MolProbity is the authors’ contribution to helping solve this problem and this article reviews its general capabilities, reports on recent enhancements and usage, and presents evidence that the resulting improvements are now beneficially affecting the global database.

  12. Upgrade of IMCA-CAT Bending Magnet Beamline 17-BM for Macromolecular Crystallography at the Advanced Photon Source

    SciTech Connect

    Koshelev, I.; Huang, R.; Graber, T.; Meron, M.; Muir, J.L.; Lavender, W.; Battaile, K.; Mulichak, A.M.; Keefe, L.J.

    2007-05-15

    Pharmaceutical research depends on macromolecular crystallography as a tool in drug design and development. To solve the de novo three-dimensional atomic structure of a protein, it is essential to know the phases of the X-rays scattered by a protein crystal. Experimental phases can be obtained from multiwavelength anomalous dispersion (MAD) experiments. Dedicated to macromolecular crystallography, the IMCA-CAT bending magnet beamline at sector 17 of the Advanced Photon Source (APS) was upgraded to provide the energy resolution required to successfully perform synchrotron radiation-based MAD phasing of protein crystal structures. A collimating mirror was inserted into the beam path upstream of a double-crystal monochromator, thus increasing the monochromatic beam throughput in a particular bandwidth without sacrificing the energy resolution of the system. The beam is focused horizontally by a sagittally bent crystal and vertically by a cylindrically bent mirror, delivering a beam at the sample of 130 {micro}m (vertically) x 250 {micro}m (horizontally) FWHM. As a result of the upgrade, the beamline now operates with an energy range of 7.5 x 17.5 keV, delivers 8 x 10{sup +11} photons/sec at 12.398 keV at the sample, and has an energy resolution of {delta}E/E = 1.45 x 10{sup -4} at 10 keV, which is suitable for MAD experiments.

  13. Upgrade of IMCA-CAT Bending Magnet Beamline 17-BM for Macromolecular Crystallography at the Advanced Photon Source

    SciTech Connect

    Koshelev, I.; Huang, R.; Muir, J. L.; Battaile, K.; Mulichak, A. M.; Keefe, L. J.; Graber, T.; Meron, M.; Lavender, W.

    2007-01-19

    Pharmaceutical research depends on macromolecular crystallography as a tool in drug design and development. To solve the de novo three-dimensional atomic structure of a protein, it is essential to know the phases of the X-rays scattered by a protein crystal. Experimental phases can be obtained from multiwavelength anomalous dispersion (MAD) experiments. Dedicated to macromolecular crystallography, the IMCA-CAT bending magnet beamline at sector 17 of the Advanced Photon Source (APS) was upgraded to provide the energy resolution required to successfully perform synchrotron radiation-based MAD phasing of protein crystal structures. A collimating mirror was inserted into the beam path upstream of a double-crystal monochromator, thus increasing the monochromatic beam throughput in a particular bandwidth without sacrificing the energy resolution of the system. The beam is focused horizontally by a sagittally bent crystal and vertically by a cylindrically bent mirror, delivering a beam at the sample of 130 {mu}m (vertically) x 250 {mu}m (horizontally) FWHM. As a result of the upgrade, the beamline now operates with an energy range of 7.5x17.5 keV, delivers 8 x 10+11 photons/sec at 12.398 keV at the sample, and has an energy resolution of {delta}E/E = 1.45 x 10-4 at 10 keV, which is suitable for MAD experiments.

  14. 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

  15. Polycapillary X-ray optics for macromolecular crystallography

    NASA Astrophysics Data System (ADS)

    Hofmann, Frank Alexander

    Macromolecules, such as proteins, nucleic acids, and carbohydrates can be found in every living cell. Nearly every chemical reaction in a cell requires the presence of enzymes, which belong to the class of proteins. In order to understand these processes, it is vital to know the three-dimensional structure of enzymes and other macromolecules. One way to find the structure is X-ray crystallography. Macromolecules are very large compared to most inorganic molecules and they have a very weak diffracting power. Structure determination with X-ray crystallography requires single crystals. Single crystals of macromolecules are difficult to grow except to very small size. Thus, intense X-ray beams are required. In order to generate intense X-ray beams, many crystallographers use synchrotrons. Intense beams can also be generated using X-ray optics. The work described includes a systematic study of the application of both collimating and slightly focusing polycapillary optics to X-ray crystallographic structure determination of egg-white lysozyme using a standard rotating anode source and a low power tabletop microfocusing source. The results summarize a series of measurements comparing duplicate data sets obtained from an individual crystal on a rotating anode source. Intensity and data quality are discussed for measurements with a pinhole collimator, a collimating polycapillary optic, and a focusing polycapillary optic. The collected data were analyzed using conventional analysis software.

  16. 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. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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

  3. In-vacuum long-wavelength macromolecular crystallography.

    PubMed

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

    2016-03-01

    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.

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

    PubMed

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

    2015-07-01

    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 pR(1)/pR(E46Q) and pR(2)/pR(CW) 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.

  5. 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

  6. Combining X-ray and neutron crystallography with spectroscopy

    PubMed Central

    Smith, Oliver

    2017-01-01

    X-ray protein crystallography has, through the determination of the three-dimensional structures of enzymes and their complexes, been essential to the understanding of biological chemistry. However, as X-rays are scattered by electrons, the technique has difficulty locating the presence and position of H atoms (and cannot locate H+ ions), knowledge of which is often crucially important for the understanding of enzyme mechanism. Furthermore, X-ray irradiation, through photoelectronic effects, will perturb the redox state in the crystal. By using single-crystal spectrophotometry, reactions taking place in the crystal can be monitored, either to trap intermediates or follow photoreduction during X-ray data collection. By using neutron crystallography, the positions of H atoms can be located, as it is the nuclei rather than the electrons that scatter neutrons, and the scattering length is not determined by the atomic number. Combining the two techniques allows much greater insight into both reaction mechanism and X-ray-induced photoreduction. PMID:28177310

  7. 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

  8. Non-contact luminescence lifetime cryothermometry for macromolecular crystallography

    PubMed Central

    Mykhaylyk, V. B.; Kraus, H.

    2017-01-01

    Temperature is a very important parameter when aiming to minimize radiation damage to biological samples during experiments that utilize intense ionizing radiation. A novel technique for remote, non-contact, in situ monitoring of the protein crystal temperature has been developed for the new I23 beamline at the Diamond Light Source, a facility dedicated to macromolecular crystallography (MX) with long-wavelength X-rays. The temperature is derived from the temperature-dependent decay time constant of luminescence from a minuscule scintillation sensor (<0.05 mm3) located in very close proximity to the sample under test. In this work the underlying principle of cryogenic luminescence lifetime thermometry is presented, the features of the detection method and the choice of temperature sensor are discussed, and it is demonstrated how the temperature monitoring system was integrated within the viewing system of the endstation used for the visualization of protein crystals. The thermometry system was characterized using a Bi4Ge3O12 crystal scintillator that exhibits good responsivity of the decay time constant as a function of temperature over a wide range (8–270 K). The scintillation sensor was calibrated and the uncertainty of the temperature measurements over the primary operation temperature range of the beamline (30–150 K) was assessed to be ±1.6 K. It has been shown that the temperature of the sample holder, measured using the luminescence sensor, agrees well with the expected value. The technique was applied to characterize the thermal performance of different sample mounts that have been used in MX experiments at the I23 beamline. The thickness of the mount is shown to have the greatest impact upon the temperature distribution across the sample mount. Altogether, these tests and findings demonstrate the usefulness of the thermometry system in highlighting the challenges that remain to be addressed for the in-vacuum MX experiment to become a reliable

  9. Non-contact luminescence lifetime cryothermometry for macromolecular crystallography.

    PubMed

    Mykhaylyk, V B; Wagner, A; Kraus, H

    2017-05-01

    Temperature is a very important parameter when aiming to minimize radiation damage to biological samples during experiments that utilize intense ionizing radiation. A novel technique for remote, non-contact, in situ monitoring of the protein crystal temperature has been developed for the new I23 beamline at the Diamond Light Source, a facility dedicated to macromolecular crystallography (MX) with long-wavelength X-rays. The temperature is derived from the temperature-dependent decay time constant of luminescence from a minuscule scintillation sensor (<0.05 mm(3)) located in very close proximity to the sample under test. In this work the underlying principle of cryogenic luminescence lifetime thermometry is presented, the features of the detection method and the choice of temperature sensor are discussed, and it is demonstrated how the temperature monitoring system was integrated within the viewing system of the endstation used for the visualization of protein crystals. The thermometry system was characterized using a Bi4Ge3O12 crystal scintillator that exhibits good responsivity of the decay time constant as a function of temperature over a wide range (8-270 K). The scintillation sensor was calibrated and the uncertainty of the temperature measurements over the primary operation temperature range of the beamline (30-150 K) was assessed to be ±1.6 K. It has been shown that the temperature of the sample holder, measured using the luminescence sensor, agrees well with the expected value. The technique was applied to characterize the thermal performance of different sample mounts that have been used in MX experiments at the I23 beamline. The thickness of the mount is shown to have the greatest impact upon the temperature distribution across the sample mount. Altogether, these tests and findings demonstrate the usefulness of the thermometry system in highlighting the challenges that remain to be addressed for the in-vacuum MX experiment to become a reliable and

  10. 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

  11. Facilities for small-molecule crystallography at synchrotron sources.

    PubMed

    Barnett, Sarah A; Nowell, Harriott; Warren, Mark R; Wilcox, Andrian; Allan, David R

    2016-01-01

    Although macromolecular crystallography is a widely supported technique at synchrotron radiation facilities throughout the world, there are, in comparison, only very few beamlines dedicated to small-molecule crystallography. This limited provision is despite the increasing demand for beamtime from the chemical crystallography community and the ever greater overlap between systems that can be classed as either small macromolecules or large small molecules. In this article, a very brief overview of beamlines that support small-molecule single-crystal diffraction techniques will be given along with a more detailed description of beamline I19, a dedicated facility for small-molecule crystallography at Diamond Light Source.

  12. 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

  13. 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.

  14. The Construction of Group Theory in Crystallography

    NASA Astrophysics Data System (ADS)

    Maitte, Bernard

    This article sets out to retrace the manner in which Group Theory evolved in crystallography. To engage in this study it is necessary to select, amongst all the approaches to crystals, those which, from the point of view of modern science, mark a step towards the establishment of our current understanding. In this way it favours our current perspective. To compensate this distortion, we recall the context in which each explanation that marks history appears. It so becomes clear that notions of triperiodic assemblages and the crystallographic laws of crystal systems and symmetrical classes do not derive from "natural" observations but were compiled and belong to precise theories.

  15. Does crystallography need a new name?

    DOE PAGES

    Argryriou, Dimitri

    2017-07-01

    The discovery of X-rays and their use in the observation of diffraction from crystals placed crystallography at the forefront of science at the beginning of the last century. The combination of this new tool, together with the emerging understanding of the symmetry of crystals, exposed the locations of atoms in matter and allowed us to start understanding macroscopic properties from an atomic perspective for the first time. These discoveries transformed physics and chemistry bringing to light new scientific fields such as materials science and structural biology.

  16. 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.

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

    DOE PAGES

    Mueller, C.; Marx, A.; Epp, S. W.; ...

    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

  18. EIGER detector: application in macromolecular crystallography

    PubMed Central

    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

    2016-01-01

    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

  19. 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.

  20. Cell-Dock: high-performance protein-protein docking.

    PubMed

    Pons, Carles; Jiménez-González, Daniel; González-Álvarez, Cecilia; Servat, Harald; Cabrera-Benítez, Daniel; Aguilar, Xavier; Fernández-Recio, Juan

    2012-09-15

    The application of docking to large-scale experiments or the explicit treatment of protein flexibility are part of the new challenges in structural bioinformatics that will require large computer resources and more efficient algorithms. Highly optimized fast Fourier transform (FFT) approaches are broadly used in docking programs but their optimal code implementation leaves hardware acceleration as the only option to significantly reduce the computational cost of these tools. In this work we present Cell-Dock, an FFT-based docking algorithm adapted to the Cell BE processor. We show that Cell-Dock runs faster than FTDock with maximum speedups of above 200×, while achieving results of similar quality. The source code is released under GNU General Public License version 2 and can be downloaded from http://mmb.pcb.ub.es/~cpons/Cell-Dock. djimenez@ac.upc.edu or juanf@bsc.es Supplementary data are available at Bioinformatics online.

  1. Outrunning free radicals in room-temperature macromolecular crystallography

    SciTech Connect

    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

    2012-06-15

    A systematic increase in lifetime is observed in room-temperature protein and virus crystals through the use of reduced exposure times and a fast detector. 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 A{sub 2A} 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.

