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Sample records for protein crystallography performance

  1. 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.

  2. 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.

  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 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.

  5. 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.

  6. 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

  7. 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.

  8. Protein Crystallography in Vaccine Research and Development

    PubMed Central

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

    2015-01-01

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

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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

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

    SciTech Connect

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

    2012-08-01

    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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

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

    SciTech Connect

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

    2015-08-04

    Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP–SFX method has recently been introduced in which microcrystals of membrane proteins are grown and delivered for SFX data collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic phase (LCP), using a special LCP microextrusion injector. Here, it is 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. Serial Femtosecond Crystallography of G Protein-Coupled Receptors

    PubMed Central

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

    2014-01-01

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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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}

  16. 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)

  17. 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.

  18. 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.

  19. 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.

  20. 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.

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

    SciTech Connect

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

    1992-12-31

    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.

  2. 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.

  3. 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.

  4. 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.

  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. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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

  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.

    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

  19. 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

  20. 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

  1. 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.

  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. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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)

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

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

    PubMed Central

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

    2016-01-01

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

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. Native Chemical Ligation at Asx-Cys, Glx-Cys: Chemical Synthesis and High Resolution X-ray Structure of ShK Toxin by Racemic Protein Crystallography

    PubMed Central

    Dang, Bobo; Kubota, Tomoya; Mandal, Kalyaneswar; Bezanilla, Francisco; Kent, Stephen B. H.

    2013-01-01

    We have re-examined the utility of native chemical ligation at −Gln/Glu-Cys− [Glx-Cys] and −Asn/Asp-Cys− [Asx-Cys] sites. Using the improved thioaryl catalyst 4-mercaptophenylacetic acid (MPAA), native chemical ligation could be performed at −Gln-Cys− and Asn-Cys− sites without side reactions. After optimization, ligation at a −Glu-Cys− could also be used as a ligation site, with minimal levels of byproduct formation. However, −Asp-Cys− is not appropriate for use as a site for native chemical ligation because of formation of significant amounts of β-linked byproduct. The feasibility of native chemical ligation at −Gln-Cys− enabled a convergent total chemical synthesis of the enantiomeric forms of the ShK toxin protein molecule. The D-ShK protein molecule was ~50,000-fold less active in blocking the Kv1.3 channel than the L-ShK protein molecule. Racemic protein crystallography was used to obtain high resolution X-ray diffraction data for ShK toxin. The structure was solved by direct methods and showed significant differences from the previously reported NMR structures in some regions of the ShK protein molecule. PMID:23919482

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

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

    SciTech Connect

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

    2012-10-23

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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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).

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

  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. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  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.

  17. 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

  18. 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

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

    PubMed Central

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

    2013-01-01

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

  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. 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.

  3. 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

  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. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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.

  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. 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.…

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

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

    PubMed Central

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

    2016-01-01

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

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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).

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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

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

    PubMed

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

    2016-08-26

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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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.

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

    SciTech Connect

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

    2015-08-18

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

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. Discovery of Leukotriene A4 Hydrolase Inhibitors Using Metabolomics Biased Fragment Crystallography

    SciTech Connect

    Davies, D.; Mamat, B; Magnusson, O; Christensen, J; Haraldsson, M; Mishra, R; Pease, B; Hansen, E; Singh, J; et. al.

    2009-01-01

    We describe a novel fragment library termed fragments of life (FOL) for structure-based drug discovery. The FOL library includes natural small molecules of life, derivatives thereof, and biaryl protein architecture mimetics. The choice of fragments facilitates the interrogation of protein active sites, allosteric binding sites, and protein-protein interaction surfaces for fragment binding. We screened the FOL library against leukotriene A4 hydrolase (LTA4H) by X-ray crystallography. A diverse set of fragments including derivatives of resveratrol, nicotinamide, and indole were identified as efficient ligands for LTA4H. These fragments were elaborated in a small number of synthetic cycles into potent inhibitors of LTA4H representing multiple novel chemotypes for modulating leukotriene biosynthesis. Analysis of the fragment-bound structures also showed that the fragments comprehensively recapitulated key chemical features and binding modes of several reported LTA4H inhibitors.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.…

  10. 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)

  11. 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.…

  12. 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…

  13. 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.

  14. 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.

  15. 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.

  16. Developing advanced x-ray scattering methods combined with crystallography and computation

    PubMed Central

    Perry, J. Jefferson P.; Tainer, John A.

    2013-01-01

    The extensive use of small angle x-ray scattering (SAXS) over the last few years is rapidly providing new insights into protein interactions, complex formation and conformational states in solution. This SAXS methodology allows for detailed biophysical quantification of samples of interest. Initial analyses provide a judgment of sample quality, revealing the potential presence of aggregation, the overall extent of folding or disorder, the radius of gyration, maximum particle dimensions and oligomerization state. Structural characterizations include ab initio approaches from SAXS data alone, and when combined with previously determined crystal/NMR, atomistic modeling can further enhance structural solutions and assess validity. This combination can provide definitions of architectures, spatial organizations of protein domains within a complex, including those not determined by crystallography or NMR, as well as defining key conformational states of a protein interaction. SAXS is not generally constrained by macromolecule size, and the rapid collection of data in a 96-well plate format provides methods to screen sample conditions. This includes screening for co-factors, substrates, differing protein or nucleotide partners or small molecule inhibitors, to more fully characterize the variations within assembly states and key conformational changes. Such analyses may be useful for screening constructs and conditions to determine those most likely to promote crystal growth of a complex under study. Moreover, these high throughput structural determinations can be leveraged to define how polymorphisms affect assembly formations and activities. This is in addition to potentially providing architectural characterizations of complexes and interactions for systems biology-based research, and distinctions in assemblies and interactions in comparative genomics. Thus, SAXS combined with crystallography/NMR and computation provides a unique set of tools that should be considered

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.« less

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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)

  18. 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

  19. 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.

  20. Serial crystallography on in vivo grown microcrystals using synchrotron radiation

    PubMed Central

    Gati, Cornelius; Bourenkov, Gleb; Klinge, Marco; Rehders, Dirk; Stellato, Francesco; Oberthür, Dominik; Yefanov, Oleksandr; Sommer, Benjamin P.; Mogk, Stefan; Duszenko, Michael; Betzel, Christian; Schneider, Thomas R.; Chapman, Henry N.; Redecke, Lars

    2014-01-01

    Crystal structure determinations of biological macromolecules are limited by the availability of sufficiently sized crystals and by the fact that crystal quality deteriorates during data collection owing to radiation damage. Exploiting a micrometre-sized X-ray beam, high-precision diffractometry and shutterless data acquisition with a pixel-array detector, a strategy for collecting data from many micrometre-sized crystals presented to an X-ray beam in a vitrified suspension is demonstrated. By combining diffraction data from 80 Trypanosoma brucei procathepsin B crystals with an average volume of 9 µm3, a complete data set to 3.0 Å resolution has been assembled. The data allowed the refinement of a structural model that is consistent with that previously obtained using free-electron laser radiation, providing mutual validation. Further improvements of the serial synchrotron crystallography technique and its combination with serial femtosecond crystallography are discussed that may allow the determination of high-resolution structures of micrometre-sized crystals. PMID:25075324

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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.

  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. Protein supplementation for military personnel: a review of the mechanisms and performance outcomes.

    PubMed

    McLellan, Tom M

    2013-11-01

    Protein supplement use is common among athletes, active adults, and military personnel. This review provides a summary of the evidence base that either supports or refutes the ergogenic effects associated with different mechanisms that have been proposed to support protein supplementation. It was clear that if carbohydrate delivery was optimal either during or after an acute bout of exercise that additional protein will not increase exercise capacity. Evidence was also weak to substantiate use of protein supplements to slow the increase in brain serotonin and onset of central fatigue. It was also evident that additional research is warranted to test whether the benefits of protein supplements for enhancing recovery of fluid balance after exercise will affect subsequent work in the heat. In contrast, with repeated exercise, use of protein supplementation was associated with reductions in muscle soreness and often a faster recovery of muscle function due to reductions in protein degradation. There was also good supportive evidence for long-term benefits of protein supplementation for gains in muscle mass and strength through accelerated rates of protein synthesis, as long as the training stimulus was of sufficient intensity, frequency, and duration. However, studies have not examined the impact of protein supplements under the combined stress of a military environment that includes repeated bouts of exercise with little opportunity for feeding and recovery, lack of sleep, and exposure to extreme environments. Both additional laboratory and field research is warranted to help provide evidence-based guidance for the choice of protein supplements to enhance soldier performance.

  6. Choice and maintenance of equipment for electron crystallography.

    PubMed

    Mills, Deryck J; Vonck, Janet

    2013-01-01

    The choice of equipment for an electron crystallography laboratory will ultimately be determined by the available budget; nevertheless, the ideal lab will have two electron microscopes: a dedicated 300 kV cryo-EM with a field emission gun and a smaller LaB(6) machine for screening. The high-end machine should be equipped with photographic film or a very large CCD or CMOS camera for 2D crystal data collection; the screening microscope needs a mid-size CCD for rapid evaluation of crystal samples. The microscope room installations should provide adequate space and a special environment that puts no restrictions on the collection of high-resolution data. Equipment for specimen preparation includes a carbon coater, glow discharge unit, light microscope, plunge freezer, and liquid nitrogen containers and storage dewars. When photographic film is to be used, additional requirements are a film desiccator, dark room, optical diffractometer, and a film scanner. Having the electron microscopes and ancillary equipment well maintained and always in optimum condition facilitates the production of high-quality data.

  7. Identification of rogue datasets in serial crystallography1

    PubMed Central

    Assmann, Greta; Brehm, Wolfgang; Diederichs, Kay

    2016-01-01

    Advances in beamline optics, detectors and X-ray sources allow new techniques of crystallographic data collection. In serial crystallography, a large number of partial datasets from crystals of small volume are measured. Merging of datasets from different crystals in order to enhance data completeness and accuracy is only valid if the crystals are isomorphous, i.e. sufficiently similar in cell parameters, unit-cell contents and molecular structure. Identification and exclusion of non-isomorphous datasets is therefore indispensable and must be done by means of suitable indicators. To identify rogue datasets, the influence of each dataset on CC1/2 [Karplus & Diederichs (2012 ▸). Science, 336, 1030–1033], the correlation coefficient between pairs of intensities averaged in two randomly assigned subsets of observations, is evaluated. The presented method employs a precise calculation of CC1/2 that avoids the random assignment, and instead of using an overall CC1/2, an average over resolution shells is employed to obtain sensible results. The selection procedure was verified by measuring the correlation of observed (merged) intensities and intensities calculated from a model. It is found that inclusion and merging of non-isomorphous datasets may bias the refined model towards those datasets, and measures to reduce this effect are suggested. PMID:27275144

  8. 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.

  9. From Recombinant Expression to Crystals: A Step-by-Step Guide to GPCR Crystallography.

    PubMed

    Shukla, Arun K; Kumari, Punita; Ghosh, Eshan; Nidhi, Kumari

    2015-01-01

    G protein-coupled receptors (GPCRs) are the primary targets of drugs prescribed for many human pathophysiological conditions such as hypertension, allergies, schizophrenia, asthma, and various types of cancer. High-resolution structure determination of GPCRs has been a key focus area in GPCR biology to understand the basic mechanism of their activation and signaling and to materialize the long-standing dream of structure-based drug design on these versatile receptors. There has been tremendous effort at this front in the past two decades and it has culminated into crystal structures of 27 different receptors so far. The recent progress in crystallization and structure determination of GPCRs has been driven by innovation and cutting-edge developments at every step involved in the process of crystallization. Here, we present a step-by-step description of various steps involved in GPCR crystallization starting from recombinant expression to obtaining diffracting crystals. We also discuss the next frontiers in GPCR biology that are likely to be a primary focus for crystallography efforts in the next decade or so.