  2. 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

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

    DOE PAGES

    Beyerlein, K. R.; Adriano, L.; Heymann, M.; ...

    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

  4. 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.

  5. 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.

  6. Towards an integrative structural biology approach: combining Cryo-TEM, X-ray crystallography, and NMR.

    PubMed

    Lengyel, Jeffrey; Hnath, Eric; Storms, Marc; Wohlfarth, Thomas

    2014-09-01

    Cryo-transmission electron microscopy (Cryo-TEM) and particularly single particle analysis is rapidly becoming the premier method for determining the three-dimensional structure of protein complexes, and viruses. In the last several years there have been dramatic technological improvements in Cryo-TEM, such as advancements in automation and use of improved detectors, as well as improved image processing techniques. While Cryo-TEM was once thought of as a low resolution structural technique, the method is currently capable of generating nearly atomic resolution structures on a routine basis. Moreover, the combination of Cryo-TEM and other methods such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics modeling are allowing researchers to address scientific questions previously thought intractable. Future technological developments are widely believed to further enhance the method and it is not inconceivable that Cryo-TEM could become as routine as X-ray crystallography for protein structure determination.

  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. 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.…

  9. Two-Dimensional Crystallization of the Ca(2+)-ATPase for Electron Crystallography.

    PubMed

    Glaves, John Paul; Primeau, Joseph O; Young, Howard S

    2016-01-01

    Electron crystallography of two-dimensional crystalline arrays is a powerful alternative for the structure determination of membrane proteins. The advantages offered by this technique include a native membrane environment and the ability to closely correlate function and dynamics with crystalline preparations and structural data. Herein, we provide a detailed protocol for the reconstitution and two-dimensional crystallization of the sarcoplasmic reticulum calcium pump (also known as Ca(2+)-ATPase or SERCA) and its regulatory subunits phospholamban and sarcolipin.

  10. Macromolecular Crystallography and Structural Biology Databases at NIST.

    PubMed

    Gilliland, G 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.

  11. 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

  12. Serial Femtosecond Crystallography Opens New Avenues for Structural Biology

    PubMed Central

    Coe, Jesse; Fromme, Petra

    2016-01-01

    Free electron lasers (FELs) provide X-ray pulses in the femtosecond time domain with up to 1012 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

  13. 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.

  14. Neutron crystallography: opportunities, challenges, and limitations.

    PubMed

    Blakeley, Matthew P; Langan, Paul; Niimura, Nobuo; Podjarny, Alberto

    2008-10-01

    Neutron crystallography has had an important, but relatively small role in structural biology over the years. In this review of recently determined neutron structures, a theme emerges of a field currently expanding beyond its traditional boundaries, to address larger and more complex problems, with smaller samples and shorter data collection times, and employing more sophisticated structure determination and refinement methods. The origin of this transformation can be found in a number of advances including first, the development of neutron image-plates and quasi-Laue methods at nuclear reactor neutron sources and the development of time-of-flight Laue methods and electronic detectors at spallation neutron sources; second, new facilities and methods for sample perdeuteration and crystallization; third, new approaches and computational tools for structure determination.

  15. CIF: the computer language of crystallography.

    PubMed

    Brown, I David; McMahon, Brian

    2002-06-01

    The Crystallographic Information File (CIF) was adopted in 1990 by the International Union of Crystallography as a file structure for the archiving and distribution of crystallographic information. The CIF standard is now well established and is in regular use for reporting crystal structure determinations to Acta Crystallographica and other journals. The structure of CIF is flexible and extensible and is compatible with other evolving standards. It is well suited to relational and object-oriented models, and is being adopted by the crystallographic databases. This paper reviews the development of CIF and describes its salient features. Future extension of the standard to include implementation of methods will allow CIF to exploit the potential of advanced information-handling software.

  16. 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.

  17. Radiation damage to nucleoprotein complexes in macromolecular crystallography

    DOE PAGES

    Bury, Charles; Garman, Elspeth F.; Ginn, Helen Mary; ...

    2015-01-30

    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. 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 themore » same controlled conditions. 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. We observed the protein at low doses and found that they were susceptible to radiation damage while the DNA was far more resistant, damage only being observed at significantly higher doses.« less

  18. 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

  19. Radiation damage to nucleoprotein complexes in macromolecular crystallography

    SciTech Connect

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

    2015-01-30

    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. 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. 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. We observed the protein at low doses and found that they were susceptible to radiation damage while the DNA was far more resistant, damage only being observed at significantly higher doses.

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

    DOE PAGES

    Jakobi, Arjen J.; Passon, Daniel M.; Knoops, Kevin; ...

    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.

  1. 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

  2. The collection of MicroED data for macromolecular crystallography

    PubMed Central

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

    2017-01-01

    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

  3. 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.

  4. Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae

    PubMed Central

    2012-01-01

    Background Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital. Results We developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies. This platform enables us to perform a screening cycle from construction to evaluation of variants within 6–7 days. We firstly confirmed the functional expression of 25 full-length class A GPCRs in this platform. Then, in order to improve the expression level and stability, we generated and evaluated the variants of the four GPCRs (hADRB2, hCHRM2, hHRH1 and hNTSR1). These stabilized receptor variants improved both functional activity and monodispersity. Finally, the expression level of the stabilized hHRH1 in Pichia pastoris was improved up to 65 pmol/mg from negligible expression of the functional full-length receptor in S. cerevisiae at first screening. The stabilized hHRH1 was able to be purified for use in crystallization trials. Conclusions We demonstrated that the S. cerevisiae system should serve as an easy-to-handle and rapid platform for the construction and evaluation of GPCR variants. This platform can be a powerful prescreening method to identify a suitable GPCR variant for crystallography. PMID:22694812

  5. Carbohydrate and carbohydrate + protein for cycling time-trial performance.

    PubMed

    Osterberg, Kristin L; Zachwieja, Jeffrey J; Smith, Johneric W

    2008-02-01

    Carbohydrate intake during endurance exercise delays the onset of fatigue and improves performance. Two recent cycling studies have reported increased time to exhaustion when protein is ingested together with carbohydrate. The purpose of the present study was to test the hypothesis that ingestion of a carbohydrate + protein beverage will lead to significant improvements in cycling time-trial performance relative to placebo and carbohydrate alone. Thirteen cyclists completed 120 min of constant-load ergometer cycling. Thereafter, participants performed a time-trial in which they completed a set amount of work (7 kJ kg(-1)) as quickly as possible. Participants completed four experimental trials, the first for familiarization and then three randomized, double-blind treatments consisting of a placebo, carbohydrate, and carbohydrate + protein. Participants received 250 ml of beverage every 15 min during the constant-load ride. Time-trial performance for carbohydrate (37.1 min, s = 3.8) was significantly (P < 0.05) faster than placebo (39.7 min, s = 4.6). Time-trial performance for carbohydrate + protein (38.8 min, s = 5.5) was not significantly different from either placebo or carbohydrate. Ingestion of a carbohydrate beverage during two hours of constant-load cycling significantly enhanced subsequent time-trial performance compared with placebo. The carbohydrate + protein beverage provided no additional performance benefit.

  6. 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

  7. Fourier-Space Crystallography as Group Cohomology

    NASA Astrophysics Data System (ADS)

    Rabson, David; Fisher, Benji

    2001-03-01

    David Mermin has argued persuasively that the theoretical significance of quasicrystals lies not so much in relaxing the requirement of periodicity as in replacing exact identity of a density function (e.g., electronic or mass) under symmetry operations with indistinguishability of correlation functions, as expressed in Fourier space.(N.D. Mermin, Phys. Stat. Sol. (a) 151), 275 (1995) and references. After reviewing the formalism of Fourier-space crystallography (phase functions and gauge transformations), we present a new formulation in the language of cohomology of groups. First we reexpress the classification of space groups in terms of a first cohomology group; we then show how recent work by König and Mermin(A. König and N.D. Mermin, Am. J. Phys. 68), 525 (2000). on band sticking in nonsymmorphic crystals derives naturally from a first homology group and discuss its connection to a second cohomology group. The new language lets us prove generally several theorems previously known only in special cases. Finally, we let the listener decide whether we're just ``speaking prose.''(N.D. Mermin, Rev. Mod. Phys. 64), 3 (1992).

  8. Phase Equilibria and Crystallography of Ceramic Oxides.

    PubMed

    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.

  9. 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.

  10. Identification of rogue datasets in serial crystallography.

    PubMed

    Assmann, Greta; Brehm, Wolfgang; Diederichs, Kay

    2016-06-01

    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.

  11. 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.

  12. Optimizing high performance computing workflow for protein functional annotation

    PubMed Central

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

    2014-01-01

    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

  13. 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.

  14. Present status of SPring-8 macromolecular crystallography beamlines

    SciTech Connect

    Kawano, Yoshiaki; Hirata, Kunio; Ueno, Go; Hikima, Takaaki; Murakami, Hironori; Maeda, Daisuke; Nisawa, Atsushi; Yamamoto, Masaki; Shimizu, Nobutaka; Baba, Seiki; Hasegawa, Kazuya; Makino, Masatomo; Mizuno, Nobuhiro; Hoshino, Takeshi; Ito, Ren; Wada, Izumi; Kumasaka, Takashi

    2010-06-23

    Seven beamlines are operated for macromolecular crystallography (MX) at SPring-8. The three undulator beamlines are developed for cutting edge target and four bending-magnet beamlines are developed for high throughput MX. The undulator beamline, BL41XU that provides the most brilliant beam, is dedicated to obtain high quality data even from small-size and weakly-diffracting crystals. The minimum beam size at sample position is achieved to 10 {mu}m diameter using a pin-hole collimator. Its photon flux at wavelength {lambda} = 1.0 A is 2.8x10{sup 11} photons/sec. This small beam coupled with irradiation point scanning method is quite useful to take diffraction dataset from small crystals by suppressing the radiation damage. These advanced technologies made a number of difficult protein structure analysis possible, (i.e. Sodium-potassium ATPase). The bending-magnet beamlines BL26B1/B2 and BL38B1 provide automatic data collection exploiting the high mobility of the beam. The beamline operation software 'BSS', sample auto-changer 'SPACE' and web-based data management software 'D-Cha' have made the automatic data collection possible. The 'Mail-in data collection system' that accepts distant users samples via courier service have made users possible to collect diffraction data without visiting SPring-8. The structural genomics research is promoted by these beamlines.

  15. Present status of SPring-8 macromolecular crystallography beamlines

    NASA Astrophysics Data System (ADS)

    Kawano, Yoshiaki; Shimizu, Nobutaka; Baba, Seiki; Hasegawa, Kazuya; Makino, Masatomo; Mizuno, Nobuhiro; Hoshino, Takeshi; Ito, Ren; Wada, Izumi; Hirata, Kunio; Ueno, Go; Hikima, Takaaki; Murakami, Hironori; Maeda, Daisuke; Nisawa, Atsushi; Kumasaka, Takashi; Yamamoto, Masaki

    2010-06-01

    Seven beamlines are operated for macromolecular crystallography (MX) at SPring-8. The three undulator beamlines are developed for cutting edge target and four bending-magnet beamlines are developed for high throughput MX. The undulator beamline, BL41XU that provides the most brilliant beam, is dedicated to obtain high quality data even from small-size and weakly-diffracting crystals. The minimum beam size at sample position is achieved to 10 μm diameter using a pin-hole collimator. Its photon flux at wavelength λ = 1.0 Å is 2.8×1011 photons/sec. This small beam coupled with irradiation point scanning method is quite useful to take diffraction dataset from small crystals by suppressing the radiation damage. These advanced technologies made a number of difficult protein structure analysis possible, (i.e. Sodium-potassium ATPase). The bending-magnet beamlines BL26B1/B2 and BL38B1 provide automatic data collection exploiting the high mobility of the beam. The beamline operation software "BSS," sample auto-changer "SPACE" and web-based data management software "D-Cha" have made the automatic data collection possible. The "Mail-in data collection system" that accepts distant users samples via courier service have made users possible to collect diffraction data without visiting SPring-8. The structural genomics research is promoted by these beamlines.

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

    DOE PAGES

    Zhang, Haitao; Unal, Hamiyet; Gati, Cornelius; ...

    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

  17. The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography

    DOE PAGES

    Edlund, Petra; Takala, Heikki; Claesson, Elin; ...

    2016-10-19

    Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 Å resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 Å resolution derived frommore » conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. As a result, the study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.« less

  18. The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography

    SciTech Connect

    Edlund, Petra; Takala, Heikki; Claesson, Elin; Henry, Léocadie; Dods, Robert; Lehtivuori, Heli; Panman, Matthijs; Pande, Kanupriya; White, Thomas; Nakane, Takanori; Berntsson, Oskar; Gustavsson, Emil; Båth, Petra; Modi, Vaibhav; Roy-Chowdhury, Shatabdi; Zook, James; Berntsen, Peter; Pandey, Suraj; Poudyal, Ishwor; Tenboer, Jason; Kupitz, Christopher; Barty, Anton; Fromme, Petra; Koralek, Jake D.; Tanaka, Tomoyuki; Spence, John; Liang, Mengning; Hunter, Mark S.; Boutet, Sebastien; Nango, Eriko; Moffat, Keith; Groenhof, Gerrit; Ihalainen, Janne; Stojković, Emina A.; Schmidt, Marius; Westenhoff, Sebastian

    2016-10-19

    Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 Å resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 Å resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. As a result, the study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.

  19. The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography

    PubMed Central

    Edlund, Petra; Takala, Heikki; Claesson, Elin; Henry, Léocadie; Dods, Robert; Lehtivuori, Heli; Panman, Matthijs; Pande, Kanupriya; White, Thomas; Nakane, Takanori; Berntsson, Oskar; Gustavsson, Emil; Båth, Petra; Modi, Vaibhav; Roy-Chowdhury, Shatabdi; Zook, James; Berntsen, Peter; Pandey, Suraj; Poudyal, Ishwor; Tenboer, Jason; Kupitz, Christopher; Barty, Anton; Fromme, Petra; Koralek, Jake D.; Tanaka, Tomoyuki; Spence, John; Liang, Mengning; Hunter, Mark S.; Boutet, Sebastien; Nango, Eriko; Moffat, Keith; Groenhof, Gerrit; Ihalainen, Janne; Stojković, Emina A.; Schmidt, Marius; Westenhoff, Sebastian

    2016-01-01

    Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 Å resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 Å resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX. PMID:27756898

  20. The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography

    NASA Astrophysics Data System (ADS)

    Edlund, Petra; Takala, Heikki; Claesson, Elin; Henry, Léocadie; Dods, Robert; Lehtivuori, Heli; Panman, Matthijs; Pande, Kanupriya; White, Thomas; Nakane, Takanori; Berntsson, Oskar; Gustavsson, Emil; Båth, Petra; Modi, Vaibhav; Roy-Chowdhury, Shatabdi; Zook, James; Berntsen, Peter; Pandey, Suraj; Poudyal, Ishwor; Tenboer, Jason; Kupitz, Christopher; Barty, Anton; Fromme, Petra; Koralek, Jake D.; Tanaka, Tomoyuki; Spence, John; Liang, Mengning; Hunter, Mark S.; Boutet, Sebastien; Nango, Eriko; Moffat, Keith; Groenhof, Gerrit; Ihalainen, Janne; Stojković, Emina A.; Schmidt, Marius; Westenhoff, Sebastian

    2016-10-01

    Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 Å resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 Å resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.

  1. 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.

  2. Zeptosens' protein microarrays: a novel high performance microarray platform for low abundance protein analysis.

    PubMed

    Pawlak, Michael; Schick, Eginhard; Bopp, Martin A; Schneider, Michael J; Oroszlan, Peter; Ehrat, Markus

    2002-04-01

    Protein microarrays are considered an enabling technology, which will significantly expand the scope of current protein expression and protein interaction analysis. Current technologies, such as two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry, allowing the identification of biologically relevant proteins, have a high resolving power, but also considerable limitations. As was demonstrated by Gygi et al. (Proc. Nat. Acad. Sci. USA 2000,97, 9390-9395), most spots in 2-DE, observed from whole cell extracts, are from high abundance proteins, whereas low abundance proteins, such as signaling molecules or kinases, are only poorly represented. Protein microarrays are expected to significantly expedite the discovery of new markers and targets of pharmaceutical interest, and to have the potential for high-throughput applications. Key factors to reach this goal are: high read-out sensitivity for quantification also of low abundance proteins, functional analysis of proteins, short assay analysis times, ease of handling and the ability to integrate a variety of different targets and new assays. Zeptosens has developed a revolutionary new bioanalytical system based on the proprietary planar waveguide technology which allows us to perform multiplexed, quantitative biomolecular interaction analysis with highest sensitivity in a microarray format upon utilizing the specific advantages of the evanescent field fluorescence detection. The analytical system, comprising an ultrasensitive fluorescence reader and microarray chips with integrated microfluidics, enables the user to generate a multitude of high fidelity data in applications such as protein expression profiling or investigating protein-protein interactions. In this paper, the important factors for developing high performance protein microarray systems, especially for targeting low abundant messengers of relevant biological information, will be discussed and the performance of the system will

  3. Performance benchmarking of four cell-free protein expression systems.

    PubMed

    Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

    2016-02-01

    Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins.

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

    PubMed

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

    2011-05-01

    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.

  5. Grain-boundary plane crystallography and energy in austenitic steel

    SciTech Connect

    Caul, M.; Randle, V.; Fiedler, J.

    1996-10-01

    The presence of grain boundaries in polycrystalline materials affects the materials properties and performance. Recently it has been realized that boundaries can be manipulated to give better properties, and the design and control of grain boundaries is now an area of strong research interest in the search for high performance engineering materials. Grain boundaries can be classified using the Coincident Site Lattice Model (CSL), which defines the periodicity, i.e., the degree of fit between the two lattices which constitute the boundary. Using this model it is possible to divide boundaries into categories: low angle (up to 15{degree} misorientation), CSL and random i.e., high angle non-CSL. Some CSL boundaries have been shown to have special properties: an example from recent research in the same program as that currently reported has shown that twin boundaries ({Sigma} = 3 in CSL notation) in High Nitrogen Austenitic Stainless Steels do not favor the formation of Cr{sub 2}N precipitates. The research presented here examines grain boundary inclinations of surface grains in austenitic steel specimens which have been isothermally aged at higher 700 C or 800 C. Grain boundary plane crystallography has also been obtained for the 800 C aged sample.

  6. Substitutional impact on biological activity of new water soluble Ni(II) complexes: Preparation, spectral characterization, X-ray crystallography, DNA/protein binding, antibacterial activity and in vitro cytotoxicity.

    PubMed

    Umadevi, C; Kalaivani, P; Puschmann, H; Murugan, S; Mohan, P S; Prabhakaran, R

    2017-02-01

    A series of new water soluble nickel(II) complexes containing triphenylphosphine and 4-methoxysalicylaldehyde-4(N)-substituted thiosemicarbazones were synthesized and characterized. Crystallographic investigations confirmed the structure of the complexes (1-4) having the general structure [Ni(4-Msal-Rtsc)(PPh3)] (Where R=H (1); CH3 (2); C2H5 (3); C6H5 (4)) which showed that thiosemicarbazone ligands coordinated to nickel(II) ion as ONS tridentate bibasic donor. DNA/BSA protein binding ability of the ligands and their new complexes were studied by taking calf-thymus DNA (CT-DNA) and Bovine serum albumin (BSA) through absorption and emission titrations. Ethidium bromide (EB) displacement study showed the intercalative binding trend of the complexes to DNA. From the albumin binding studies, the mechanism of quenching was found as static and the alterations in the secondary structure of BSA by the compounds were confirmed with synchronous spectral studies. The binding affinity of the complexes to CT-DNA and BSA has the order of [Ni(4-Msal-etsc)(PPh3)] (3) >[Ni(4-Msal-mtsc)(PPh3)] (2) >[Ni(4-Msal-tsc)(PPh3)] (1) >[Ni(4-Msal-ptsc)(PPh3)] (4). In vitro cytotoxicity of the complexes was tested on human lung cancer cells (A549), human cervical cancer cells (HeLa), human liver carcinoma cells (Hep G2). All the complexes exhibited significant activity against three cancer cells. Among them, complex 4 exhibited almost 2.5 fold activity than cisplatin in A549 and HepG2 cell lines. In HeLa cell line, the complexes exhibited significant activity which is less than cisplatin. While comparing the activity of the complexes in A549 and HepG2 cell lines it falls in the order 4>1>2>3>cisplatin. The results obtained from DNA, protein binding and cytotoxicity studies, it is concluded that the cytotoxicity of the complexes as determined by MTT assay were not unduly influenced by the complexes having different binding efficiency with DNA and protein. The complexes exhibited good spectrum

  7. Toward chaperone-assisted crystallography: Protein engineering enhancement of crystal packing and X-ray phasing capabilities of a camelid single-domain antibody (V[subscript H]H) scaffold

    SciTech Connect

    Tereshko, Valentina; Uysal, Serdar; Koide, Akiko; Margalef, Katrina; Koide, Shohei; Kossiakoff, Anthony A.

    2008-07-28

    A crystallization chaperone is an auxiliary protein that binds to a target of interest, enhances and modulates crystal packing, and provides high-quality phasing information. We critically evaluated the effectiveness of a camelid single-domain antibody (V{sub H}H) as a crystallization chaperone. By using a yeast surface display system for V{sub H}H, we successfully introduced additional Met residues in the core of the V{sub H}H scaffold. We identified a set of SeMet-labeled V{sub H}H variants that collectively produced six new crystal forms as the complex with the model antigen, RNase A. The crystals exhibited monoclinic, orthorhombic, triclinic, and tetragonal symmetry and have one or two complexes in the asymmetric unit, some of which diffracted to an atomic resolution. The phasing power of the Met-enriched V{sub H}H chaperone allowed for auto-building the entire complex using single-anomalous dispersion technique (SAD) without the need for introducing SeMet into the target protein. We show that phases produced by combining SAD and VHH model-based phases are accurate enough to easily solve structures of the size reported here, eliminating the need to collect multiple wavelength multiple-anomalous dispersion (MAD) data. Together with the presence of high-throughput selection systems (e.g., phage display libraries) for V{sub H}H, the enhanced V{sub H}H domain described here will be an excellent scaffold for producing effective crystallization chaperones.

  8. A new high-performance thin layer chromatography-based assay of detergents and surfactants commonly used in membrane protein studies.

    PubMed

    Barret, Laurie-Anne; Polidori, Ange; Bonneté, Françoise; Bernard-Savary, Pierre; Jungas, Colette

    2013-03-15

    The hydrophobic nature of membrane proteins (MPs) necessitates the use of detergents for their extraction, solubilization and purification. Because the concentration of amphiphiles is crucial in the crystallization process, detergent quantification is essential to routine analysis. Here we describe a quantitative high-performance thin-layer chromatography (HPTLC) method we developed for the detection of small quantities of detergent bound to solubilized MPs. After optimization of aqueous deposit conditions, we show that most detergents widely used in membrane protein crystallography display distinctive mobilities in a mixture of dichloromethane, methanol and acetic acid 32:7.6:0.4 (v/v/v). Migration and derivatization conditions were optimized with n-dodecyl-β-D-maltoside (DDM), the most popular detergent for membrane protein crystallization. A linear calibration curve very well fits our data from 0.1 to 1.6 μg of DDM in water with a limit of detection of 0.05 μg. This limit of detection is the best achieved to date for a routine detergent assay, being not modified by the addition of NaCl, commonly used in protein buffers. With these chromatographic conditions, no prior treatment is required to assess the quantities of detergent bound to purified MPs, thus enabling the quantification of close structure detergents via a single procedure. This HPTLC method, which is fast and requires low sample volume, is fully suitable for routine measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Exploring ribozyme conformational changes with X-ray crystallography.

    PubMed

    Spitale, Robert C; Wedekind, Joseph E

    2009-10-01

    Relating three-dimensional fold to function is a central challenge in RNA structural biology. Toward this goal, X-ray crystallography has long been considered the "gold standard" for structure determinations at atomic resolution, although NMR spectroscopy has become a powerhouse in this arena as well. In the area of dynamics, NMR remains the dominant technique to probe the magnitude and timescales of molecular motion. Although the latter area remains largely unassailable by conventional crystallographic methods, inroads have been made on proteins using Laue radiation on timescales of ms to ns. Proposed 'fourth generation' radiation sources, such as free-electron X-ray lasers, promise ps- to fs-timescale resolution, and credible evidence is emerging that supports the feasibility of single molecule imaging. At present however, the preponderance of RNA structural information has been derived from timescale and motion insensitive crystallographic techniques. Importantly, developments in computing, automation and high-flux synchrotron sources have propelled the rapidity of 'conventional' RNA crystal structure determinations to timeframes of hours once a suitable set of phases is obtained. With a sufficient number of crystal structures, it is possible to create a structural ensemble that can provide insight into global and local molecular motion characteristics that are relevant to biological function. Here we describe techniques to explore conformational changes in the hairpin ribozyme, a representative non-protein-coding RNA catalyst. The approaches discussed include: (i) construct choice and design using prior knowledge to improve X-ray diffraction; (ii) recognition of long-range conformational changes and (iii) use of single-base or single-atom changes to create ensembles. The methods are broadly applicable to other RNA systems.