  10. Getting More From Less: Correlated Single-Crystal Spectroscopy and X-ray Crystallography at the NSLS (456th Brookhaven Lecture)

    SciTech Connect

    Orville, Allen

    2010-04-21

    By integrating different techniques to collect complementary data at beam line X26C of the National Syncrotron Light Source (NSLS), Allen Orville and his colleagues of the Macromolecular Crystallography Research Resource are providing new insights into the structures of macromolecules. During the 456th Brookhaven Lecture, on Wednesday, April 21st, Orville will describe his approach and his findings in a talk entitled “Getting More From Less: Correlated Singe-Crystal Spectroscopy and X-Ray Crystallography at the NSLS,” beginning at 4 p.m. in Berkner Hall. Refreshments will be offered before and after the lecture. During his talk, Orville will discuss his field of protein crystallography, reviewing its landmark discoveries and explaining new ways of using the NSLS and, in the future, the NSLS-II, to build on those discoveries. Allen Orville took his Ph.D. in biology from the University of Minnesota in 1997. After completing a postdoc with the Howard Hughes Medical Institute at the University of Oregon, 1997-2000, he began an assistant professor at the Georgia Institute of Technology. Orville joined BNL's Biology Department in 2006 as an associate biophysicist and was promoted to biophysicist in 2008.

  11. Challenges of sulfur SAD phasing as a routine method in macromolecular crystallography

    SciTech Connect

    Doutch, James; Hough, Michael A.; Hasnain, S. Samar; Strange, Richard W.

    2011-11-29

    The sulfur SAD phasing method allows the determination of protein structuresde novowithout reference to derivatives such as Se-methionine. The feasibility for routine automated sulfur SAD phasing using a number of current protein crystallography beamlines at several synchrotrons was examined using crystals of trimericAchromobacter cycloclastesnitrite reductase (AcNiR), which contains a near average proportion of sulfur-containing residues and two Cu atoms per subunit. Experiments using X-ray wavelengths in the range 1.9–2.4 Å show that we are not yet at the level where sulfur SAD is routinely successful forautomatedstructure solution and model building using existing beamlines and current software tools. On the other hand, experiments using the shortest X-ray wavelengths available on existing beamlines could be routinely exploited to solve and produce unbiased structural models using the similarly weak anomalous scattering signals from the intrinsic metal atoms in proteins. The comparison of long-wavelength phasing (the Bijvoet ratio for nine S atoms and two Cu atoms is ~1.25% at ~2 Å) and copper phasing (the Bijvoet ratio for two Cu atoms is 0.81% at ~0.75Å) forAcNiR suggests that lower data multiplicity than is currently required for success should in general be possible for sulfur phasing if appropriate improvements to beamlines and data collection strategies can be implemented.

  12. Impaired cognitive performance in neuronal nitric oxide synthase knockout mice is associated with hippocampal protein derangements.

    PubMed

    Kirchner, Liselotte; Weitzdoerfer, Rachel; Hoeger, Harald; Url, Angelika; Schmidt, Peter; Engelmann, Mario; Villar, Santiago Rosell; Fountoulakis, Michael; Lubec, Gert; Lubec, Barbara

    2004-12-01

    Nitric oxide is implicated in modulation of memory and pharmacological as well as genetic inhibition of neuronal nitric oxide synthase (nNOS) leads to impaired cognitive function. We therefore decided to study learning and memory functions and cognitive flexibility in the Morris water maze (MWM) in 1-month-old male mice lacking nNOS (nNOS KO). Hippocampal protein profiling was carried out to possibly link protein derangement to impaired cognitive function. Two-dimensional gel electrophoresis with in-gel digestion of spots and subsequent MALDI-TOF identification of proteins and quantification of proteins using specific software was applied. In the memory as well as in the relearning task of the MWM, most of the nNOS KO failed to find the submerged platform within a given time. Proteomic evaluation of hippocampus, the main anatomical structure computing cognitive functions, revealed aberrant expression of a synaptosomal associated protein of the exocytotic machinery (NSF), glycolytic enzymes, chaperones 78 kDa glucose-regulated protein, T-complex protein 1; the signaling structure guanine nucleotide-binding protein G(I)/G(S)/G(T) and heterogeneous nuclear ribonucleoprotein H of the splicing machinery. We conclude that nNOS knockout mice show impaired spatial performance in the MWM, a finding that may be either linked to direct effects of nNOS/NO and/or to specific hippocampal protein derangements.

  13. Crystal structure of the TLDc domain of oxidation resistance protein 2 from zebrafish.

    PubMed

    Blaise, Mickaël; Alsarraf, Husam M A B; Wong, Jaslyn E M M; Midtgaard, Søren Roi; Laroche, Fabrice; Schack, Lotte; Spaink, Herman; Stougaard, Jens; Thirup, Søren

    2012-06-01

    The oxidation resistance proteins (OXR) help to protect eukaryotes from reactive oxygen species. The sole C-terminal domain of the OXR, named TLDc is sufficient to perform this function. However, the mechanism by which oxidation resistance occurs is poorly understood. We present here the crystal structure of the TLDc domain of the oxidation resistance protein 2 from zebrafish. The structure was determined by X-ray crystallography to atomic resolution (0.97Å) and adopts an overall globular shape. Two antiparallel β-sheets form a central β-sandwich, surrounded by two helices and two one-turn helices. The fold shares low structural similarity to known structures.

  14. Substrate specificity of pyrimidine nucleoside phosphorylases of NP-II family probed by X-ray crystallography and molecular modeling

    NASA Astrophysics Data System (ADS)

    Balaev, V. V.; Lashkov, A. A.; Prokofev, I. I.; Gabdulkhakov, A. G.; Seregina, T. A.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.

    2016-09-01

    Pyrimidine nucleoside phosphorylases, which are widely used in the biotechnological production of nucleosides, have different substrate specificity for pyrimidine nucleosides. An interesting feature of these enzymes is that the three-dimensional structure of thymidine-specific nucleoside phosphorylase is similar to the structure of nonspecific pyrimidine nucleoside phosphorylase. The three-dimensional structures of thymidine phosphorylase from Salmonella typhimurium and nonspecific pyrimidine nucleoside phosphorylase from Bacillus subtilis in complexes with a sulfate anion were determined for the first time by X-ray crystallography. An analysis of the structural differences between these enzymes demonstrated that Lys108, which is involved in the phosphate binding in pyrimidine nucleoside phosphorylase, corresponds to Met111 in thymidine phosphorylases. This difference results in a decrease in the charge on one of the hydroxyl oxygens of the phosphate anion in thymidine phosphorylase and facilitates the catalysis through SN2 nucleophilic substitution. Based on the results of X-ray crystallography, the virtual screening was performed for identifying a potent inhibitor (anticancer agent) of nonspecific pyrimidine nucleoside phosphorylase, which does not bind to thymidine phosphorylase. The molecular dynamics simulation revealed the stable binding of the discovered compound—2-pyrimidin-2-yl-1H-imidazole-4-carboxylic acid—to the active site of pyrimidine nucleoside phosphorylase.

  15. A short history of structure based research on the photocycle of photoactive yellow protein

    PubMed Central

    Schmidt, Marius

    2017-01-01

    The goals of time-resolved macromolecular crystallography are to extract the molecular structures of the reaction intermediates and the reaction dynamics from time-resolved X-ray data alone. To develop the techniques of time-resolved crystallography, biomolecules with special properties are required. The Photoactive Yellow Protein is the most sparkling of these. PMID:28191482

  16. Microsequence analysis of electroblotted proteins. II. Comparison of sequence performance on different types of PVDF membranes.

    PubMed

    Reim, D F; Speicher, D W

    1992-11-15

    The influence of different types of polyvinylidene difluoride (PVDF) membranes on gas phase sequence performance has been evaluated. These PVDF membranes have been classified as either high retention (Trans-Blot and ProBlott) or low retention membranes (Immobilon-P) based on their ability to bind proteins during electroblotting from gels. Initial yields, repetitive yields, and extraction efficiency of the anilinothiazolinone amino acid derivatives have been compared for several standard proteins that have been either electroblotted or loaded onto PVDF membranes by direct adsorption. These results show that the major differences in initial sequence yields between membranes arise from differences in the amount of protein actually transferred to the membrane rather than sequencer-related factors. In contrast to several previous observations from other laboratories, more tightly bound proteins do not sequence with lower initial yields and initial yields are not affected by the ratio of surface area to protein. The stronger binding on high retention PVDF membranes does not adversely affect recoveries of difficult to extract, or very hydrophobic, amino acid derivatives. Several amino acids, especially tryptophan, are actually recovered in dramatically higher yield on high retention membranes compared with either Immobilon or glass filters. At the same time, the protein and peptide binding properties of high retention membranes will frequently improve the repetitive yield by minimizing sample extraction during the sequencer cycle. Stronger protein binding together with improved electroblotting yields offer substantially improved sequence performance when high retention PVDF membranes are used.

  17. Effect of whey protein hydrolysate on performance and recovery of top-class orienteering runners.

    PubMed

    Hansen, Mette; Bangsbo, Jens; Jensen, Jørgen; Bibby, Bo Martin; Madsen, Klavs

    2015-04-01

    This trial aimed to examine the effect of whey protein hydrolysate intake before and after exercise sessions on endurance performance and recovery in elite orienteers during a training camp. Eighteen elite orienteers participated in a randomized controlled intervention trial during a 1-week training camp (13 exercise sessions). Half of the runners (PRO-CHO) ingested a protein drink before (0.3 g kg(-1)) and a protein-carbohydrate drink after (0.3 g protein kg(-1) and 1 g carbohydrate kg(-1)) each exercise session. The others ingested energy and time-matched carbohydrate drinks (CHO). A 4-km run-test with 20 control points was performed before and on the last day of the intervention. Blood and saliva were obtained in the mornings, before and after run-tests, and after the last training session. During the intervention, questionnaires were fulfilled regarding psychological sense of performance capacity and motivation. PRO-CHO and not CHO improved performance in the 4-km run-test (interaction p < .05). An increase in serum creatine kinase was observed during the week, which was greater in CHO than PRO-CHO (interaction p < .01). Lactate dehydrogenase (p < .001) and cortisol (p = .057) increased during the week, but the change did not differ between groups. Reduction in sense of performance capacity during the intervention was greater in CHO (p < .05) than PRO-CHO. In conclusion, ingestion of whey protein hydrolysate before and after each exercise session improves performance and reduces markers of muscle damage during a strenuous 1-week training camp. The results indicate that protein supplementation in conjunction with each exercise session facilitates the recovery from strenuous training in elite orienteers.

  18. HPTLC-aptastaining – Innovative protein detection system for high-performance thin-layer chromatography

    NASA Astrophysics Data System (ADS)

    Morschheuser, Lena; Wessels, Hauke; Pille, Christina; Fischer, Judith; Hünniger, Tim; Fischer, Markus; Paschke-Kratzin, Angelika; Rohn, Sascha

    2016-05-01

    Protein analysis using high-performance thin-layer chromatography (HPTLC) is not commonly used but can complement traditional electrophoretic and mass spectrometric approaches in a unique way. Due to various detection protocols and possibilities for hyphenation, HPTLC protein analysis is a promising alternative for e.g., investigating posttranslational modifications. This study exemplarily focused on the investigation of lysozyme, an enzyme which is occurring in eggs and technologically added to foods and beverages such as wine. The detection of lysozyme is mandatory, as it might trigger allergenic reactions in sensitive individuals. To underline the advantages of HPTLC in protein analysis, the development of innovative, highly specific staining protocols leads to improved sensitivity for protein detection on HPTLC plates in comparison to universal protein derivatization reagents. This study aimed at developing a detection methodology for HPTLC separated proteins using aptamers. Due to their affinity and specificity towards a wide range of targets, an aptamer based staining procedure on HPTLC (HPTLC-aptastaining) will enable manifold analytical possibilities. Besides the proof of its applicability for the very first time, (i) aptamer-based staining of proteins is applicable on different stationary phase materials and (ii) furthermore, it can be used as an approach for a semi-quantitative estimation of protein concentrations.

  19. HPTLC-aptastaining – Innovative protein detection system for high-performance thin-layer chromatography

    PubMed Central

    Morschheuser, Lena; Wessels, Hauke; Pille, Christina; Fischer, Judith; Hünniger, Tim; Fischer, Markus; Paschke-Kratzin, Angelika; Rohn, Sascha

    2016-01-01

    Protein analysis using high-performance thin-layer chromatography (HPTLC) is not commonly used but can complement traditional electrophoretic and mass spectrometric approaches in a unique way. Due to various detection protocols and possibilities for hyphenation, HPTLC protein analysis is a promising alternative for e.g., investigating posttranslational modifications. This study exemplarily focused on the investigation of lysozyme, an enzyme which is occurring in eggs and technologically added to foods and beverages such as wine. The detection of lysozyme is mandatory, as it might trigger allergenic reactions in sensitive individuals. To underline the advantages of HPTLC in protein analysis, the development of innovative, highly specific staining protocols leads to improved sensitivity for protein detection on HPTLC plates in comparison to universal protein derivatization reagents. This study aimed at developing a detection methodology for HPTLC separated proteins using aptamers. Due to their affinity and specificity towards a wide range of targets, an aptamer based staining procedure on HPTLC (HPTLC-aptastaining) will enable manifold analytical possibilities. Besides the proof of its applicability for the very first time, (i) aptamer-based staining of proteins is applicable on different stationary phase materials and (ii) furthermore, it can be used as an approach for a semi-quantitative estimation of protein concentrations. PMID:27220270

  20. Advance chromatin extraction improves capture performance of protein A affinity chromatography.

    PubMed

    Nian, Rui; Zhang, Wei; Tan, Lihan; Lee, Jeremy; Bi, Xeuzhi; Yang, Yuansheng; Gan, Hui Theng; Gagnon, Pete

    2016-01-29

    Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise. Protein A ligand leakage from columns loaded with chromatin-extracted harvest was half the level observed on protein A columns loaded with non-extracted harvest. Columns loaded with chromatin-extracted harvest were cleaned more effectively by 50-100mM NaOH than columns loaded with non-extracted harvest that were cleaned with 250-500mM NaOH. Two protein A media with IgG capacities in excess of 50g/L were loaded with chromatin-extracted harvest, washed with 2.0M NaCl before elution, and the eluted IgG fraction titrated to pH 5.5 before microfiltration. Host protein contamination in the filtrate was reduced to <1ppm, DNA to <1ppb, protein A leakage to 0.5ppm, and aggregates to 1.0%. Caprylic acid and allantoin were both reduced below 5ppm. Step recovery of IgG was 99.4%. Addition of a single polishing step reduced residual protein A beneath the level of detection and aggregates to <0.1%. Overall process recovery including chromatin extraction was 90%.

  1. Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals

    SciTech Connect

    Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Lyubimov, Artem Y.; Hattne, Johan; Brewster, Aaron S.; Sauter, Nicholas K.; Brunger, Axel T.; Weis, William I.

    2015-03-17

    There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as the resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited.

  2. Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals.

    PubMed

    Uervirojnangkoorn, Monarin; Zeldin, Oliver B; Lyubimov, Artem Y; Hattne, Johan; Brewster, Aaron S; Sauter, Nicholas K; Brunger, Axel T; Weis, William I

    2015-03-17

    There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as the resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited.

  3. Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals

    DOE PAGES

    Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Lyubimov, Artem Y.; ...

    2015-03-17

    There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as themore » resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited.« less

  4. Caspase-3 binds diverse P4 residues in peptides as revealed by crystallography and structural modeling.

    SciTech Connect

    Fang, Bin; Fu, Guoxing; Agniswamy, Johnson; Harrison, Robert W.; Weber, Irene T.

    2009-03-31

    Caspase-3 recognition of various P4 residues in its numerous protein substrates was investigated by crystallography, kinetics, and calculations on model complexes. Asp is the most frequent P4 residue in peptide substrates, although a wide variety of P4 residues are found in the cellular proteins cleaved by caspase-3. The binding of peptidic inhibitors with hydrophobic P4 residues, or no P4 residue, is illustrated by crystal structures of caspase-3 complexes with Ac-IEPD-Cho, Ac-WEHD-Cho, Ac-YVAD-Cho, and Boc-D(OMe)-Fmk at resolutions of 1.9-2.6 {angstrom}. The P4 residues formed favorable hydrophobic interactions in two separate hydrophobic regions of the binding site. The side chains of P4 Ile and Tyr form hydrophobic interactions with caspase-3 residues Trp206 and Trp214 within a non-polar pocket of the S4 subsite, while P4 Trp interacts with Phe250 and Phe252 that can also form the S5 subsite. These interactions of hydrophobic P4 residues are distinct from those for polar P4 Asp, which indicates the adaptability of caspase-3 for binding diverse P4 residues. The predicted trends in peptide binding from molecular models had high correlation with experimental values for peptide inhibitors. Analysis of structural models for the binding of 20 different amino acids at P4 in the aldehyde peptide Ac-XEVD-Cho suggested that the majority of hydrophilic P4 residues interact with Phe250, while hydrophobic residues interact with Trp206, Phe250, and Trp214. Overall, the S4 pocket of caspase-3 exhibits flexible adaptation for different residues and the new structures and models, especially for hydrophobic P4 residues, will be helpful for the design of caspase-3 based drugs.

  5. Goniometer-based femtosecond crystallography with X-ray free electron lasers

    SciTech Connect

    Cohen, Aina E.; Soltis, S. Michael; González, Ana; Aguila, Laura; Alonso-Mori, Roberto; Barnes, Christopher O.; Baxter, Elizabeth L.; Brehmer, Winnie; Brewster, Aaron S.; Brunger, Axel T.; Calero, Guillermo; Chang, Joseph F.; Chollet, Matthieu; Ehrensberger, Paul; Eriksson, Thomas L.; Feng, Yiping; Hattne, Johan; Hedman, Britt; Hollenbeck, Michael; Holton, James M.; Keable, Stephen; Kobilka, Brian K.; Kovaleva, Elena G.; Kruse, Andrew C.; Lemke, Henrik T.; Lin, Guowu; Lyubimov, Artem Y.; Manglik, Aashish; Mathews, Irimpan I.; McPhillips, Scott E.; Nelson, Silke; Peters, John W.; Sauter, Nicholas K.; Smith, Clyde A.; Song, Jinhu; Stevenson, Hilary P.; Tsai, Yingssu; Uervirojnangkoorn, Monarin; Vinetsky, Vladimir; Wakatsuki, Soichi; Weis, William I.; Zadvornyy, Oleg A.; Zeldin, Oliver B.; Zhu, Diling; Hodgson, Keith O.

    2014-10-31

    The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. With smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.

  6. Goniometer-based femtosecond crystallography with X-ray free electron lasers

    PubMed Central

    Cohen, Aina E.; Soltis, S. Michael; González, Ana; Aguila, Laura; Alonso-Mori, Roberto; Barnes, Christopher O.; Baxter, Elizabeth L.; Brehmer, Winnie; Brewster, Aaron S.; Brunger, Axel T.; Calero, Guillermo; Chang, Joseph F.; Chollet, Matthieu; Ehrensberger, Paul; Eriksson, Thomas L.; Feng, Yiping; Hattne, Johan; Hedman, Britt; Hollenbeck, Michael; Holton, James M.; Keable, Stephen; Kobilka, Brian K.; Kovaleva, Elena G.; Kruse, Andrew C.; Lemke, Henrik T.; Lin, Guowu; Lyubimov, Artem Y.; Manglik, Aashish; Mathews, Irimpan I.; McPhillips, Scott E.; Nelson, Silke; Peters, John W.; Sauter, Nicholas K.; Smith, Clyde A.; Song, Jinhu; Stevenson, Hilary P.; Tsai, Yingssu; Uervirojnangkoorn, Monarin; Vinetsky, Vladimir; Wakatsuki, Soichi; Weis, William I.; Zadvornyy, Oleg A.; Zeldin, Oliver B.; Zhu, Diling; Hodgson, Keith O.

    2014-01-01

    The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources. PMID:25362050

  7. Goniometer-based femtosecond crystallography with X-ray free electron lasers.

    PubMed

    Cohen, Aina E; Soltis, S Michael; González, Ana; Aguila, Laura; Alonso-Mori, Roberto; Barnes, Christopher O; Baxter, Elizabeth L; Brehmer, Winnie; Brewster, Aaron S; Brunger, Axel T; Calero, Guillermo; Chang, Joseph F; Chollet, Matthieu; Ehrensberger, Paul; Eriksson, Thomas L; Feng, Yiping; Hattne, Johan; Hedman, Britt; Hollenbeck, Michael; Holton, James M; Keable, Stephen; Kobilka, Brian K; Kovaleva, Elena G; Kruse, Andrew C; Lemke, Henrik T; Lin, Guowu; Lyubimov, Artem Y; Manglik, Aashish; Mathews, Irimpan I; McPhillips, Scott E; Nelson, Silke; Peters, John W; Sauter, Nicholas K; Smith, Clyde A; Song, Jinhu; Stevenson, Hilary P; Tsai, Yingssu; Uervirojnangkoorn, Monarin; Vinetsky, Vladimir; Wakatsuki, Soichi; Weis, William I; Zadvornyy, Oleg A; Zeldin, Oliver B; Zhu, Diling; Hodgson, Keith O

    2014-12-02

    The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.

  8. Goniometer-based femtosecond crystallography with X-ray free electron lasers

    DOE PAGES

    Cohen, Aina E.; Soltis, S. Michael; González, Ana; ...

    2014-10-31

    The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. With smaller crystals, high-density grids were usedmore » to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.« less

  9. Effects of different extraction buffers on peanut protein detectability and lateral flow device (LFD) performance.

    PubMed

    Rudolf, J; Ansari, P; Kern, C; Ludwig, T; Baumgartner, S

    2012-01-01

    The accidental uptake of peanuts can cause severe health reactions in allergic individuals. Reliable determination of traces of peanuts in food products is required to support correct labelling and therefore minimise consumers' risk. The immunoanalytical detectability of potentially allergenic peanut proteins is dependent on previous heat treatment, the extraction capacity of the applied buffer and the specificity of the antibody. In this study a lateral flow device (LFD) for the detection of peanut protein was developed and the capacity of 30 different buffers to extract proteins from mildly and strongly roasted peanut samples as well as their influence on the test strip performance were investigated. Most of the tested buffers showed good extraction capacity for putative Ara h 1 from mildly roasted peanuts. Protein extraction from dark-roasted samples required denaturing additives, which were proven to be incompatible with LFD performance. High-pH buffers increased the protein yield but inhibited signal generation on the test strip. Overall, the best results were achieved using neutral phosphate buffers but equal detectability of differently altered proteins due to food processing cannot be assured yet for immunoanalytical methods.