  10. Probing the Complex Architecture of Multimodular Carbohydrate-Active Enzymes Using a Combination of Small Angle X-Ray Scattering and X-Ray Crystallography.

    PubMed

    Czjzek, Mirjam; Ficko-Blean, Elizabeth

    2017-01-01

    The various modules in multimodular carbohydrate-active enzymes (CAZymes) may function in catalysis, carbohydrate binding, protein-protein interactions or as linkers. Here, we describe how combining the biophysical techniques of Small Angle X-ray Scattering (SAXS) and macromolecular X-ray crystallography (XRC) provides a powerful tool for examination into questions related to overall structural organization of ultra multimodular CAZymes.

  11. Macromolecular crystallography with a large format CMOS detector

    SciTech Connect

    Nix, Jay C.

    2016-07-27

    Recent advances in CMOS technology have allowed the production of large surface area detectors suitable for macromolecular crystallography experiments [1]. The Molecular Biology Consortium (MBC) Beamline 4.2.2 at the Advanced Light Source in Berkeley, CA, has installed a 2952 x 2820 mm RDI CMOS-8M detector with funds from NIH grant S10OD012073. The detector has a 20nsec dead pixel time and performs well with shutterless data collection strategies. The sensor obtains sharp point response and minimal optical distortion by use of a thin fiber-optic plate between the phosphor and sensor module. Shutterless data collections produce high-quality redundant datasets that can be obtained in minutes. The fine-sliced data are suitable for processing in standard crystallographic software packages (XDS, HKL2000, D*TREK, MOSFLM). Faster collection times relative to the previous CCD detector have resulted in a record number of datasets collected in a calendar year and de novo phasing experiments have resulted in publications in both Science and Nature [2,3]. The faster collections are due to a combination of the decreased overhead requirements of shutterless collections combined with exposure times that have decreased by over a factor of 2 for images with comparable signal to noise of the NOIR-1 detector. The overall increased productivity has allowed the development of new beamline capabilities and data collection strategies.

  12. 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

  13. Average protein density is a molecular-weight-dependent function.

    PubMed

    Fischer, Hannes; Polikarpov, Igor; Craievich, Aldo F

    2004-10-01

    The mass density of proteins is a relevant basic biophysical quantity. It is also a useful input parameter, for example, for three-dimensional structure determination by protein crystallography and studies of protein oligomers in solution by analytic ultracentrifugation. We have performed a critical analysis of published, theoretical, and experimental investigations about this issue and concluded that the average density of proteins is not a constant as often assumed. For proteins with a molecular weight below 20 kDa, the average density exhibits a positive deviation that increases for decreasing molecular weight. A simple molecular-weight-depending function is proposed that provides a more accurate estimate of the average protein density.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. Status of the crystallography beamlines at PETRA III

    NASA Astrophysics Data System (ADS)

    Burkhardt, Anja; Pakendorf, Tim; Reime, Bernd; Meyer, Jan; Fischer, Pontus; Stübe, Nicolas; Panneerselvam, Saravanan; Lorbeer, Olga; Stachnik, Karolina; Warmer, Martin; Rödig, Philip; Göries, Dennis; Meents, Alke

    2016-03-01

    Since 2013, three beamlines for macromolecular crystallography are available to users at the third-generation synchrotron PETRA III in Hamburg: P11, P13 and P14, the latter two operated by EMBL. Beamline P11 is operated by DESY and is equipped with a Pilatus 6M detector. Together with the photon flux of 2× 10^{13} ph/s provided by the very brilliant X-ray source of PETRA III, a full data set can be typically collected in less than 2min. P11 provides state-of-the-art microfocusing capabilities with beam sizes down to 1× 1 μ m2, which makes the beamline ideally suited for investigation of microcrystals and serial crystallography experiments. An automatic sample changer allows fast sample exchange in less than 20s, which enables high-throughput crystallography and fast crystal screening. For sample preparation, an S2 biosafety laboratory is available in close proximity to the beamline.

  19. Temperature-dependent macromolecular X-ray crystallography

    SciTech Connect

    Weik, Martin Colletier, Jacques-Philippe

    2010-04-01

    The dynamical behaviour of crystalline macromolecules and their surrounding solvent as a function of cryo-temperature is reviewed. X-ray crystallography provides structural details of biological macromolecules. Whereas routine data are collected close to 100 K in order to mitigate radiation damage, more exotic temperature-controlled experiments in a broader temperature range from 15 K to room temperature can provide both dynamical and structural insights. Here, the dynamical behaviour of crystalline macromolecules and their surrounding solvent as a function of cryo-temperature is reviewed. Experimental strategies of kinetic crystallography are discussed that have allowed the generation and trapping of macromolecular intermediate states by combining reaction initiation in the crystalline state with appropriate temperature profiles. A particular focus is on recruiting X-ray-induced changes for reaction initiation, thus unveiling useful aspects of radiation damage, which otherwise has to be minimized in macromolecular crystallography.

  20. 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

  1. Dietary protein level and performance of growing Baladi kids.

    PubMed

    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.

  2. Dietary protein effects on hen performance and nitrogen excretion.

    PubMed

    Latshaw, J D; Zhao, L

    2011-01-01

    Because dietary nitrogen intake affects nitrogen content in manure, diet management has been recognized as a means to reduce ammonia emissions from poultry operations. The objectives of the present research were 1) to determine the extent to which the CP content of laying diets can be reduced, based on performance criteria, and 2) to determine how ash:nitrogen ratios of manure, eggs, and hens are affected by dietary protein changes. Egg-type hens were fed equal daily amounts of essential amino acids in diets that provided 13, 15, or 17 g of protein/d. Each diet was fed to 20 hens, with 2 hens/cage. The planned digestible lysine intake was 0.71 g/hen per day. Ratios of other digestible amino acids to lysine were methionine plus cysteine, 0.83; threonine, 0.68; and isoleucine, 0.94. The experiment began when hens were 29 wk old and continued until they were 57 wk old. Egg production averaged approximately 90%, and daily protein intake caused no effects on egg production or grams of egg per hen per day. Feed intake was higher for hens fed 13 g of protein than for hens in the other 2 treatments (P < 0.01). Average feed intake for the experiment was approximately 95 g/d. Composition of the eggs was not affected by protein intake. Average values were DM, 30.5%; ash, 31.0% of DM; and nitrogen, 6.31% of DM. The average manure DM production was 25.9 g/hen per day, with an ash content of 25.5% of DM. Manure nitrogen content ranged from 3.98% of DM for hens fed 13 g of protein to 5.68% for those fed 17 g of protein (P < 0.01). A method is outlined that uses the analysis of fresh manure and manure leaving the poultry operation to estimate the loss of nitrogen as ammonia.

  3. Mixing injector enables time-resolved crystallography with high hit rate at X-ray free electron lasers.

    PubMed

    Calvey, George D; Katz, Andrea M; Schaffer, Chris B; Pollack, Lois

    2016-09-01

    Knowledge of protein structure provides essential insight into function, enhancing our understanding of diseases and enabling new treatment development. X-ray crystallography has been used to solve the structures of more than 100 000 proteins; however, the vast majority represent long-lived states that do not capture the functional motions of these molecular machines. Reactions triggered by the addition of a ligand can be the most challenging to detect with crystallography because of the difficulty of synchronizing reactions to create detectable quantities of transient states. The development of X-ray free electron lasers (XFELs) and serial femtosecond crystallography (SFX) enables new approaches for solving protein structures following the rapid diffusion of ligands into micron sized protein crystals. Conformational changes occurring on millisecond timescales can be detected and time-resolved. Here, we describe a new XFEL injector which incorporates a microfluidic mixer to rapidly combine reactant and sample milliseconds before the sample reaches the X-ray beam. The mixing injector consists of bonded, concentric glass capillaries. The fabrication process, employing custom laser cut centering spacers and UV curable epoxy, ensures precise alignment of capillaries for repeatable, centered sample flow and dependable mixing. Crystal delivery capillaries are 50 or 75 μm in diameter and can contain an integrated filter depending on the demands of the experiment. Reaction times can be varied from submillisecond to several hundred milliseconds. The injector features rapid and uniform mixing, low sample dilution, and high hit rates. It is fully compatible with existing SFX beamlines.

  4. Macromolecular crystallography beamline X25 at the NSLS.

    PubMed

    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

    2014-05-01

    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.

  5. Crystallography Open Databases and Preservation: a World-wide Initiative

    NASA Astrophysics Data System (ADS)

    Chateigner, Daniel

    In 2003, an international team of crystallographers proposed the Crystallography Open Database (COD), a fully-free collection of crystal structure data, in the aim of ensuring their preservation. With nearly 250000 entries, this database represents a large open set of data for crystallographers, academics and industrials, located at five different places world-wide, and included in Thomson-Reuters’ ISI. As a large step towards data preservation, raw data can now be uploaded along with «digested» structure files, and COD can be questioned by most of the crystallography-linked industrial software. The COD initiative work deserves several other open developments.

  6. 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

  7. Cryo-crystallography: diffraction at low temperature and more.

    PubMed

    Macchi, Piero

    2012-01-01

    This chapter comments on the motivations and the methods of crystallographic studies at low temperature. Cry-crystallography is a brunch of Crystallography, a science that is too often confused with a technique. On the other hand, the scientific background to study crystal phases at low temperature is here provided, together with a survey of many possible techniques that provide complementary or supplementary information. Several applications are discussed, in particular in relation with highly accurate studies like electron density determination or phase transition mechanisms.

  8. 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.

  9. 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

  10. The kinetic dose limit in room-temperature time-resolved macromolecular crystallography

    SciTech Connect

    Schmidt, M.; Srajer, V.; Purwar, N.; Tripathi, S.

    2012-05-24

    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.

  11. Nanosecond pump-probe device for time-resolved serial femtosecond crystallography developed at SACLA.

    PubMed

    Kubo, Minoru; Nango, Eriko; Tono, Kensuke; Kimura, Tetsunari; Owada, Shigeki; Song, Changyong; Mafuné, Fumitaka; Miyajima, Ken; Takeda, Yoshihiro; Kohno, Jun Ya; Miyauchi, Naoya; Nakane, Takanori; Tanaka, Tomoyuki; Nomura, Takashi; Davidsson, Jan; Tanaka, Rie; Murata, Michio; Kameshima, Takashi; Hatsui, Takaki; Joti, Yasumasa; Neutze, Richard; Yabashi, Makina; Iwata, So

    2017-09-01

    X-ray free-electron lasers (XFELs) have opened new opportunities for time-resolved X-ray crystallography. Here a nanosecond optical-pump XFEL-probe device developed for time-resolved serial femtosecond crystallography (TR-SFX) studies of photo-induced reactions in proteins at the SPring-8 Angstrom Compact free-electron LAser (SACLA) is reported. The optical-fiber-based system is a good choice for a quick setup in a limited beam time and allows pump illumination from two directions to achieve high excitation efficiency of protein microcrystals. Two types of injectors are used: one for extruding highly viscous samples such as lipidic cubic phase (LCP) and the other for pulsed liquid droplets. Under standard sample flow conditions from the viscous-sample injector, delay times from nanoseconds to tens of milliseconds are accessible, typical time scales required to study large protein conformational changes. A first demonstration of a TR-SFX experiment on bacteriorhodopsin in bicelle using a setup with a droplet-type injector is also presented.

  12. Radiation damage within nucleoprotein complexes studied by macromolecular X-ray crystallography

    NASA Astrophysics Data System (ADS)

    Bury, Charles S.; Carmichael, Ian; McGeehan, John E.; Garman, Elspeth F.

    2016-11-01

    In X-ray crystallography, for the determination of the 3-D structure of macromolecules, radiation damage is still an inherent problem at modern third generation synchrotron sources, even when utilising cryo-crystallographic techniques (sample held at 100 K). At doses of just several MGy, at which a typical diffraction dataset is collected, site-specific radiation-induced chemical changes are known to manifest within protein crystals, and a wide body of literature is now devoted to understanding the mechanisms behind such damage. Far less is known regarding radiation-induced damage to crystalline nucleic acids and the wider class of nucleoprotein complexes during macromolecular X-ray crystallography (MX) data collection. As the MX structural biology community now strives to solve structures for increasingly larger and complex macromolecular assemblies, it essential to understand how such structures are affected by the X-ray radiation used to solve them. The purpose of this review is to summarise advances in the field of specific damage to nucleoprotein complexes and to present case studies of MX damage investigations on both protein-DNA (C.Esp1396I) and protein-RNA (TRAP) complexes. To motivate further investigations into MX damage mechanisms within nucleoprotein complexes, current and emerging protocols for investigating specific damage within Fobs(n)-Fobs(1) electron density difference maps are discussed.