  10. Effects of protein and carbohydrate meals on mood and performance: interactions with sex and age.

    PubMed

    Spring, B; Maller, O; Wurtman, J; Digman, L; Cozolino, L

    Normal adult subjects (n = 184) consumed a high-protein or high-carbohydrate meal. Two hours later their mood and performance were tested. The effects of meal composition on mood were different for men and women, and for older and younger subjects. Females, but not males, reported greater sleepiness after a carbohydrate as opposed to a protein meal. Male subjects, but not females, reported greater calmness after a carbohydrate as opposed to a protein meal. Older subjects responded differently to meals depending upon the time of day when these were consumed. When meals were eaten for breakfast (but not for lunch) individuals 40 yr of age or older felt more tense and less calm after a protein than after a carbohydrate meal. Although older subjects reported subjective discomfort after a morning protein meal, they displayed objective performance impairments after a carbohydrate lunch. Subjects 40 yr of age or older were impaired on a test of sustained selective attention (dichotic shadowing) after consuming a high-carbohydrate lunch. The shadowing impairment after carbohydrate consumption was as pronounced without distraction as with distraction and resulted mostly from increased omission errors. Our findings suggest negative effects on concentration when older subjects consume a high-carbohydrate, low-protein lunch. These negative effects of carbohydrate consumption appear to arise predominantly from lapses of attention rather than from intrusion of distractors.

  11. Protein structure prediction provides comparable performance to crystallographic structures in docking-based virtual screening.

    PubMed

    Du, Hongying; Brender, Jeffrey R; Zhang, Jian; Zhang, Yang

    2015-01-01

    Structure based virtual screening has largely been limited to protein targets for which either an experimental structure is available or a strongly homologous template exists so that a high-resolution model can be constructed. The performance of state of the art protein structure predictions in virtual screening in systems where only weakly homologous templates are available is largely untested. Using the challenging DUD database of structural decoys, we show here that even using templates with only weak sequence homology (<30% sequence identity) structural models can be constructed by I-TASSER which achieve comparable enrichment rates to using the experimental bound crystal structure in the majority of the cases studied. For 65% of the targets, the I-TASSER models, which are constructed essentially in the apo conformations, reached 70% of the virtual screening performance of using the holo-crystal structures. A correlation was observed between the success of I-TASSER in modeling the global fold and local structures in the binding pockets of the proteins versus the relative success in virtual screening. The virtual screening performance can be further improved by the recognition of chemical features of the ligand compounds. These results suggest that the combination of structure-based docking and advanced protein structure modeling methods should be a valuable approach to the large-scale drug screening and discovery studies, especially for the proteins lacking crystallographic structures.

  12. Ribosomal crystallography: peptide bond formation, chaperone assistance and antibiotics activity.

    PubMed

    Yonath, Ada

    2005-08-31

    The peptidyl transferase center (PTC) is located in a protein free environment, thus confirming that the ribosome is a ribozyme. This arched void has dimensions suitable for accommodating the 3' ends of the A-and the P-site tRNAs, and is situated within a universal sizable symmetry-related region that connects all ribosomal functional centers involved in amino-acid polymerization. The linkage between the elaborate PTC architecture and the A-site tRNA position revealed that the A- to P-site passage of the tRNA 3' end is performed by a rotatory motion, which leads to stereochemistry suitable for peptide bond formation and for substrate mediated catalysis, thus suggesting that the PTC evolved by gene-fusion. Adjacent to the PTC is the entrance of the protein exit tunnel, shown to play active roles in sequence-specific gating of nascent chains and in responding to cellular signals. This tunnel also provides a site that may be exploited for local co-translational folding and seems to assist in nascent chain trafficking into the hydrophobic space formed by the first bacterial chaperone, the trigger factor. Many antibiotics target ribosomes. Although the ribosome is highly conserved, subtle sequence and/or conformational variations enable drug selectivity, thus facilitating clinical usage. Comparisons of high-resolution structures of complexes of antibiotics bound to ribosomes from eubacteria resembling pathogens, to an archaeon that shares properties with eukaryotes and to its mutant that allows antibiotics binding, demonstrated the unambiguous difference between mere binding and therapeutical effectiveness. The observed variability in antibiotics inhibitory modes, accompanied by the elucidation of the structural basis to antibiotics mechanism justifies expectations for structural based improved properties of existing compounds as well as for the development of novel drugs.

  13. De novo protein crystal structure determination from X-ray free-electron laser data.

    PubMed

    Barends, Thomas R M; Foucar, Lutz; Botha, Sabine; Doak, R Bruce; Shoeman, Robert L; Nass, Karol; Koglin, Jason E; Williams, Garth J; Boutet, Sébastien; Messerschmidt, Marc; Schlichting, Ilme

    2014-01-09

    The determination of protein crystal structures is hampered by the need for macroscopic crystals. X-ray free-electron lasers (FELs) provide extremely intense pulses of femtosecond duration, which allow data collection from nanometre- to micrometre-sized crystals in a 'diffraction-before-destruction' approach. So far, all protein structure determinations carried out using FELs have been based on previous knowledge of related, known structures. Here we show that X-ray FEL data can be used for de novo protein structure determination, that is, without previous knowledge about the structure. Using the emerging technique of serial femtosecond crystallography, we performed single-wavelength anomalous scattering measurements on microcrystals of the well-established model system lysozyme, in complex with a lanthanide compound. Using Monte-Carlo integration, we obtained high-quality diffraction intensities from which experimental phases could be determined, resulting in an experimental electron density map good enough for automated building of the protein structure. This demonstrates the feasibility of determining novel protein structures using FELs. We anticipate that serial femtosecond crystallography will become an important tool for the structure determination of proteins that are difficult to crystallize, such as membrane proteins.

  14. Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids

    PubMed Central

    Hesse, Almut

    2016-01-01

    Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports. PMID:27559481

  15. Effects of rumen undegradable protein supplementation on productive performance and indicators of protein and energy metabolism in Holstein fresh cows.

    PubMed

    Amanlou, H; Farahani, T Amirabadi; Farsuni, N Eslamian

    2017-03-08

    The objective of this study was to determine the effects of feeding increased dietary crude protein (CP) on productive performance and indicators of protein and energy metabolism during 21 d postpartum. Thirty multiparous Holstein dairy cows were balanced by previous lactation milk yield, body condition score (BCS) at calving, and parity and randomly allocated to 1 of 3 dietary treatments from calving until 21 d postpartum. Dietary treatments were 16.0% CP with 5.0% rumen undegradable protein (RUP) based on dry matter (DM) (16CP), 18.7% CP with 7.0% RUP based on DM (19CP), and 21.4% CP with 9.0% RUP based on DM (21CP). Diets were similar in net energy for lactation (approximately 1.7 Mcal/kg of DM) and CP levels were increased with corn gluten meal and fish meal. Dry matter intake (DMI) was increased by increasing dietary CP levels from 16.0 to 19.0% of DM, but dietary CP beyond 19.0% had no effect on DMI. Milk yields were 4.7 and 6.5 kg/d greater in cows fed the 19CP and 21CP diets versus those fed the 16CP diet, whereas 4% fat-corrected milk was greater for cows fed the 21CP than the 16CP diet (36.0 vs. 31.4 kg/d). Milk protein content and yield, lactose yield, and milk urea nitrogen were elevated by increased dietary CP. Milk lactose content and fat yield were not different among dietary treatments, but milk fat content tended to decline with increasing content of CP in diets. High CP levels increased milk N secretion but decreased milk N efficiency. Apparent digestibility of DM, CP, and neutral detergent fiber was greater on the 19CP and 21CP diets compared with the 16CP diet. Cows fed the 19CP and 21CP diets lost less body condition relative to those fed the 16CP diet over 21 d postpartum. Feeding higher CP levels increased the concentrations of serum albumin, albumin to globulin ratio, and urea nitrogen and decreased aspartate aminotransferase, nonesterified fatty acids, and β-hydroxybutyrate, but had no effect on globulin, glucose, cholesterol, or

  16. Leucine-protein supplemented recovery feeding enhances subsequent cycling performance in well-trained men.

    PubMed

    Thomson, Jasmine S; Ali, Ajmol; Rowlands, David S

    2011-04-01

    The purpose of this study was to determine whether a practical leucine-protein, high-carbohydrate postexercise feeding regimen could improve recovery, as measured by subsequent cycling performance and mechanistic markers, relative to control feeding. In a crossover, 10 male cyclists performed 2- to 2.5-h interval training bouts on 3 consecutive evenings, ingesting either leucine-protein, high-carbohydrate nutrition (0.1/0.4/1.2/0.2 g·kg(-1)·h(-1); leucine, protein, carbohydrate, fat, respectively) or isocaloric control (0.06/1.6/0.2 g·kg(-1)·h(-1); protein, carbohydrate, fat, respectively) nutrition for 1.5 h postexercise. Throughout the experimental period diet was controlled, energy and macronutrient intake balanced, and protein intake clamped at 1.6 g·kg(-1)·day(-1). The alternate supplement was provided the next morning, thereby isolating the postexercise nutrition effect. Following 39 h of recovery, cyclists performed a repeat-sprint performance test. Postexercise leucine-protein ingestion improved mean sprint power by 2.5% (99% confidence limit, ±2.6%; p = 0.013) and reduced perceived overall tiredness during the sprints by 13% (90% confidence limit, ±9.2%), but perceptions of leg tiredness and soreness were unaffected. Before exercise, creatine-kinase concentration was lowered by 19% (90% confidence limits, ±18%), but lactate dehydrogenase and pressure-pain threshold were unaltered. There was a small reduction in anger (25% ± 18%), but other moods were unchanged. Plasma leucine (3-fold) and essential amino acid (47%) concentrations were elevated postexercise. Net nitrogen balance trended mildly negative in both conditions (mean ± SD: leucine-protein, -20 ± 46 mg·kg(-1) per 24 h; control, -25 ± 36 mg·kg(-1) per 24 h). The ingestion of a leucine-protein supplement along with other high-carbohydrate food following intense training on consecutive days enhances subsequent high-intensity endurance performance and may attenuate

  17. Development of optimized mobile phases for protein separation by high performance thin layer chromatography.

    PubMed

    Biller, Julia; Morschheuser, Lena; Riedner, Maria; Rohn, Sascha

    2015-10-09

    In recent years, protein chemistry tends inexorably toward the analysis of more complex proteins, proteoforms, and posttranslational protein modifications. Although mass spectrometry developed quite fast correspondingly, sample preparation and separation of these analytes is still a major issue and quite challenging. For many years, electrophoresis seemed to be the method of choice; nonetheless its variance is limited to parameters such as size and charge. When taking a look at traditional (thin-layer) chromatography, further parameters such as polarity and different mobile and stationary phases can be utilized. Further, possibilities of detection are manifold compared to electrophoresis. Similarly, two-dimensional separation can be also performed with thin-layer chromatography (TLC). As the revival of TLC developed enormously in the last decade, it seems to be also an alternative to use high performance thin-layer chromatography (HPTLC) for the separation of proteins. The aim of this study was to establish an HPTLC separation system that allows a separation of protein mixtures over a broad polarity range, or if necessary allowing to modify the separation with only few steps to improve the separation for a specific scope. Several layers and solvent systems have been evaluated to reach a fully utilized and optimized separation system.

  18. New paradigm for macromolecular crystallography experiments at SSRL: automated crystal screening and remote data collection

    PubMed Central

    Soltis, S. Michael; Cohen, Aina E.; Deacon, Ashley; Eriksson, Thomas; González, Ana; McPhillips, Scott; Chui, Hsui; Dunten, Pete; Hollenbeck, Michael; Mathews, Irimpan; Miller, Mitch; Moorhead, Penjit; Phizackerley, R. Paul; Smith, Clyde; Song, Jinhu; van dem Bedem, Henry; Ellis, Paul; Kuhn, Peter; McPhillips, Timothy; Sauter, Nicholas; Sharp, Kenneth; Tsyba, Irina; Wolf, Guenter

    2008-01-01

    Complete automation of the macromolecular crystallography experiment has been achieved at SSRL through the combination of robust mechanized experimental hardware and a flexible control system with an intuitive user interface. These highly reliable systems have enabled crystallography experiments to be carried out from the researchers’ home institutions and other remote locations while retaining complete control over even the most challenging systems. A breakthrough component of the system, the Stanford Auto-Mounter (SAM), has enabled the efficient mounting of cryocooled samples without human intervention. Taking advantage of this automation, researchers have successfully screened more than 200 000 samples to select the crystals with the best diffraction quality for data collection as well as to determine optimal crystallization and cryocooling conditions. These systems, which have been deployed on all SSRL macromolecular crystallography beamlines and several beamlines worldwide, are used by more than 80 research groups in remote locations, establishing a new paradigm for macromolecular crystallo­graphy experimentation. PMID:19018097

  19. CHARACTERIZATION OF DRUG INTERACTIONS WITH SERUM PROTEINS BY USING HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe; Barnaby, Omar; Jackson, Abby; Yoo, Michelle J.; Papastavros, Efthimia; Pfaunmiller, Erika; Sobansky, Matt; Tong, Zenghan

    2011-01-01

    The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding. PMID:21395530

  20. Atomic structure of the 75 MDa extremophile Sulfolobus turreted icosahedral virus determined by CryoEM and X-ray crystallography

    PubMed Central

    Veesler, David; Ng, Thiam-Seng; Sendamarai, Anoop K.; Eilers, Brian J.; Lawrence, C. Martin; Lok, Shee-Mei; Young, Mark J.; Johnson, John E.; Fu, Chi-yu

    2013-01-01

    Sulfolobus turreted icosahedral virus (STIV) was isolated in acidic hot springs where it infects the archeon Sulfolobus solfataricus. We determined the STIV structure using near-atomic resolution electron microscopy and X-ray crystallography allowing tracing of structural polypeptide chains and visualization of transmembrane proteins embedded in the viral membrane. We propose that the vertex complexes orchestrate virion assembly by coordinating interactions of the membrane and various protein components involved. STIV shares the same coat subunit and penton base protein folds as some eukaryotic and bacterial viruses, suggesting that they derive from a common ancestor predating the divergence of the three kingdoms of life. One architectural motif (β-jelly roll fold) forms virtually the entire capsid (distributed in three different gene products), indicating that a single ancestral protein module may have been at the origin of its evolution. PMID:23520050

  1. Nonspecific interactions of chromatin with immunoglobulin G and protein A, and their impact on purification performance.

    PubMed

    Gagnon, Pete; Nian, Rui; Lee, Jeremy; Tan, Lihan; Latiff, Sarah Maria Abdul; Lim, Chiew Ling; Chuah, Cindy; Bi, Xuezhi; Yang, Yuansheng; Zhang, Wei; Gan, Hui Theng

    2014-05-02

    Chromatin released from dead host cells during in vitro production of IgG monoclonal antibodies exists mostly in complex hetero-aggregates consisting of nucleosomal arrays (DNA+histone proteins), non-histone proteins, and aberrant forms of IgG. They bind immobilized protein A more aggressively than IgG, through their nucleosomal histone components, and hinder access of IgG to Fc-specific binding sites, thereby reducing dynamic binding capacity. The majority of host cell contaminants in eluted IgG are leachates from chromatin hetero-aggregates that remain bound to protein A. Formation of turbidity in eluted IgG during pH titration is caused by neutral-pH insolubility of chromatin hetero-aggregates. NaOH is required at 500 mM to remove accumulated chromatin. A chromatin-directed clarification method removed 99% of histones, 90% of non-histone proteins, achieved a 6 log reduction of DNA, 4 log reduction of lipid-enveloped virus, and 5 log reduction of non-enveloped retrovirus, while conserving 98% of the native IgG. This suspended most of performance compromises imposed on protein A. IgG binding capacity increased ~20%. Host protein contamination was reduced about 100-fold compared to protein A loaded with harvest clarified by centrifugation and microfiltration. Aggregates were reduced to less than 0.05%. Turbidity of eluted IgG upon pH neutralization was nearly eliminated. Column cleaning was facilitated by minimizing the accumulation of chromatin.

  2. Using Dali for structural comparison of proteins.

    PubMed

    Holm, Liisa; Kääriäinen, Sakari; Wilton, Chris; Plewczynski, Dariusz

    2006-07-01

    The Dali program is widely used for carrying out automatic comparisons of protein structures determined by X-ray crystallography or NMR. The most familiar version is the Dali server, which performs a database search comparing a query structure supplied by the user against the database of known structures (PDB) and returns the list of structural neighbors by e-mail. The more recently introduced DaliLite server compares two structures against each other and visualizes the result interactively. The Dali database is a structural classification based on precomputed all-against-all structural similarities within the PDB. The resulting hierarchical classification can be browsed on the Web and is linked to protein sequence classification resources. All Dali resources use an identical algorithm for structure comparison. Users may run Dali using the Web, or the program may be downloaded to be run locally on Linux computers.

  3. Remote Access to the PXRR Macromolecular Crystallography Facilities at the NSLS

    SciTech Connect

    Soares, A.S.; Schneider, D. K.; Skinner, J. M.; Cowan, M.; Buono, R.; Robinson, H. H.; Heroux, A.; Carlucci-Dayton, M.; Saxena, A.; Sweet, R. M.

    2008-09-01

    The most recent surge of innovations that have simplified and streamlined the process of determining macromolecular structures by crystallography owes much to the efforts of the structural genomics community. However, this was only the last step in a long evolution that saw the metamorphosis of crystallography from an heroic effort that involved years of dedication and skill into a straightforward measurement that is occasionally almost trivial. Many of the steps in this remarkable odyssey involved reducing the physical labor that is demanded of experimenters in the field. Other steps reduced the technical expertise required for conducting those experiments.

  4. Remote Access to the PXRR Macromolecular Crystallography Facilities at the NSLS

    SciTech Connect

    A Soares; D Schneider; J Skinner; M Cowan; R Buono; H Robinson; A Heroux; M Carlucci-Dayton; A Saxena; R Sweet

    2011-12-31

    The most recent surge of innovations that have simplified and streamlined the process of determining macromolecular structures by crystallography owes much to the efforts of the structural genomics community. However, this was only the last step in a long evolution that saw the metamorphosis of crystallography from an heroic effort that involved years of dedication and skill into a straightforward measurement that is occasionally almost trivial. Many of the steps in this remarkable odyssey involved reducing the physical labor that is demanded of experimenters in the field. Other steps reduced the technical expertise required for conducting those experiments.

  5. Optimized multi-step NMR-crystallography approach for structural characterization of a stable quercetin solvate.

    PubMed

    Filip, Xenia; Miclaus, Maria; Martin, Flavia; Filip, Claudiu; Grosu, Ioana Georgeta

    2017-01-31

    Herein we report the preparation and solid state structural investigation of the 1,4-dioxane-quercetin solvate. NMR crystallography methods were employed for crystal structure determination of the solvate from microcrystalline powder. The stability of the compound relative to other reported quercetin solvates is discussed and found to be in perfect agreement with the hydrogen bonding networks/supra-molecular architectures formed in each case. It is also clearly shown that NMR crystallography represents an ideal analytical tool in such cases when hydrogen-bonding networks are required to be constrained at a high accuracy level.

  6. Effect of salmon protein hydrolysate and spray-dried plasma protein on growth performance of weanling pigs.

    PubMed

    Tucker, J L; Naranjo, V D; Bidner, T D; Southern, L L

    2011-05-01

    Two experiments, each consisting of 2 trials, were conducted to determine the effect of salmon protein hydrolysate (SPH) and spray-dried plasma protein (SDPP) fed during the first week postweaning and their subsequent effect on the growth performance of weanling pigs. Pigs were fed in a 3-phase feeding program with durations of 7 d for phase 1 in both Exp. 1 and 2; 14 or 15 d for phase 2 in Exp. 1 and 2, respectively; and 7 or 8 d for phase 3 in Exp. 1 and 2, respectively. Dietary treatments were fed only during phase 1, whereas the same diet was fed to all pigs in phases 2 and 3. Pigs were blocked by initial BW and sex, and littermates were balanced across treatments. Data from the 2 trials within each experiment were combined and analyzed together; no treatment × trial interactions (P > 0.10) were observed. In Exp. 1, a total of 324 weanling pigs (10 replications of 5 or 6 pigs per pen) with an average initial BW of 6.4 ± 1.3 kg were assigned to 1) a control diet with no SPH or SDPP, 2) 1.5% SPH, 3) 3.0% SPH, 4) 1.5% SDPP, 5) 3.0% SDPP, or 6) 1.5% SPH + 1.5% SDPP. Experiment 2 was similar to Exp. 1, but red blood cells were removed from all diets to reduce diet complexity. In Exp. 2, weanling pigs (n = 320, 14 replications of 5 or 6 pigs per pen) with an average initial BW of 5.4 ± 1.2 kg were assigned to 1) a control diet with no SPH or SDPP, 2) 1.5% SPH, 3) 1.5% SDPP, or 4) 1.5% SPH + 1.5% SDPP. Three batches of SPH were used, and each batch was analyzed for AA composition. In Exp. 1, the inclusion of SDPP or SPH during phase 1 did not affect (P > 0.10) ADG, ADFI, or G:F compared with those of pigs fed the control diet. No carryover effects on growth performance were observed in any of the subsequent phases. Overall, G:F was greater (P = 0.08) in pigs fed the 1.5% diets compared with those fed the 3.0% diets. In Exp. 2, no differences (P > 0.10) were observed in ADG, ADFI, or G:F among pigs fed the SPH or SDPP diets compared with those of pigs fed the

  7. Self-assembled monolayers as a base for immunofunctionalisation: unequal performance for protein and bacteria detection.

    PubMed

    Baldrich, Eva; Laczka, Olivier; del Campo, F Javier; Muñoz, Francesc Xavier

    2008-03-01

    Biosensor development strongly depends on the optimisation of surface functionalisation strategies. When gold surfaces are considered, immunofunctionalisation by modification of self-assembled monolayers (SAMs) is one of the preferred approaches. In this respect, SAM-based antibody (Ab) incorporation has shown better performance than Ab physisorption for the detection of proteins and small targets. Reports on bacteria detection are less frequent. In this work, we assess the performance of various SAM-based gold immunofunctionalisation strategies, currently applied to protein detection, in the field of bacteria determination. We present the results for Ab chemical conjugation on mercaptopropanoic acid and mercaptoundecanoic acid SAMs, as well as on a dextranized cysteamine SAM. All the modified surfaces studied were shown to be appropriate for the direct detection of an enzyme-labelled protein, but none succeeded in detecting a bacterial target in a sandwich assay format. Conversely, gold functionalised by Ab physisorption allowed E. coli detection when a sandwich enzyme-linked assay was carried out. The implications of bacteria size and wall complexity are discussed. These results indicate that immunofunctionalisation strategies appropriate for protein detection are not necessarily transferable to work with more complex targets such as bacteria. In this respect, Ab physisorption appears to be a suitable alternative to SAM-based gold functionalisation for bacteria detection.

  8. Improvement of hydrophobic integral membrane protein identification by mild performic acid oxidation-assisted digestion.

    PubMed

    Cao, Rui; Liu, Yisong; Chen, Ping; Lv, Rong; Song, Qin; Sheng, Tingting; He, Quanyuan; Wang, Yin; Wang, Xianchun; Liang, Songping

    2010-12-15

    Integral membrane proteins (IMPs) are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of IMPs make them difficult to analyze. In proteomic analyses, hydrophobic peptides including transmembrane domains are often underrepresented, and this reduces the sequence coverage and reliability of the identified IMPs. Here we report a new strategy, mild performic acid oxidation treatment (mPAOT), for improvement of IMP identification. In the mPAOT strategy, the hydrophobicity of IMPs is significantly decreased by oxidizing their methionine and cysteine residues with performic acid, thereby improving the solubility and enzymolysis of these proteins. The application of the mPAOT strategy to the analysis of IMPs from human nasopharyngeal carcinoma CNE1 cell line demonstrated that many IMPs, including those with high hydrophobicity, could be reliably identified.