  13. 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.

  14. 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

  15. 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

  16. 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)

  17. 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)

  18. 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.…

  19. 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…

  20. 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.…

  1. 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…

  2. Facilitating structure determination: workshop on robotics andautomation in macromolecular crystallography

    SciTech Connect

    Ralston, Corie; Cork, C.W.; McDermott, G.; Earnest, T.N.

    2006-03-28

    As part of the annual Advanced Light Source (ALS) andStanford Synchrotron Radiation Laboratory (SSRL) Users' Meeting inOctober of this year, the macromolecular crystallography staff at bothsynchrotrons held a joint hands-on workshop to address automation issuesin crystal mounting and data collection at the beamline. This paperdescribes the ALS portion of the workshop, while the accompanying paperreviews the SSRL workshop.

  3. 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.…

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

    SciTech Connect

    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.

  5. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7.

    PubMed

    Langer, Gerrit; 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 nonexpert 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.

  6. Dose, exposure time, and resolution in Serial X-ray Crystallography

    SciTech Connect

    Starodub, D; Rez, P; Hembree, G; Howells, M; Shapiro, D; Chapman, H N; Fromme, P; Schmidt, K; Weierstall, U; Doak, R B; Spence, J C

    2007-03-22

    Using detailed simulation and analytical models, the exposure time is estimated for serial crystallography, where hydrated laser-aligned proteins are sprayed across a continuous synchrotron beam. The resolution of X-ray diffraction microscopy is limited by the maximum dose that can be delivered prior to sample damage. In the proposed Serial Crystallography method, the damage problem is addressed by distributing the total dose over many identical hydrated macromolecules running continuously in a single-file train across a continuous X-ray beam, and resolution is then limited only by the available fluxes of molecules and X-rays. Orientation of the diffracting molecules is achieved by laser alignment. We evaluate the incident X-ray fluence (energy/area) required to obtain a given resolution from (1) an analytical model, giving the count rate at the maximum scattering angle for a model protein, (2) explicit simulation of diffraction patterns for a GroEL-GroES protein complex, and (3) the frequency cut off of the transfer function following iterative solution of the phase problem, and reconstruction of a density map in the projection approximation. These calculations include counting shot noise and multiple starts of the phasing algorithm. The results indicate the number of proteins needed within the beam at any instant for a given resolution and X-ray flux. We confirm an inverse fourth power dependence of exposure time on resolution, with important implications for all coherent X-ray imaging. We find that multiple single-file protein beams will be needed for sub-nanometer resolution on current third generation synchrotrons, but not on fourth generation designs, where reconstruction of secondary protein structure at a resolution of 7 {angstrom} should be possible with short (below 100 s) exposures.

  7. Combining Electron Crystallography and X-ray Crystallography to Study the MlotiK1 Cyclic Nucleotide-Regulated Potassium Channel

    SciTech Connect

    Clayton, G.; Aller, S; Wang, J; Unger, V; Morais-Cabral, J

    2009-01-01

    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 {angstrom} 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.

  8. Is too 'creative' language acceptable in crystallography?

    PubMed

    Wlodawer, Alexander; Lubkowski, Jacek; Minor, Wladek; Jaskolski, Mariusz

    2010-09-01

    While figures of speech are often useful and even educational, flashy titles combined with hyperbolae and imprecise language can mislead or deceive non-specialist readers and should therefore be avoided. The possibility of such confusion exists when poorly defined terms like ;structure quality' or ;super-resolution' are used to describe a protein structure.

  9. 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

  10. High-performance cation-exchange chromatofocusing of proteins.

    PubMed

    Kang, Xuezhen; Frey, Douglas D

    2003-03-28

    Chromatofocusing using high-performance cation-exchange column packings, as opposed to the more commonly used anion-exchange column packings, is investigated with regard to the performance achieved and the range of applications possible. Linear or convex gradients in the range from pH 2.6 to 9 were formed using a variety of commercially available column packings that provide a buffering capacity in different pH ranges, and either polyampholytes or simple mixtures having a small number (three or fewer) of buffering species as the elution buffer. The resolutions achieved using cation-exchange or anion-exchange chromatofocusing were in general comparable, although for certain pairs of proteins better resolution could be achieved using one type of packing as compared to the other, evidently due to the way electrostatic charges are distributed on the protein surface. Several chromatofocusing methods were investigated that take advantage of the acid-base properties of commercially available cation-exchange column packings. These include the use of gradients with a composite shape, the use of very low pH ranges, and the use of elution buffers containing a single buffering species. The advantages of chromatofocusing over ion-exchange chromatography using a salt gradient at constant pH were illustrated by employing the former method and a cation-exchange column packing to separate beta-lactoglobulins A and B, which is a separation reported to be impossible using the latter method and a cation-exchange column packing. Trends in the apparent isoelectric points determined using cation- and anion-exchange chromatofocusing were interpreted using applicable theories. Results of this study indicate that cation-exchange chromatofocusing is a useful technique which is complementary to anion-exchange chromatofocusing and isoelectric focusing for separating proteins at both the analytical and preparative scales.

  11. 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

  12. Protein crystallization under microgravity conditions. Analysis of the results of Russian experiments performed on the International Space Station in 2005-2015

    NASA Astrophysics Data System (ADS)

    Boyko, K. M.; Timofeev, V. I.; Samygina, V. R.; Kuranova, I. P.; Popov, V. O.; Koval'chuk, M. V.

    2016-09-01

    Conditions of mass transport to growing crystals have a considerable effect on the crystal size and quality. The reduction of convective transport can help improve the quality of crystals for X-ray crystallography. One approach to minimizing convective transport is crystallization in a microgravity environment, in particular, in space. The data obtained by our research team in protein crystallization experiments on the International Space Station are surveyed and analyzed.

  13. PILATUS: a two-dimensional X-ray detector for macromolecular crystallography

    NASA Astrophysics Data System (ADS)

    Eikenberry, E. F.; Brönnimann, Ch.; Hülsen, G.; Toyokawa, H.; Horisberger, R.; Schmitt, B.; Schulze-Briese, C.; Tomizaki, T.

    2003-03-01

    A large quantum-limited area X-ray detector for protein crystallography is under development at the Swiss Light Source. The final detector will be 2k×2k pixels covering 40×40 cm2. A three-module prototype with 1120×157 pixels covering an active area of 24.3×3.4 cm2 has been tested. X-rays above 6 keV with peak count rates exceeding 5×10 5 X-ray/pixel/s could be detected in single photon counting mode. Statistics of module production and results of threshold trimming are presented. To demonstrate the potential of this new detector, protein crystal data were collected at beamline 6S of the SLS.

  14. Adenovirus Structure as Revealed by X-Ray Crystallography, Electron Microscopy, and Difference Imaging

    NASA Astrophysics Data System (ADS)

    Stewart, Phoebe L.; Burnett, Roger M.

    1993-03-01

    The three-dimensional structure of human type 2 adenovirus was studied by combining X-ray crystallography and electron microscopy in a novel way. The 2.9 Å crystal structure of the major capsid protein, hexon, was positioned into a three-dimensional image reconstruction of the intact virus that was derived from cryo-electron micrographs. A three-dimensional difference map was generated by subtracting 240 copies of the crystallographic hexon from the density of the intact virus. This map revealed several minor structural proteins acting as “cement” to stabilize the assembly. The current state of structural knowledge concerning the location of the polypeptide components and the viral DNA is presented.

  15. Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography

    PubMed Central

    Foadi, James; Aller, Pierre; Alguel, Yilmaz; Cameron, Alex; Axford, Danny; Owen, Robin L.; Armour, Wes; Waterman, David G.; Iwata, So; Evans, Gwyndaf

    2013-01-01

    The availability of intense microbeam macromolecular crystallography beamlines at third-generation synchrotron sources has enabled data collection and structure solution from microcrystals of <10 µm in size. The increased likelihood of severe radiation damage where microcrystals or particularly sensitive crystals are used forces crystallographers to acquire large numbers of data sets from many crystals of the same protein structure. The associated analysis and merging of multi-crystal data is currently a manual and time-consuming step. Here, a computer program, BLEND, that has been written to assist with and automate many of the steps in this process is described. It is demonstrated how BLEND has successfully been used in the solution of a novel membrane protein. PMID:23897484

  16. Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography.

    PubMed

    Foadi, James; Aller, Pierre; Alguel, Yilmaz; Cameron, Alex; Axford, Danny; Owen, Robin L; Armour, Wes; Waterman, David G; Iwata, So; Evans, Gwyndaf

    2013-08-01

    The availability of intense microbeam macromolecular crystallography beamlines at third-generation synchrotron sources has enabled data collection and structure solution from microcrystals of <10 µm in size. The increased likelihood of severe radiation damage where microcrystals or particularly sensitive crystals are used forces crystallographers to acquire large numbers of data sets from many crystals of the same protein structure. The associated analysis and merging of multi-crystal data is currently a manual and time-consuming step. Here, a computer program, BLEND, that has been written to assist with and automate many of the steps in this process is described. It is demonstrated how BLEND has successfully been used in the solution of a novel membrane protein.

  17. Semiautomated model building for RNA crystallography using a directed rotameric approach.

    PubMed

    Keating, Kevin S; Pyle, Anna Marie

    2010-05-04

    Structured RNA molecules play essential roles in a variety of cellular processes; however, crystallographic studies of such RNA molecules present a large number of challenges. One notable complication arises from the low resolutions typical of RNA crystallography, which results in electron density maps that are imprecise and difficult to interpret. This problem is exacerbated by the lack of computational tools for RNA modeling, as many of the techniques commonly used in protein crystallography have no equivalents for RNA structure. This leads to difficulty and errors in the model building process, particularly in modeling of the RNA backbone, which is highly error prone due to the large number of variable torsion angles per nucleotide. To address this, we have developed a method for accurately building the RNA backbone into maps of intermediate or low resolution. This method is semiautomated, as it requires a crystallographer to first locate phosphates and bases in the electron density map. After this initial trace of the molecule, however, an accurate backbone structure can be built without further user intervention. To accomplish this, backbone conformers are first predicted using RNA pseudotorsions and the base-phosphate perpendicular distance. Detailed backbone coordinates are then calculated to conform both to the predicted conformer and to the previously located phosphates and bases. This technique is shown to produce accurate backbone structure even when starting from imprecise phosphate and base coordinates. A program implementing this methodology is currently available, and a plugin for the Coot model building program is under development.

  18. PRIGo: a new multi-axis goniometer for macromolecular crystallography.

    PubMed

    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

    2015-07-01

    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.

  19. 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

  20. 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.

  1. 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.

  2. Towards a compact and precise sample holder for macromolecular crystallography.

    PubMed

    Papp, Gergely; Rossi, Christopher; Janocha, Robert; Sorez, Clement; Lopez-Marrero, Marcos; Astruc, Anthony; McCarthy, Andrew; Belrhali, Hassan; Bowler, Matthew W; Cipriani, Florent

    2017-10-01

    Most of the sample holders currently used in macromolecular crystallography offer limited storage density and poor initial crystal-positioning precision upon mounting on a goniometer. This has now become a limiting factor at high-throughput beamlines, where data collection can be performed in a matter of seconds. Furthermore, this lack of precision limits the potential benefits emerging from automated harvesting systems that could provide crystal-position information which would further enhance alignment at beamlines. This situation provided the motivation for the development of a compact and precise sample holder with corresponding pucks, handling tools and robotic transfer protocols. The development process included four main phases: design, prototype manufacture, testing with a robotic sample changer and validation under real conditions on a beamline. Two sample-holder designs are proposed: NewPin and miniSPINE. They share the same robot gripper and allow the storage of 36 sample holders in uni-puck footprint-style pucks, which represents 252 samples in a dry-shipping dewar commonly used in the field. The pucks are identified with human- and machine-readable codes, as well as with radio-frequency identification (RFID) tags. NewPin offers a crystal-repositioning precision of up to 10 µm but requires a specific goniometer socket. The storage density could reach 64 samples using a special puck designed for fully robotic handling. miniSPINE is less precise but uses a goniometer mount compatible with the current SPINE standard. miniSPINE is proposed for the first implementation of the new standard, since it is easier to integrate at beamlines. An upgraded version of the SPINE sample holder with a corresponding puck named SPINEplus is also proposed in order to offer a homogenous and interoperable system. The project involved several European synchrotrons and industrial companies in the fields of consumables and sample-changer robotics. Manual handling of mini

  3. 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.

  4. Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography

    DOE PAGES

    Keedy, Daniel A.; Kenner, Lillian R.; Warkentin, Matthew; ...

    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. Altogether, 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. 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.

  7. 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.

  8. Predicting the protein targets for athletic performance-enhancing substances.

    PubMed

    Mavridis, Lazaros; Mitchell, John Bo

    2013-06-25

    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. 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. 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 it to identify the protein targets

  9. NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity

    PubMed Central

    2016-01-01

    Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5′-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallography—the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry—to interrogate a carbanionic/quinonoid intermediate analogue in the β-subunit active site of the PLP-requiring enzyme tryptophan synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established. Most significantly, we find that a deprotonated pyridine nitrogen on PLP precludes formation of a true quinonoid species and that there is an equilibrium between the phenolic and protonated Schiff base tautomeric forms of this intermediate. Natural bond orbital analysis indicates that the latter builds up negative charge at the substrate Cα and positive charge at C4′ of the cofactor, consistent with its role as the catalytic tautomer. These findings support the hypothesis that the specificity for β-elimination/replacement versus transamination is dictated in part by the protonation states of ionizable groups on PLP and the reacting substrates and underscore the essential role that NMR crystallography can play in characterizing both chemical structure and dynamics within functioning enzyme active sites. PMID:27779384

  10. Mixing injector enables time-resolved crystallography with high hit rate at X-ray free electron lasers

    PubMed Central

    Calvey, George D.; Katz, Andrea M.; Schaffer, Chris B.; Pollack, Lois

    2016-01-01

    Knowledge of protein structure provides essential insight into function, enhancing our understanding of diseases and enabling new treatment development. X-ray crystallography has been used to solve the structures of more than 100 000 proteins; however, the vast majority represent long-lived states that do not capture the functional motions of these molecular machines. Reactions triggered by the addition of a ligand can be the most challenging to detect with crystallography because of the difficulty of synchronizing reactions to create detectable quantities of transient states. The development of X-ray free electron lasers (XFELs) and serial femtosecond crystallography (SFX) enables new approaches for solving protein structures following the rapid diffusion of ligands into micron sized protein crystals. Conformational changes occurring on millisecond timescales can be detected and time-resolved. Here, we describe a new XFEL injector which incorporates a microfluidic mixer to rapidly combine reactant and sample milliseconds before the sample reaches the X-ray beam. The mixing injector consists of bonded, concentric glass capillaries. The fabrication process, employing custom laser cut centering spacers and UV curable epoxy, ensures precise alignment of capillaries for repeatable, centered sample flow and dependable mixing. Crystal delivery capillaries are 50 or 75 μm in diameter and can contain an integrated filter depending on the demands of the experiment. Reaction times can be varied from submillisecond to several hundred milliseconds. The injector features rapid and uniform mixing, low sample dilution, and high hit rates. It is fully compatible with existing SFX beamlines. PMID:27679802

  11. Double-flow focused liquid injector for efficient serial femtosecond crystallography

    PubMed Central

    Oberthuer, Dominik; Knoška, Juraj; Wiedorn, Max O.; Beyerlein, Kenneth R.; Bushnell, David A.; Kovaleva, Elena G.; Heymann, Michael; Gumprecht, Lars; Kirian, Richard A.; Barty, Anton; Mariani, Valerio; Tolstikova, Aleksandra; Adriano, Luigi; Awel, Salah; Barthelmess, Miriam; Dörner, Katerina; Xavier, P. Lourdu; Yefanov, Oleksandr; James, Daniel R.; Nelson, Garrett; Wang, Dingjie; Calvey, George; Chen, Yujie; Schmidt, Andrea; Szczepek, Michael; Frielingsdorf, Stefan; Lenz, Oliver; Snell, Edward; Robinson, Philip J.; Šarler, Božidar; Belšak, Grega; Maček, Marjan; Wilde, Fabian; Aquila, Andrew; Boutet, Sébastien; Liang, Mengning; Hunter, Mark S.; Scheerer, Patrick; Lipscomb, John D.; Weierstall, Uwe; Kornberg, Roger D.; Spence, John C. H.; Pollack, Lois; Chapman, Henry N.; Bajt, Saša

    2017-01-01

    Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Moreover, the double flow-focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improved operation and characteristics of these devices. PMID:28300169

  12. Double-flow focused liquid injector for efficient serial femtosecond crystallography

    DOE PAGES

    Oberthuer, Dominik; Knoška, Juraj; Wiedorn, Max O.; ...

    2017-03-16

    Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Furthermore, the double flow-focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improvedmore » operation and characteristics of these devices.« less

  13. Double-flow focused liquid injector for efficient serial femtosecond crystallography

    NASA Astrophysics Data System (ADS)

    Oberthuer, Dominik; Knoška, Juraj; Wiedorn, Max O.; Beyerlein, Kenneth R.; Bushnell, David A.; Kovaleva, Elena G.; Heymann, Michael; Gumprecht, Lars; Kirian, Richard A.; Barty, Anton; Mariani, Valerio; Tolstikova, Aleksandra; Adriano, Luigi; Awel, Salah; Barthelmess, Miriam; Dörner, Katerina; Xavier, P. Lourdu; Yefanov, Oleksandr; James, Daniel R.; Nelson, Garrett; Wang, Dingjie; Calvey, George; Chen, Yujie; Schmidt, Andrea; Szczepek, Michael; Frielingsdorf, Stefan; Lenz, Oliver; Snell, Edward; Robinson, Philip J.; Šarler, Božidar; Belšak, Grega; Maček, Marjan; Wilde, Fabian; Aquila, Andrew; Boutet, Sébastien; Liang, Mengning; Hunter, Mark S.; Scheerer, Patrick; Lipscomb, John D.; Weierstall, Uwe; Kornberg, Roger D.; Spence, John C. H.; Pollack, Lois; Chapman, Henry N.; Bajt, Saša

    2017-03-01

    Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Moreover, the double flow-focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improved operation and characteristics of these devices.

  14. Free RCK arrangement in Kch, a putative escherichia coli potassium channel, as suggested by electron crystallography.

    PubMed

    Kuang, Qie; Purhonen, Pasi; Jegerschöld, Caroline; Koeck, Philip J B; Hebert, Hans

    2015-01-06

    The ligand-gated potassium channels are stimulated by various kinds of messengers. Previous studies showed that ligand-gated potassium channels containing RCK domains (the regulator of the conductance of potassium ion) form a dimer of tetramer structure through the RCK octameric gating ring in the presence of detergent. Here, we have analyzed the structure of Kch, a channel of this type from Escherichia coli, in a lipid environment using electron crystallography. By combining information from the 3D map of the transmembrane part of the protein and docking of an atomic model of a potassium channel, we conclude that the RCK domains face the solution and that an RCK octameric gating ring arrangement does not form under our crystallization condition. Our findings may be applied to other potassium channels that have an RCK gating ring arrangement.

  15. Improving protein array performance: focus on washing and storage conditions.

    PubMed

    Nath, Nidhi; Hurst, Robin; Hook, Brad; Meisenheimer, Poncho; Zhao, Kate Q; Nassif, Nadine; Bulleit, Robert F; Storts, Douglas R

    2008-10-01

    For protein microarrays, maintaining protein stability during the slide processing steps of washing, drying, and storage is of major concern. Although several studies have focused on the stability of immobilized antibodies in antibody microarrays, studies on protein-protein interaction arrays and enzyme arrays are lacking. In this paper we used five bait-prey protein interaction pairs and three enzymes to optimize the washing, drying, and storage conditions for protein arrays. The protein arrays for the study were fabricated by combining HaloTag technology and cell-free protein expression. The HaloTag technology, in combination with cell-free expression, allowed rapid expression and immobilization of fusion proteins on hydrogel-coated glass slides directly from cell extracts without any prior purification. Experimental results indicate enzyme captured on glass slides undergoes significant loss of activity when washed and spin-dried using only phosphate buffer, as is typically done with antibody arrays. The impact of washing and spin-drying in phosphate buffer on protein-protein interaction arrays was minimal. However, addition of 5% glycerol to the wash buffer helps retain enzyme activity during washing and drying. We observed significant loss of enzyme activity when slides were stored dry at 4 degrees C, however immobilized enzymes remained active for 30 days when stored at -20 degrees C in 50% glycerol. We also found that cell-free extract containing HaloTag-fused enzymes could undergo multiple freeze/thaw cycles without any adverse impact on enzyme activity. The findings indicate that for large ongoing studies, proteins of interest expressed in cell-free extract can be stored at -70 degrees C and repeatedly used to print small batches of protein array slides to be used over a few weeks.

  16. Development of an online UV-visible microspectrophotometer for a macromolecular crystallography beamline.

    PubMed

    Shimizu, Nobutaka; Shimizu, Tetsuya; Baba, Seiki; Hasegawa, Kazuya; Yamamoto, Masaki; Kumasaka, Takashi

    2013-11-01

    Measurement of the UV-visible absorption spectrum is a convenient technique for detecting chemical changes of proteins, and it is therefore useful to combine spectroscopy and diffraction studies. An online microspectrophotometer for the UV-visible region was developed and installed on the macromolecular crystallography beamline, BL38B1, at SPring-8. This spectrophotometer is equipped with a difference dispersive double monochromator, a mercury-xenon lamp as the light source, and a photomultiplier as the detector. The optical path is mostly constructed using mirrors, in order to obtain high brightness in the UV region, and the confocal optics are assembled using a cross-slit diaphragm like an iris to eliminate stray light. This system can measure optical densities up to a maximum of 4.0. To study the effect of radiation damage, preliminary measurements of glucose isomerase and thaumatin crystals were conducted in the UV region. Spectral changes dependent on X-ray dose were observed at around 280 nm, suggesting that structural changes involving Trp or Tyr residues occurred in the protein crystal. In the case of the thaumatin crystal, a broad peak around 400 nm was also generated after X-ray irradiation, suggesting the cleavage of a disulfide bond. Dose-dependent spectral changes were also observed in cryo-solutions alone, and these changes differed with the composition of the cryo-solution. These responses in the UV region are informative regarding the state of the sample; consequently, this device might be useful for X-ray crystallography.

  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. 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

  19. Crystallography of lath martensite and stabilization of retained austenite

    SciTech Connect

    Sarikaya. M.

    1982-10-01

    TEM was used to study the morphology and crystallography of lath martensite in low and medium carbon steels in the as-quenched and 200/sup 0/C tempered conditions. The steels have microduplex structures of dislocated lath martensite and continuous thin films of retained austenite at the lath interfaces. Stacks of laths form the packets which are derived from different (111) variants of the same austenite grain. The residual parent austenite enables microdiffraction experiments with small electron beam spot sizes for the orientation relationships (OR) between austenite and martensite. All three most commonly observed ORs, namely Kurdjumov-Sachs, Nishiyama-Wassermann, and Greninger-Troiano, operate within the same sample.

  20. Low-dose fixed-target serial synchrotron crystallography

    PubMed Central

    Owen, Robin L.; Axford, Danny; Sherrell, Darren A.; Kuo, Anling; Ernst, Oliver P.; Schulz, Eike C.; Miller, R. J. Dwayne; Mueller-Werkmeister, Henrike M.

    2017-01-01

    The development of serial crystallography has been driven by the sample requirements imposed by X-ray free-electron lasers. Serial techniques are now being exploited at synchrotrons. Using a fixed-target approach to high-throughput serial sampling, it is demonstrated that high-quality data can be collected from myoglobin crystals, allowing room-temperature, low-dose structure determination. The combination of fixed-target arrays and a fast, accurate translation system allows high-throughput serial data collection at high hit rates and with low sample consumption. PMID:28375148

  1. Low-dose fixed-target serial synchrotron crystallography.

    PubMed

    Owen, Robin L; Axford, Danny; Sherrell, Darren A; Kuo, Anling; Ernst, Oliver P; Schulz, Eike C; Miller, R J Dwayne; Mueller-Werkmeister, Henrike M

    2017-04-01

    The development of serial crystallography has been driven by the sample requirements imposed by X-ray free-electron lasers. Serial techniques are now being exploited at synchrotrons. Using a fixed-target approach to high-throughput serial sampling, it is demonstrated that high-quality data can be collected from myoglobin crystals, allowing room-temperature, low-dose structure determination. The combination of fixed-target arrays and a fast, accurate translation system allows high-throughput serial data collection at high hit rates and with low sample consumption.