  9. Which strategy for a protein crystallization project?

    NASA Technical Reports Server (NTRS)

    Kundrot, C. E.

    2004-01-01

    The three-dimensional, atomic-resolution protein structures produced by X-ray crystallography over the past 50+ years have led to tremendous chemical understanding of fundamental biochemical processes. The pace of discovery in protein crystallography has increased greatly with advances in molecular biology, crystallization techniques, cryocrystallography, area detectors, synchrotrons and computing. While the methods used to produce single, well-ordered crystals have also evolved over the years in response to increased understanding and advancing technology, crystallization strategies continue to be rooted in trial-and-error approaches. This review summarizes the current approaches in protein crystallization and surveys the first results to emerge from the structural genomics efforts.

  10. Effects of protein supplementation on muscular performance and resting hormonal changes in college football players.

    PubMed

    Hoffman, Jay R; Ratamess, Nicholas A; Kang, Jie; Falvo, Michael J; Faigenbaum, Avery D

    2007-01-01

    The effect of protein supplementation on athletic performance and hormonal changes was examined in 21 experienced collegiate strength/power athletes participating in a 12-week resistance training program. Subjects were randomly assigned to either a protein supplement (PR; n = 11) or a placebo (PL; n = 10) group. During each testing session subjects were assessed for strength (one repetition maximum [1-RM] bench press and squat), power (Wingate anaerobic power test) and body composition. Resting blood samples were analyzed at weeks 0 (PRE), 6 (MID) and 12 (POST) for total testosterone, cortisol, growth hormone, and IGF-1. No difference was seen in energy intake between PR and PL (3034 ± 209 kcal and 3130 ± 266 kcal, respectively), but a significant difference in daily protein intake was seen between PR (2.00 g·kg body mass[BM](-1)·d(-1)) and PL (1.24 g·kgBM(-1)·d(-1)). A greater change (p < 0.05) in the ∆ 1-RM squat was seen in PR (23.5 ± 13.6 kg) compared to PL (9.1 ± 11.9 kg). No other significant strength or power differences were seen between the groups. Cortisol concentrations were significantly lower at MID for PL and this difference was significantly different than PR. No significant changes were noted in resting growth hormone or IGF-1 concentrations in either group. Although protein supplementation appeared to augment lower body strength development, similar upper body strength, anaerobic power and lean tissue changes do not provide clear evidence supporting the efficacy of a 12-week protein supplementation period in experienced resistance trained athletes. Key pointsCollegiate strength/power athletes may not meet daily recommended energy or protein needs.When athletes are provided a protein supplement they appear to meet the recommended daily protein intake for strength/power athletes.Protein supplementation did augment lower body strength development in experienced strength/power athletes.Results of upper body strength, anaerobic power and lean

  11. Protein crystal growth and the International Space Station

    NASA Technical Reports Server (NTRS)

    DeLucas, L. J.; Moore, K. M.; Long, M. M.

    1999-01-01

    Protein structural information plays a key role in understanding biological structure-function relationships and in the development of new pharmaceuticals for both chronic and infectious diseases. The Center for Macromolecular Crystallography (CMC) has devoted considerable effort studying the fundamental processes involved in macromolecular crystal growth both in a 1-g and microgravity environment. Results from experiments performed on more than 35 U.S. space shuttle flights have clearly indicated that microgravity can provide a beneficial environment for macromolecular crystal growth. This research has led to the development of a new generation of pharmaceuticals that are currently in preclinical or clinical trials for diseases such as cutaneous T-cell lymphoma, psoriasis, rheumatoid arthritis, AIDS, influenza, stroke and other cardiovascular complications. The International Space Station (ISS) provides an opportunity to have complete crystallographic capability on orbit, which was previously not possible with the space shuttle orbiter. As envisioned, the x-ray Crystallography Facility (XCF) will be a complete facility for growing protein crystals; selecting, harvesting, and mounting sample crystals for x-ray diffraction; cryo-freezing mounted crystals if necessary; performing x-ray diffraction studies; and downlinking the data for use by crystallographers on the ground. Other advantages of such a facility include crystal characterization so that iterations in the crystal growth conditions can be made, thereby optimizing the final crystals produced in a three month interval on the ISS.

  12. Performance characteristics needed for protein crystal diffraction x-ray detectors.

    SciTech Connect

    Westbrook, E. M.

    1999-09-21

    During the 1990's, macromolecular crystallography became progressively more dependent on synchrotrons X-ray sources for diffraction data collection. Detectors of this diffraction data at synchrotrons beamlines have evolved over the decade, from film to image phosphor plates, and then to CCD systems. These changes have been driven by the data quality and quantity improvements each newer detector technology provided. The improvements have been significant. It is likely that newer detector technologies will be adopted at synchrotron beamlines for crystallographic diffraction data collection in the future, but these technologies will have to compete with existing CCD detector systems which are already excellent and are getting incrementally better in terms of size, speed, efficiency, and resolving power. Detector development for this application at synchrotrons must concentrate on making systems which are bigger and faster than CCDs and which can capture weak data more efficiently. And there is a need for excellent detectors which are less expensive than CCD systems.

  13. Contributions of NIST/NBS Researchers to the Crystallography of Construction Materials

    PubMed Central

    Stutzman, Paul

    2001-01-01

    For more than 100 years, the primary theme underlying the NBS/NIST staff contribution to the crystallography of building materials has been the development of an improved understanding of concrete materials performance. Over that time period, portland cement concrete has become one of the most important of our construction materials for roads, buildings, and other large municipal structures. At the beginning of the 20th century our understanding of portland cement composition, performance, use in concrete, and how the concrete performs in harsh environments was lacking. The efforts of NIST have served to advance construction materials science and technology through the combined efforts of experimental, field study, and theoretical computational materials science. One major achievement in the late 1920s, derived from studies on phase equilibria in cement clinker, allows calculation of potential cement clinker composition. Known as the Bogue calculation, this continues to be an essential tool in cement plant process control to this day. Additionally, contributions of NIST scientists to our knowledge of the chemistry and nature of cement hydration products have been crucial in our understanding of cement hydration and concrete durability. Today, computational materials science is a rapidly developing discipline, and NIST is developing tools incorporating predictive models aided by empirical studies. Examples include a computer-integrated knowledge system for prediction and optimization of performance and life-cycle cost of high performance concrete and the Virtual Cement and Concrete Testing Laboratory. Understanding the relationships between material and performance properties has not been confined only to portland cements. One of the longest running experiments at NIST, the stone test wall, has stood for over 50 years as one of the world’s largest single collections of building stone, and is invaluable for studying weathering effects associated with stone

  14. Frontiers of Crystallography: A Project-Based Research-Led Learning Exercise

    ERIC Educational Resources Information Center

    Wilson, Chick C.; Parkin, Andrew; Thomas, Lynne H.

    2012-01-01

    A highly interactive research-led learning session for chemistry undergraduates is described, which aims to lead students to an awareness of the applications of crystallography technique through a mentored hands-on crystal structure solution and refinement session. The research-based environment is inherent throughout the 4.5 h program and is…

  15. Celebrating the International Year of Crystallography with a Wisconsin High School Crystal Growing Competition

    ERIC Educational Resources Information Center

    Guzei, Ilia A.

    2014-01-01

    In honor of the 2014 International Year of Crystallography, the first Wisconsin Crystal Growing Competition was successfully organized and conducted. High school students from 26 schools across the state competed for prizes by growing large crystals of CuSO[subscript4]·5(H[subscript2]O). This paper describes how the event was planned and carried…

  16. Using the Plan View to Teach Basic Crystallography in General Chemistry

    ERIC Educational Resources Information Center

    Cushman, Cody V.; Linford, Matthew R.

    2015-01-01

    The plan view is used in crystallography and materials science to show the positions of atoms in crystal structures. However, it is not widely used in teaching general chemistry. In this contribution, we introduce the plan view, and show these views for the simple cubic, body-centered cubic, face-centered cubic, hexagonal close packed, CsCl, NaCl,…

  17. 100 Years later: Celebrating the contributions of x-ray crystallography to allergy and clinical immunology.

    PubMed

    Pomés, Anna; Chruszcz, Maksymilian; Gustchina, Alla; Minor, Wladek; Mueller, Geoffrey A; Pedersen, Lars C; Wlodawer, Alexander; Chapman, Martin D

    2015-07-01

    Current knowledge of molecules involved in immunology and allergic disease results from the significant contributions of x-ray crystallography, a discipline that just celebrated its 100th anniversary. The histories of allergens and x-ray crystallography are intimately intertwined. The first enzyme structure to be determined was lysozyme, also known as the chicken food allergen Gal d 4. Crystallography determines the exact 3-dimensional positions of atoms in molecules. Structures of molecular complexes in the disciplines of immunology and allergy have revealed the atoms involved in molecular interactions and mechanisms of disease. These complexes include peptides presented by MHC class II molecules, cytokines bound to their receptors, allergen-antibody complexes, and innate immune receptors with their ligands. The information derived from crystallographic studies provides insights into the function of molecules. Allergen function is one of the determinants of environmental exposure, which is essential for IgE sensitization. Proteolytic activity of allergens or their capacity to bind LPSs can also contribute to allergenicity. The atomic positions define the molecular surface that is accessible to antibodies. In turn, this surface determines antibody specificity and cross-reactivity, which are important factors for the selection of allergen panels used for molecular diagnosis and the interpretation of clinical symptoms. This review celebrates the contributions of x-ray crystallography to clinical immunology and allergy, focusing on new molecular perspectives that influence the diagnosis and treatment of allergic diseases.

  18. Effects of growth rate on cell extract performance in cell-free protein synthesis.

    PubMed

    Zawada, James; Swartz, James

    2006-07-05

    Cell-free protein synthesis is a useful research tool and now stands poised to compete with in vivo expression for commercial production of proteins. However, both the extract preparation and protein synthesis procedures must be scaled up. A key challenge is producing the required amount of biomass that also results in highly active cell-free extracts. In this work, we show that the growth rate of the culture dramatically affects extract performance. Extracts prepared from cultures with a specific growth rate of 0.7/h or higher produced approximately 0.9 mg/mL of chloramphenicol acetyl transferase (CAT) in a batch reaction. In contrast, when the source culture growth rate was 0.3/h, the resulting extract produced only 0.5 mg/mL CAT. Examination of the ribosome content in the extracts revealed that the growth rate of the source cells strongly influenced the final ribosome concentration. Polysome analysis of cell-free protein synthesis reactions indicated that about 22% of the total 70S ribosomes are in polysomes for all extracts regardless of growth rate. Furthermore, the overall specific production from the 70S ribosomes is about 22 CAT proteins per ribosome over the course of the reaction in all cases. It appears that rapid culture growth rates are essential for producing a productive extract. However, growth rate does not seem to influence specific ribosome activity. Rather, the increase in extract productivity is a result of a higher ribosome concentration. These results are important for cell-free technology and also suggest an assay for intrinsic in vivo protein synthesis activity.

  19. Performance of the Red-shifted Fluorescent Proteins in deep-tissue molecular imaging applications

    PubMed Central

    Deliolanis, Nikolaos C.; Kasmieh, Randa; Würdinger, Thomas; Tannous, Bakhos A.; Shah, Khalid; Ntziachristos, Vasilis

    2009-01-01

    The discovery of new fluorescent proteins (FPs) that emit in the far-red part of the spectrum, where light absorption from tissue is significantly lower than in the visible, offers the possibility for non-invasive biological interrogation at the entire organ or small animal level in vivo. The performance of FPs in deep-tissue imaging depends not only on their optical characteristics, but also on the wavelength-dependent tissue absorption and the depth of the fluorescence activity. In order to determine the optimal choice of FP and illumination wavelength we compared the performance of five of the most promising FPs, i.e. tdTomato, mCherry, mRaspberry, mPlum, and Katushka. We experimentally measured the signal strength through mice and employed theoretical predictions to obtain an understanding on the performance of different illumination scenarios, especially as it pertains to tomographic imaging. It was found that the appropriate combination of red-shifted proteins and illumination wavelengths can improve detection sensitivity in small animals by at least 2 orders of magnitude compared to Green FP (GFP). It is also shown that the steep attenuation change of the hemoglobin spectrum around the 600nm range may significantly affect the detection sensitivity and necessitates the careful selection of illumination wavelengths for optimal imaging performance. PMID:19021336

  20. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    SciTech Connect

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; Drennan, Catherine L.; Baker, David; Ting, Alice Y.

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.

  1. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    DOE PAGES

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; ...

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of themore » intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.« less

  2. Effect of wet corn gluten feed, supplemental protein, and tallow on steer finishing performance.

    PubMed

    Richards, C J; Stock, R A; Klopfenstein, T J; Shain, D H

    1998-02-01

    Two trials evaluated the effects of level of wet corn gluten feed (WCGF), type of supplemental protein, and supplemental tallow on steer finishing performance. In Trial 1, WCGF was fed at 0, 25 (two diets), or 50% of the dietary DM replacing dry-rolled corn (DRC), molasses, and a portion of the supplement. The DRC control diet and one 25% WCGF diet were supplemented with a combination of protein sources. The second 25% WCGF diet was supplemented with urea alone. The 50% WCGF diet contained no additional protein supplementation. No differences in DMI (P > .10) were observed. Calves fed 25% WCGF plus a combination of protein sources or 50% WCGF gained faster (P < .10) and more efficiently (P < .10) than calves fed the DRC control. Calves fed 25% WCGF plus urea gained faster (P < .10) and tended (P = .14) to be more efficient than calves fed the DRC control. In Trial 2, WCGF was fed to replace 0 or 50% of the DRC and the molasses-urea supplement (DM basis). Both diets were fed with or without 3% tallow. Steers fed WCGF gained faster (P < .01) and more efficiently (P < .01) than steers fed DRC. Inclusion of 3% tallow increased gain (P < .05) and improved efficiency (P < .05). Feed efficiency is improved by the addition of WCGF or tallow to DRC finishing diets.

  3. Analysis of protein-glutathione mixed disulfides by high performance liquid chromatography.

    PubMed

    Meredith, M J

    1983-06-01

    After precipitation of proteins; serum, hepatocytes, or glutathione-derivatized bovine serum albumin, by perchloric acid, dithiothreitol was used to reduce glutathione-protein mixed disulfides in the ether-washed, resuspended pellet. Following neutralization and S-carboxymethylation of free sulfhydral groups in the acid soluble fraction by iodoacetic acid. 2,4-dinitrophenyl derivatives of released compounds were produced by addition of ethanolic fluorodinitrobenzene. The 2,4-dinitrophenyl derivative of S-carboxymethylglutathione was measured by high-performance liquid chromatography. The method was found to be reproducible and limited only by the sensitivity of the glutathione analysis (about 10 pmol/sample). Quantitation of protein-bound glutathione was shown to be independent of the ratio of bound to soluble glutathione as well as the protein concentration in the sample. This method was found to produce glutathione values identical to those measured after borohydride reduction without the problems of foaming, sample loss, and the need of continuous pH adjustment during reduction.

  4. Newly engineered cyan fluorescent proteins with enhanced performances for live cell FRET imaging.

    PubMed

    Mérola, Fabienne; Fredj, Asma; Betolngar, Dahdjim-Benoît; Ziegler, Cornelia; Erard, Marie; Pasquier, Hélène

    2014-02-01

    Cyan fluorescent proteins (CFPs) derived from Aequorea victoria green fluorescent protein are the most widely used Förster resonant energy transfer (FRET) donors in genetically encoded biosensors for live-cell imaging and bioassays. However, the weak and complex fluorescence emission of cyan variants, such as enhanced cyan fluorescent protein (ECFP) or Cerulean, has long remained a major bottleneck in these FRET techniques. Recently, several CFPs with greatly improved performances, including mTurquoise, mTurquoise2, mCerulean3, and Aquamarine, have been engineered through a mixture of site-directed and large-scale random mutagenesis. This review summarizes the engineering and relative merits of these new cyan donors, which can readily replace popular CFPs in FRET imaging protocols, while reaching fluorescence quantum yields close to 90%, and unprecedented long, near-single fluorescence lifetimes of about 4 ns. These variants display an increased general photostability and much reduced environmental sensitivity, notably towards acid pH. These new, bright, and robust CFPs now open up exciting outlooks for fluorescence lifetime imaging microscopy and advanced quantitative FRET analyses in living cells. In addition, the stepwise engineering of Aquamarine shows that only two critical mutations in ECFP, and one in Cerulean, are required to achieve these performances, which brings new insights into the structural bases of their photophysical properties.

  5. Effects of prebiotic, protein level, and stocking density on performance, immunity, and stress indicators of broilers.

    PubMed

    Houshmand, M; Azhar, K; Zulkifli, I; Bejo, M H; Kamyab, A

    2012-02-01

    An experiment was conducted to determine the effects of period on the performance, immunity, and some stress indicators of broilers fed 2 levels of protein and stocked at a normal or high stocking density. Experimental treatments consisted of a 2 × 2 × 2 factorial arrangement with 2 levels of prebiotic (with or without prebiotic), 2 levels of dietary CP [NRC-recommended or low CP level (85% of NRC-recommended level)], and 2 levels of stocking density (10 birds/m(2) as the normal density or 16 birds/m(2) as the high density), for a total of 8 treatments. Each treatment had 5 replicates (cages). Birds were reared in 3-tiered battery cages with wire floors in an open-sided housing system under natural tropical conditions. Housing and general management practices were similar for all treatment groups. Starter and finisher diets in mash form were fed from 1 to 21 d and 22 to 42 d of age, respectively. Supplementation with a prebiotic had no significant effect on performance, immunity, and stress indicators (blood glucose, cholesterol, corticosterone, and heterophil:lymphocyte ratio). Protein level significantly influenced broiler performance but did not affect immunity or stress indicators (except for cholesterol level). The normal stocking density resulted in better FCR and also higher antibody titer against Newcastle disease compared with the high stocking density. However, density had no significant effect on blood levels of glucose, cholesterol, corticosterone, and the heterophil:lymphocyte ratio. Significant interactions between protein level and stocking density were observed for BW gain and final BW. The results indicated that, under the conditions of this experiment, dietary addition of a prebiotic had no significant effect on the performance, immunity, and stress indicators of broilers.

  6. Effects of dietary protein concentration on performance and nutrient digestibility in Pekin ducks during aflatoxicosis.

    PubMed

    Chen, X; Murdoch, R; Zhang, Q; Shafer, D J; Applegate, T J

    2016-04-01

    A 14-d study was conducted to determine the impact of dietary crude protein concentration on performance, serum biochemistry, and nutrient digestive functions in Pekin ducklings during aflatoxicosis. A total of 144 male Pekin ducklings were randomly allotted to 4 dietary treatments arranged in a 2×2 factorial with 2 crude protein (CP) (20 and 24% on an analyzed basis) with or without 0.2 mg/kg aflatoxin B1 (AFB1) (0.21 mg/kg analyzed). The AFB1 reduced BW gain, feed intake, and breast muscle weight by 33 to 43% (P<0.0001). Serum concentration of protein, glucose, and Ca were also decreased by AFB1 (P≤0.0015), while pancreatic activities of amylase and lipase were increased by AFB1 (P<0.005). Apparent N digestibility was not affected by dietary treatment, whereas apparent ileal digestible energy was reduced 7.6% by AFB1 (P=0.0003). Higher dietary CP improved BW gain, gain:feed ratio, and breast muscle weight (P≤0.021), and tended to improve feed intake (P=0.094), but did not improve serum measures, digestive enzyme activity, or nutrient digestibility. No statistical interaction of AFB1 by CP was observed for any measures. Results from the current study suggest that AFB1 at low concentration can significantly impair performance of Pekin ducklings primarily through inhibited feed intake, as well as influence nutrient digestion processes (jejunum morphology, digestive enzyme activity, and apparent energy digestibility). Higher dietary CP can improve growth performance of ducklings regardless of AF exposure, but did not interact with dietary AFB1 on performance, serum biochemistry, or nutrient digestion in Pekin ducklings from hatch to 14 d.

  7. Macromolecular Crystallography conventional and high-throughput methods

    SciTech Connect

    Wasserman, Stephen R.; Smith, David W.; D'Amico, Kevin L.; Koss, John W.; Morisco, Laura L.; Burley, Stephen K.

    2007-09-27

    High-throughput data collection requires the seamless interoperation of various hardware components. User-supplied descriptions of protein crystals must also be directly linked with the diffraction data. Such linkages can be achieved efficiently with computer databases. A database that tracks production of the protein samples, crystallization, and diffraction from the resultant crystals serves as the glue that holds the entire gene-to-structure process together. This chapter begins by discussing data collection processes and hardware. It then illustrates how a well-constructed database ensures information flow through the steps of data acquisition. Such a database allows synchrotron beamline measurements to be directly and efficiently integrated into the process of protein crystallographic structure determination.

  8. Scientist prepare Lysozyme Protein Crystal

    NASA Technical Reports Server (NTRS)

    1996-01-01

    Dan Carter and Charles Sisk center a Lysozyme Protein crystal grown aboard the USML-2 shuttle mission. Protein isolated from hen egg-white and functions as a bacteriostatic enzyme by degrading bacterial cell walls. First enzyme ever characterized by protein crystallography. It is used as an excellent model system for better understanding parameters involved in microgravity crystal growth experiments. The goal is to compare kinetic data from microgravity experiments with data from laboratory experiments to study the equilibrium.