  2. 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.

  3. 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.

  4. JBluIce-EPICS control system for macromolecular crystallography.

    PubMed

    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

    2011-03-01

    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.

  5. 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.

  6. Review: Serial Femtosecond Crystallography: A Revolution in Structural Biology

    PubMed Central

    Martin-Garcia, Jose M.; Conrad, Chelsie E.; Coe, Jesse; Roy-Chowdhury, Shatabdi; Fromme, Petra

    2016-01-01

    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

  7. Canadian macromolecular crystallography facility: a suite of fully automated beamlines.

    PubMed

    Grochulski, Pawel; Fodje, Michel; Labiuk, Shaunivan; Gorin, James; Janzen, Kathryn; Berg, Russ

    2012-06-01

    The Canadian light source is a 2.9 GeV national synchrotron radiation facility located on the University of Saskatchewan campus in Saskatoon. The small-gap in-vacuum undulator illuminated beamline, 08ID-1, together with the bending magnet beamline, 08B1-1, constitute the Canadian Macromolecular Crystallography Facility (CMCF). The CMCF provides service to more than 50 Principal Investigators in Canada and the United States. Up to 25% of the beam time is devoted to commercial users and the general user program is guaranteed up to 55% of the useful beam time through a peer-review process. CMCF staff provides "Mail-In" crystallography service to users with the highest scored proposals. Both beamlines are equipped with very robust end-stations including on-axis visualization systems, Rayonix 300 CCD series detectors and Stanford-type robotic sample auto-mounters. MxDC, an in-house developed beamline control system, is integrated with a data processing module, AutoProcess, allowing full automation of data collection and data processing with minimal human intervention. Sample management and remote monitoring of experiments is enabled through interaction with a Laboratory Information Management System developed at the facility.

  8. Structural study of piracetam polymorphs and cocrystals: crystallography redetermination and quantum mechanics calculations.

    PubMed

    Tilborg, Anaëlle; Jacquemin, Denis; Norberg, Bernadette; Perpète, Eric; Michaux, Catherine; Wouters, Johan

    2011-12-01

    Pharmaceutical compounds are mostly developed as solid dosage forms containing a single-crystal form. It means that the selection of a particular crystal state for a given molecule is an important step for further clinical outlooks. In this context, piracetam, a pharmaceutical molecule known since the sixties for its nootropic properties, is considered in the present work. This molecule is analyzed using several experimental and theoretical approaches. First, the conformational space of the molecule has been systematically explored by performing a quantum mechanics scan of the two most relevant dihedral angles of the lateral chain. The predicted stable conformations have been compared to all the reported experimental geometries retrieved from the Cambridge Structural Database (CSD) covering polymorphs and cocrystals structures. In parallel, different batches of powders have been recrystallized. Under specific conditions, single crystals of polymorph (III) of piracetam have been obtained, an outcome confirmed by crystallographic analysis. © 2011 International Union of Crystallography. Printed in Singapore – all rights reserved.

  9. Cryo-electron microscopy and X-ray crystallography: complementary approaches to structural biology and drug discovery.

    PubMed

    Vénien-Bryan, Catherine; Li, Zhuolun; Vuillard, Laurent; Boutin, Jean Albert

    2017-04-01

    The invention of the electron microscope has greatly enhanced the view scientists have of small structural details. Since its implementation, this technology has undergone considerable evolution and the resolution that can be obtained for biological objects has been extended. In addition, the latest generation of cryo-electron microscopes equipped with direct electron detectors and software for the automated collection of images, in combination with the use of advanced image-analysis methods, has dramatically improved the performance of this technique in terms of resolution. While calculating a sub-10 Å resolution structure was an accomplishment less than a decade ago, it is now common to generate structures at sub-5 Å resolution and even better. It is becoming possible to relatively quickly obtain high-resolution structures of biological molecules, in particular large ones (>500 kDa) which, in some cases, have resisted more conventional methods such as X-ray crystallography or nuclear magnetic resonance (NMR). Such newly resolved structures may, for the first time, shed light on the precise mechanisms that are essential for cellular physiological processes. The ability to attain atomic resolution may support the development of new drugs that target these proteins, allowing medicinal chemists to understand the intimacy of the relationship between their molecules and targets. In addition, recent developments in cryo-electron microscopy combined with image analysis can provide unique information on the conformational variability of macromolecular complexes. Conformational flexibility of macromolecular complexes can be investigated using cryo-electron microscopy and multiconformation reconstruction methods. However, the biochemical quality of the sample remains the major bottleneck to routine cryo-electron microscopy-based determination of structures at very high resolution.

  10. Thermotolerance and Human Performance: Role of Heat Shock Proteins

    DTIC Science & Technology

    2009-10-01

    and provide a mechanistic basis for the requirement of HSF1 in the regulation of life span and establish a role for SIRT1 in protein homeostasis and...Stress-Inducible Regulation of Heat Shock Factor 1 by the Deacetylase SIRT1 . Science. 2009; 323: 1063-1066. Wilson N, Gisolfi C, Farber J, Hinrichs D

  11. 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)

  12. 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)

  13. 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

  14. Serum protein determination by high-performance gel-permeation chromatography.

    PubMed

    Hayakawa, K; Masuko, M; Mineta, M; Yoshikawa, K; Yamauchi, K; Hirano, M; Katsumata, N; Tanaka, T

    1997-08-15

    A general high-performance gel-permeation chromatography (HPGPC) method was developed to determine protein in human serum with improved sensitivity and speed. The optimum UV wavelength for protein detection was found to be 210 nm, by comparing the protein values obtained by varying the UV wavelength of the HPLC detection system with the protein values obtained from spectrophotometric protein assays, i.e., the bicinchoninic acid (BCA) method and the biuret method. The analysis time was less than 1 min. Since this HPGPC serum protein assay method is simple and rapid, it is expected to be particularly well adapted for use in clinical laboratories.

  15. A history of experimental phasing in macromolecular crystallography.

    PubMed

    Isaacs, Neil

    2016-03-01

    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.

  16. Crystallography of Co/Pt multilayers and nanostructures

    SciTech Connect

    Zhang, Bing; Krishnan, K.M.; Farrow, R.F.C.

    1992-04-01

    Atomically engineered nanostructures and multilayers of Co/Pt exhibit strong perpendicular anisotropy. This unique property, that determines their potential as a magneto-optic recording media, is dependent on a variety of microstructural parameters that include the overall crystallography, thickness of the layers, orientation, defect formation, interface reactions etc. A series of Co/Pt multilayer samples with different thickness of the Co layer were studied by electron diffraction. It has been determined that the Co layers persists in the fcc structure up to a thickness of 50 {Angstrom}. As the thickness is varied fmm 3{Angstrom} to 50{Angstrom} in the multilayers, the Co film gradually relaxed to its bulk lattice parameter. (111) twinning and lattice strain at the interfaces between Pt and Co layers are also observed. The symmetry forbidden reflections observed at 1/3 (224) positions in (111) zone diffraction patterns of the multilayer are due to (111) twinning and compositional modulations along the multilayer growth direction.

  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. Lens-Coupled CCD Detector for X-ray Crystallography

    PubMed Central

    Madden, Timothy J.; McGuigan, William; Molitsky, Michael J.; Naday, Istvan; McArthur, Alan; Westbrook, Edwin M.

    2007-01-01

    An x-ray crystallography detector (Blue-1) has been built based upon a Fairchild 486 back-illuminated CCD and a custom lens system designed by Optics One Inc. The advantages of our Blue-1 lens system over more conventional fiber-optic tapers are: lower noise and higher efficiency; improved point spread function; negligible spatial distortion; and lack of “chicken-wire” patterns. Also, the engineering is simpler because the CCD is not bonded to the fiber-optic taper. A unique mechanical design has been employed to accurately focus the image on the CCD. The detector software is based on MATLAB and takes advantage of its powerful imaging and signal processing libraries. The CCD timing can be updated on the fly by using a “CCD controller language” to specify timing. PMID:18185837

  19. Crystallography and elasticity of individual GaN nanotubes

    NASA Astrophysics Data System (ADS)

    Liu, Baodan; Bando, Yoshio; Wang, Mingsheng; Tang, Chengchun; Mitome, Masanori; Golberg, Dmitri

    2009-05-01

    High-purity, crystalline [001]-oriented GaN nanotubes with outer diameters of 200 nm or more, rough surfaces and irregular internal channels were synthesized under epitaxial growth on [001]-oriented sapphire substrates. Elastic property measurements on free-standing individual GaN nanotubes, using the in situ transmission electron microscopy (TEM) electromechanical resonance technique, pointed at an average Young's modulus E of 37 GPa and minimum quality factor of 320. These numbers are notably lower than those for previously reported GaN nanowires. The crystallography and chemistry of the GaN nanotubes were analyzed using TEM and energy dispersion x-ray spectroscopy (EDS). It is suggested that the lowered Young's modulus and quality factor of the nanotubes are mainly due to the surface roughness and defectiveness.

  20. Seeing the chemistry in biology with neutron crystallography.

    PubMed

    Langan, Paul; Chen, Julian C-H

    2013-09-07

    New developments in macromolecular neutron crystallography have led to an increasing number of structures published over the last decade. Hydrogen atoms, normally invisible in most X-ray crystal structures, become visible with neutrons. Using X-rays allows one to see structure, while neutrons allow one to reveal the chemistry inherent in these macromolecular structures. A number of surprising and sometimes controversial results have emerged; because it is difficult to see or predict hydrogen atoms in X-ray structures, when they are seen by neutrons they can be in unexpected locations with important chemical and biological consequences. Here we describe examples of chemistry seen with neutrons for the first time in biological macromolecules over the past few years.

  1. Status of the crystallography beamlines at synchrotron SOLEIL⋆

    NASA Astrophysics Data System (ADS)

    Coati, A.; Chavas, L. M. G.; Fontaine, P.; Foos, N.; Guimaraes, B.; Gourhant, P.; Legrand, P.; Itie, J.-P.; Fertey, P.; Shepard, W.; Isabet, T.; Sirigu, S.; Solari, P.-L.; Thiaudiere, D.; Thompson, A.

    2017-04-01

    Synchrotron SOLEIL (www.synchrotron-soleil.fr) is the French national centre for synchrotron radiation research, and has been open, for user applications, since 2008. Operating at an energy of 2.75GeV, injected current of 500mA, with an excellent beam stability of < 1 micron at the source position and beam "top up", and with a high proportion of straight sections for the ring circumference, SOLEIL offers a wide range of possibilities for diffraction experiments. The beamlines serving crystallographic communities are listed, along with their area of expertise and available equipment. A detailed description of the design of the PROXIMA 1 beamline for macromolecular crystallography is given, and an example of an (unsuccessful but significant) experiment is given.

  2. Seeing the chemistry in biology with neutron crystallography

    PubMed Central

    Langan, Paul; Chen, Julian C.-H.

    2017-01-01

    New developments in macromolecular neutron crystallography have led to an increasing number of structures published over the last decade. Hydrogen atoms, normally invisible in most X-ray crystal structures, become visible in neutron structures. Using X-rays allows one to see structure, while neutrons allow one to reveal the chemistry inherent in these macromolecular structures. A number of surprising and sometimes controversial results have emerged from recent neutron structures; because it is difficult to see or predict hydrogen atoms in X-ray structures, when they are seen by neutrons they can be in unexpected locations with important chemical and biological consequences. Here we describe examples of chemistry seen with neutrons for the first time in biological macromolecules over the past few years. PMID:23852376

  3. 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.

  4. 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.

  5. Energy optimization of a regular macromolecular crystallography beamline for ultra-high-resolution crystallography

    DOE PAGES

    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

  6. Affinity crystallography reveals the bioactive compounds of industrial juicing byproducts of Punica granatum for glycogen phosphorylase.

    PubMed

    Stravodimos, George A; Kantsadi, Anastassia L; Apostolou, Anna; Kyriakis, Efthimios; Kafaski-Kanelli, Vassiliki-Nafsika; Solovou, Theodora G A; Gatzona, Pagona; Liggri, Panagiota Cv; Theofanous, Stavroula; Gorgogietas, Vyron A; Kissa, Apostolia; Psachoula, Chariklia; Chatzileontiadou, Demetra S M; Lemonakis, Angelos; Psarra, Anna-Maria G; Skamnaki, Vassiliki T; Haroutounian, Serkos; Leonidas, Demetres D

    2017-06-18

    Glycogen phosphorylase (GP) is a pharmaceutical target for the discovery of new antihyperglycaemic agents. Punica granatum is a well-known plant for its potent antioxidant and antimicrobial activities but so far has not been examined for antihyperglycaemic activity. To examine the inhibitory potency of eighteen polyphenolic extracts obtained from Punica granatum fruits and industrial juicing byproducts against GP and discover their most bioactive ingredients. Kinetic experiments were conducted to measure the IC50 values of the extracts while affinity crystallography was used to identify the most bioactive ingredient. The inhibitory effect of one of the polyphenolic extracts was also verified ex vivo, in HepG2 cells. All extracts exhibit significant in vitro inhibitory potency (IC50 values in the range of low μg/mL). Affinity crystallography revealed that the most bioactive ingredients of the extracts were chlorogenic and ellagic acids, found bound in the active and the inhibitor site of GP, respectively. While ellagic acid is an established GP inhibitor, the inhibition of chlorogenic acid is reported for the first time. Kinetic analysis indicated that chlorogenic acid is an inhibitor with Ki=2.5 x 10-3 M that acts synergistically with ellagic acid. Our study provides the first evidence for a potential antidiabetic usage of Punica granatum extracts as antidiabetic food supplements. Although, more in vivo studies have to be performed before these extracts reach the stage of antidiabetic food supplements our study provides a first positive step towards this process. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  7. Application of multigrain crystallography to mineralogy of the deep Earth

    NASA Astrophysics Data System (ADS)

    Zhang, L.; Meng, Y.; Popov, D.; Wang, J.; Mao, H. K.

    2014-12-01

    Laser-heated diamond anvil cell (DAC) technology coupled with x-ray diffraction (XRD) probe has become a powerful tool to explore the mineralogy of the deep Earth. However, powder diffraction alone has the intrinsic limitations when the sample consists of multiple different phases; only a couple of low angle diffraction rings are unique to each of the phases; most overlapping diffraction rings cannot be used for unique identification or accurate determination of crystallographic parameters. Such powder refinements at extreme P-T conditions also suffer from unreliable intensity measurements caused by texturing of the sample and spottiness due to crystal growth under high T. In addition, new structures and minor phases are often overshadowed by the diffraction peaks of major phases. The crystal structure and unit-cell parameters of Fe-bearing minerals change with pressure/temperature, spin transition, and Fe redistribution. Precise structure analysis is required to understand the role of Fe in the geophysical and geochemical processes in the deep Earth. To overcome the difficulties, we introduced a multigrain crystallography method to determine crystallographic orientations of individual crystallites within a polycrystalline sample in-situ in a DAC. Using this method, we are able to separate individual crystallites and treat them as individual single crystals. The high-brilliance x-ray beam available at the 3rd generation synchrotron facilities has made it possible to collect diffraction spots in a powder sample comprised of hundreds of submicron-sized crystallites. Once separated, the data set for each crystallite can be treated with the standard single-crystal refinement program, resulting in excellent statistics in refinement and maximal coverage of the reciprocal space. The combination of powder diffraction and multigrain crystallography is very powerful in unequivocal determination of symmetry and unit cells, solving new structures, and picking out minor phases

  8. 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

  9. Structural studies of Neurospora crassa LPMO9D and redox partner CDHIIA using neutron crystallography and small-angle scattering.

    PubMed

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

    2017-08-07

    Sensitivity to hydrogen/deuterium and lack of observable radiation damage makes cold neutrons an ideal probe the structural studies of proteins with highly photosensitive groups such as the copper center of lytic polysaccharide monooxygenases (LPMOs) and flavin adenine dinucleotide (FAD) and heme redox cofactors of cellobiose dehydrogenases (CDHs). Here, neutron crystallography and small-angle neutron scattering are used to investigate Neurospora crassa LPMO9D (NcLPMO9D) and CDHIIA (NcCDHIIA), respectively. The presence of LPMO greatly enhances the efficiency of commercial glycoside hydrolase cocktails in the depolymerization of cellulose. LPMOs can receive electrons from CDHs to activate molecular dioxygen for the oxidation of cellulose resulting in chain cleavage and disruption of local crystallinity. Using neutron protein crystallography, the hydrogen/deuterium atoms of NcLPMO9D could be located throughout the structure. At the copper active site, the protonation states of the side chains of His1, His84, His157 and Tyr168, and the orientation of water molecules could be determined. Small-angle neutron scattering measurements provided low resolution models of NcCDHIIA with both the dehydrogenase and cytochrome domains in oxidized states that exhibited elongated conformations. This work demonstrates the suitability of neutron diffraction and scattering for characterizing enzymes critical to oxidative cellulose deconstruction. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. 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.

  11. A putative GTP binding protein homologous to interferon-inducible Mx proteins performs an essential function in yeast protein sorting.

    PubMed

    Rothman, J H; Raymond, C K; Gilbert, T; O'Hara, P J; Stevens, T H

    1990-06-15

    Members of the Mx protein family promote interferon-inducible resistance to viral infection in mammals and act by unknown mechanisms. We identified an Mx-like protein in yeast and present genetic evidence for its cellular function. This protein, the VPS1 product, is essential for vacuolar protein sorting, normal organization of intracellular membranes, and growth at high temperature, implying that Mx-like proteins are engaged in fundamental cellular processes in eukaryotes. Vps1p contains a tripartite GTP binding motif, which suggests that binding to GTP is essential to its role in protein sorting. Vps1p-specific antibody labels punctate cytoplasmic structures that condense to larger structures in a Golgi-accumulating sec7 mutant; thus, Vps1p may associate with an intermediate organelle of the secretory pathway.

  12. Whey Protein Supplementation Enhances Whole Body Protein Metabolism and Performance Recovery after Resistance Exercise: A Double-Blind Crossover Study.

    PubMed

    West, Daniel W D; Abou Sawan, Sidney; Mazzulla, Michael; Williamson, Eric; Moore, Daniel R

    2017-07-11

    No study has concurrently measured changes in free-living whole body protein metabolism and exercise performance during recovery from an acute bout of resistance exercise. We aimed to determine if whey protein ingestion enhances whole body net protein balance and recovery of exercise performance during overnight (10 h) and 24 h recovery after whole body resistance exercise in trained men. In a double-blind crossover design, 12 trained men (76 ± 8 kg, 24 ± 4 years old, 14% ± 5% body fat; means ± standard deviation (SD)) performed resistance exercise in the evening prior to consuming either 25 g of whey protein (PRO; MuscleTech 100% Whey) or an energy-matched placebo (CHO) immediately post-exercise (0 h), and again the following morning (~10 h of recovery). A third randomized trial, completed by the same participants, involving no exercise and no supplement served as a rested control trial (Rest). Participants ingested [(15)N]glycine to determine whole body protein kinetics and net protein balance over 10 and 24 h of recovery. Performance was assessed pre-exercise and at 0, 10, and 24 h of recovery using a battery of tests. Net protein balance tended to improve in PRO (P = 0.064; effect size (ES) = 0.61, PRO vs. CHO) during overnight recovery. Over 24 h, net balance was enhanced in PRO (P = 0.036) but not in CHO (P = 0.84; ES = 0.69, PRO vs. CHO), which was mediated primarily by a reduction in protein breakdown (PRO < CHO; P < 0.01. Exercise decreased repetitions to failure (REP), maximal strength (MVC), peak and mean power, and countermovement jump performance (CMJ) at 0 h (all P < 0.05 vs. Pre). At 10 h, there were small-to-moderate effects for enhanced recovery of the MVC (ES = 0.56), mean power (ES = 0.49), and CMJ variables (ES: 0.27-0.49) in PRO. At 24 h, protein supplementation improved MVC (ES = 0.76), REP (ES = 0.44), and peak power (ES = 0.55). In conclusion, whey protein supplementation enhances whole body anabolism, and may improve acute recovery of

  13. Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography

    SciTech Connect

    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; Bath, 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; Chapman, Henry N.; Fromme, Petra; Frank, Matthias; Abela, Rafael; Boutet, Sebastien; Barty, Anton; White, Thomas A.; Weierstall, Uwe; Spence, John; Neutze, Richard; Schertler, Gebhard; Standfuss, Jorg

    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 the 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.

  14. 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

  15. Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography

    DOE PAGES

    Nogly, Przemyslaw; Panneels, Valerie; Nelson, Garrett; ...

    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

  16. 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

    2016-08-22

    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 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.

  17. 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.

  18. X-Ray Crystallography as a Tool to Determine Three-Dimensional Structures of Commercial Enzymes Subjected to Treatment in Pressurized Fluids.

    PubMed

    Feiten, Mirian Cristina; Di Luccio, Marco; Santos, Karine F; de Oliveira, Débora; Oliveira, J Vladimir

    2017-06-01

    The study of enzyme function often involves a multi-disciplinary approach. Several techniques are documented in the literature towards determining secondary and tertiary structures of enzymes, and X-ray crystallography is the most explored technique for obtaining three-dimensional structures of proteins. Knowledge of three-dimensional structures is essential to understand reaction mechanisms at the atomic level. Additionally, structures can be used to modulate or improve functional activity of enzymes by the production of small molecules that act as substrates/cofactors or by engineering selected mutants with enhanced biological activity. This paper presentes a short overview on how to streamline sample preparation for crystallographic studies of treated enzymes. We additionally revise recent developments on the effects of pressurized fluid treatment on activity and stability of commercial enzymes. Future directions and perspectives on the the role of crystallography as a tool to access the molecular mechanisms underlying enzymatic activity modulation upon treatment in pressurized fluids are also addressed.

  19. 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

  20. 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.

  1. 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.

  2. Associations between milk protein concentration at various stages of lactation and reproductive performance in dairy cows.

    PubMed

    Morton, J M; Auldist, M J; Douglas, M L; Macmillan, K L

    2016-12-01

    Milk protein concentration has been positively associated with a range of measures of reproductive performance in dairy cows. These beneficial associations are most likely due to factors affecting both milk protein concentration and reproductive performance possibly being mediated, in part, by energy balance during early lactation. However, it is likely that factors other than energy balance are also involved in these relationships. A retrospective single cohort study was conducted using subsets of data collected from 74 dairy herds with seasonal or split calving patterns. Associations between milk protein concentration at various stages of lactation and reproductive performance in Holstein dairy cows were assessed using random effects logistic regression and survival analysis with milk protein concentration during the cow's breeding period fitted as a time-varying covariate. The beneficial associations between milk protein concentration and each of the 4 selected indices for measuring reproductive performance were evident when milk protein concentration was derived for each 30-d period from calving up to 300d in milk. For the first 150d of lactation the adjusted odds ratios were highest from 31 to 60d and only slightly lower for all periods up to 150d of lactation. Estimated associations for 31 to 60d were stronger than for 0 to 30d. In addition, milk protein concentration during a cow's breeding period was positively associated with the subsequent daily hazard of conception, even after adjusting for milk protein concentration in the cow's first or second month of lactation. Milk protein concentrations from 0 to 30d of lactation were less closely correlated with concentrations measured at subsequent 30-d intervals; correlations were closer between other periods in lactation. These results indicate that the association between milk protein concentration and reproductive performance is partly due to factors other than the extent of negative energy balance in early

  3. 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.

  4. UV-Visible Absorption Spectroscopy Enhanced X-ray Crystallography at Synchrotron and X-ray Free Electron Laser Sources.

    PubMed

    Cohen, Aina E; Doukov, Tzanko; Soltis, Michael S

    2016-01-01

    This review describes the use of single crystal UV-Visible Absorption micro-Spectrophotometry (UV-Vis AS) to enhance the design and execution of X-ray crystallography experiments for structural investigations of reaction intermediates of redox active and photosensitive proteins. Considerations for UV-Vis AS measurements at the synchrotron and associated instrumentation are described. UV-Vis AS is useful to verify the intermediate state of an enzyme and to monitor the progression of reactions within crystals. Radiation induced redox changes within protein crystals may be monitored to devise effective diffraction data collection strategies. An overview of the specific effects of radiation damage on macromolecular crystals is presented along with data collection strategies that minimize these effects by combining data from multiple crystals used at the synchrotron and with the X-ray free electron laser.

  5. Lipid monolayer and sparse matrix screening for growing two-dimensional crystals for electron crystallography: methods and examples.

    PubMed

    Yeager, Mark; Dryden, Kelly A; Ganser-Pornillos, Barbie K

    2013-01-01

    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 Ni(2+) 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.

  6. 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.

  7. Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography

    SciTech Connect

    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.

  8. Structure Determination Algorithms in Computational X-Ray Crystallography.

    NASA Astrophysics Data System (ADS)

    Chang, Chun-Shi

    This dissertation focuses on the development and implementation of practical algorithms for solving the phase problem of X-ray