  9. A High Performance Cloud-Based Protein-Ligand Docking Prediction Algorithm

    PubMed Central

    Chen, Jui-Le; Yang, Chu-Sing

    2013-01-01

    The potential of predicting druggability for a particular disease by integrating biological and computer science technologies has witnessed success in recent years. Although the computer science technologies can be used to reduce the costs of the pharmaceutical research, the computation time of the structure-based protein-ligand docking prediction is still unsatisfied until now. Hence, in this paper, a novel docking prediction algorithm, named fast cloud-based protein-ligand docking prediction algorithm (FCPLDPA), is presented to accelerate the docking prediction algorithm. The proposed algorithm works by leveraging two high-performance operators: (1) the novel migration (information exchange) operator is designed specially for cloud-based environments to reduce the computation time; (2) the efficient operator is aimed at filtering out the worst search directions. Our simulation results illustrate that the proposed method outperforms the other docking algorithms compared in this paper in terms of both the computation time and the quality of the end result. PMID:23762864

  10. Automated High Throughput Protein Crystallization Screening at Nanoliter Scale and Protein Structural Study on Lactate Dehydrogenase

    SciTech Connect

    Li, Fenglei

    2006-08-09

    The purposes of our research were: (1) To develop an economical, easy to use, automated, high throughput system for large scale protein crystallization screening. (2) To develop a new protein crystallization method with high screening efficiency, low protein consumption and complete compatibility with high throughput screening system. (3) To determine the structure of lactate dehydrogenase complexed with NADH by x-ray protein crystallography to study its inherent structural properties. Firstly, we demonstrated large scale protein crystallization screening can be performed in a high throughput manner with low cost, easy operation. The overall system integrates liquid dispensing, crystallization and detection and serves as a whole solution to protein crystallization screening. The system can dispense protein and multiple different precipitants in nanoliter scale and in parallel. A new detection scheme, native fluorescence, has been developed in this system to form a two-detector system with a visible light detector for detecting protein crystallization screening results. This detection scheme has capability of eliminating common false positives by distinguishing protein crystals from inorganic crystals in a high throughput and non-destructive manner. The entire system from liquid dispensing, crystallization to crystal detection is essentially parallel, high throughput and compatible with automation. The system was successfully demonstrated by lysozyme crystallization screening. Secondly, we developed a new crystallization method with high screening efficiency, low protein consumption and compatibility with automation and high throughput. In this crystallization method, a gas permeable membrane is employed to achieve the gentle evaporation required by protein crystallization. Protein consumption is significantly reduced to nanoliter scale for each condition and thus permits exploring more conditions in a phase diagram for given amount of protein. In addition

  11. Automatic recovery of missing amplitudes and phases in tilt-limited electron crystallography of two-dimensional crystals

    NASA Astrophysics Data System (ADS)

    Gipson, Bryant R.; Masiel, Daniel J.; Browning, Nigel D.; Spence, John; Mitsuoka, Kaoru; Stahlberg, Henning

    2011-07-01

    Electron crystallography of 2D protein crystals provides a powerful tool for the determination of membrane protein structure. In this method, data is acquired in the Fourier domain as randomly sampled, uncoupled, amplitudes and phases. Due to physical constraints on specimen tilting, those Fourier data show a vast un-sampled “missing cone” of information, producing resolution loss in the direction perpendicular to the membrane plane. Based on the flexible language of projection onto sets, we provide a full solution for these problems with a projective constraint optimization algorithm that, for sufficiently oversampled data, produces complete recovery of unmeasured data in the missing cone. We apply this method to an experimental data set of Bacteriorhodopsin and show that, in addition to producing superior results compared to traditional reconstruction methods, full, reproducible, recovery of the missing cone from noisy data is possible. Finally, we present an automatic implementation of the refinement routine as open source, freely distributed, software that will be included in our 2dx software package.

  12. Automatic recovery of missing amplitudes and phases in tilt-limited electron crystallography of two-dimensional crystals

    SciTech Connect

    Gipson, Bryant R.; Stahlberg, Henning; Masiel, Daniel J.; Browning, Nigel D.; Spence, John; Mitsuoka, Kaoru

    2011-07-15

    Electron crystallography of 2D protein crystals provides a powerful tool for the determination of membrane protein structure. In this method, data is acquired in the Fourier domain as randomly sampled, uncoupled, amplitudes and phases. Due to physical constraints on specimen tilting, those Fourier data show a vast un-sampled ''missing cone'' of information, producing resolution loss in the direction perpendicular to the membrane plane. Based on the flexible language of projection onto sets, we provide a full solution for these problems with a projective constraint optimization algorithm that, for sufficiently oversampled data, produces complete recovery of unmeasured data in the missing cone. We apply this method to an experimental data set of Bacteriorhodopsin and show that, in addition to producing superior results compared to traditional reconstruction methods, full, reproducible, recovery of the missing cone from noisy data is possible. Finally, we present an automatic implementation of the refinement routine as open source, freely distributed, software that will be included in our 2dx software package.

  13. Functional Consequences of the Open Distal Pocket of Dehaloperoxidase-Hemoglobin Observed by Time-Resolved X-ray Crystallography

    PubMed Central

    Zhao, Junjie; Srajer, Vukica; Franzen, Stefan

    2014-01-01

    Using time-resolved X-ray crystallography, we contrast a bifunctional dehaloperoxidase-hemoglobin (DHP) with previously studied examples of myoglobin and hemoglobin in order to understand the functional role of the distal pocket of globins. One key functional difference between the DHP and other globins is the requirement that H2O2 enter the distal pocket of oxyferrous DHP in order to displace O2 from the heme Fe atom and thereby activate the heme for the peroxidase function. The open architecture of DHP permits more than one molecule to simultaneously enter the distal pocket of the protein above the heme in order to facilitate the unique peroxidase cycle starting from the oxyferrous state. The time-resolved X-ray data show that the distal pocket of DHP lacks a protein valve found in the two other globins that have been studied previously. The photolyzed CO ligand trajectory in DHP does not have a docking site. Rather the CO moves immediately to the Xe-binding site. From there CO can escape, but also recombine an order of magnitude more rapidly than in other globins. The contrast with DHP dynamics and function more precisely defines the functional role of the multiple conformational states of myoglobin. Taken together with the high reduction potential of DHP, the open distal site helps to explain how a globin can also function as a peroxidase. PMID:24116924

  14. Exploring the speed and performance of molecular replacement with AMPLE using QUARK ab initio protein models.

    PubMed

    Keegan, Ronan M; Bibby, Jaclyn; Thomas, Jens; Xu, Dong; Zhang, Yang; Mayans, Olga; Winn, Martyn D; Rigden, Daniel J

    2015-02-01

    AMPLE clusters and truncates ab initio protein structure predictions, producing search models for molecular replacement. Here, an interesting degree of complementarity is shown between targets solved using the different ab initio modelling programs QUARK and ROSETTA. Search models derived from either program collectively solve almost all of the all-helical targets in the test set. Initial solutions produced by Phaser after only 5 min perform surprisingly well, improving the prospects for in situ structure solution by AMPLE during synchrotron visits. Taken together, the results show the potential for AMPLE to run more quickly and successfully solve more targets than previously suspected.

  15. The ingestion of protein with a maltodextrin and fructose beverage on substrate utilisation and exercise performance.

    PubMed

    Tarpey, Michael D; Roberts, Justin D; Kass, Lindsy S; Tarpey, Richard J; Roberts, Michael G

    2013-12-01

    The study investigated the ingestion of maltodextrin, fructose, and protein on exogenous carbohydrate oxidation (CHOEXO) and exercise performance. Seven trained cyclists and (or) triathletes (maximal oxygen consumption, 59.20 ± 9.00 mL · kg(-1) · min(-1)) performed 3 exercise trials that consisted of 150 min of cycling at 50% maximal power output (160 ± 11 W), followed by a 60-km time trial. One of 3 beverages were randomly assigned during each trial and consumed at 15-min intervals: (i) 0.84 g · min(-1) maltodextrin + 0.52 g · min(-1) fructose + 0.34 g · min(-1) protein (MD+F+P); (ii) 1.10 g · min(-1) maltodextrin + 0.60 g · min(-1) fructose (MD+F); or (iii) 1.70 g · min(-1) maltodextrin (MD). CHO(EXO) and fuel utilisation were assessed via measurement of expired air (13)C content and indirect calorimetry, respectively. Mean total CHO oxidation (CHOTOT) rates were 2.35 ± 0.18, 2.76 ± 0.08, and 2.61 ± 0.17 g · min(-1) with MD, MD+F, and MD+F+P, respectively, although not significantly different. Peak CHO(EXO) rates with MD+F were significantly greater by 41.4% (p = 0.001) and 45.4% (p = 0.0001) compared with MD+F+P and MD, respectively (1.57 ± 0.22 g · min(-1), 1.11 ± 0.08 g · min(-1), and 1.08 ± 0.11 g · min(-1), respectively). Performance times were 2.2% and 5.0% faster with MD+F compared with MD+F+P and MD, respectively; however, they were not statistically significant. Ingestion of an MD-fructose-protein commercial sports beverage significantly reduced peak and mean CHO(EXO) rates compared with MD+F, but did not significantly influence CHOTOT. The addition of protein to an MD+F beverage did not enhance performance times.

  16. Fabrication and characterization of aligned macroporous monolith for high-performance protein chromatography.

    PubMed

    Du, Kaifeng; Zhang, Qi; Dan, Shunmin; Yang, Min; Zhang, Yongkui; Chai, Dezhi

    2016-04-22

    In the present study, a freeze casting method combined with particle accumulation was applied to fabricate the aligned macroporous monolith for high-performance protein chromatography. For the preparation, the reactive colloids were first prepared by using glycidyl methacrylate and ethylene glycol dimethacrylate as monomers. Subsequently, these colloids accumulated regularly and polymerized into the aligned macroporous monolith. The aligned porous structure of the monolith was characterized by SEM, mercury intrusion, and flow hydrodynamics. The results revealed that the generated monolith was possessed of aligned macropores in size of about 10 μm and high column permeability. Finally, after being modified with sulfonated groups, the monolith was evaluated for its chromatographic performance. It demonstrated that the aligned macropores endowed the monolith with excellent adsorption capacity and high column efficiency.

  17. Effect of four processed animal proteins in the diet on digestibility and performance in laying hens.

    PubMed

    van Krimpen, M M; Veldkamp, T; Binnendijk, G P; de Veer, R

    2010-12-01

    An experiment was performed to investigate the effect of animal vs. vegetable protein sources in the diet of laying hens on the development of hen performance. A diet containing protein sources of only vegetable origin was compared with 4 diets, each containing 1 of 4 processed animal proteins (PAP). Two PAP (Daka-58 and Sonac-60) were classified as meat meals, and the remaining 2 (Daka-40 and Sonac-50) were classified as meat and bone meals. First, fecal digestibility of nutrients in the PAP was determined in Lohmann Brown layers. Hens (n = 132) were housed in 22 cages (6 hens/cage) and allotted to 5 dietary treatments. In the PAP diets (4 replicates/treatment), 100 g/kg of CP of animal origin was added, replacing soybean meal and corn (Zea mays) in the basal diet (6 replicates/treatment). The PAP sources differed largely in chemical composition and digestibility coefficients. Energy content (AME(n)) varied from 1,817 (Daka-40) to 3,107 kcal/kg (Sonac-60), and digestible lysine varied from 15.4 (Daka-40) to 28.3 g/kg (Sonac-50). Subsequently, the effect of a control diet (without PAP) vs. 4 PAP diets (50 g/kg of CP of animal origin from the same batches as used in the digestibility study) on performance was determined. All diets were isocaloric (AME(n) = 2,825 kcal/kg) and isonitrogenous (digestible lysine = 6.8 g/kg). Hens were housed in 40 floor pens (12 hens/pen, 8 pens/treatment) from 20 to 40 wk of age. Feed intake levels of the hens fed the meat and bone meal diets were reduced compared with those of hens fed the meat meal diets, whereas the feed intake level of hens fed the control diet was intermediate. Laying hen performance differed between treatments, being was most favorable for the Sonac-50 treatment and most adverse for the Daka-40 treatment. Differences in laying hen performance seemed to be related partly to differences in feed intake and corresponding amino acid intake.

  18. Protein source and dietary structure influence growth performance, gut morphology, and hindgut fermentation characteristics in broilers.

    PubMed

    Qaisrani, S N; Moquet, P C A; van Krimpen, M M; Kwakkel, R P; Verstegen, M W A; Hendriks, W H

    2014-12-01

    An experiment with 210 male (Ross 308) 1-d-old broilers was conducted to test the hypothesis that a coarse diet improves performance of broilers fed a poorly digestible protein source. A highly digestible diet based on soybean meal was gradually replaced by a low digestible diet based on rapeseed meal (RSM) in 5 steps (RSM-0%, RSM-25%, RSM-50%, RSM-75%, and RSM-100%). Two diet structures (fine and coarse) were used as an additional factor. These 2 factors and their interactions were tested at different ages in a factorial arrangement with 10 dietary treatments. An increase in indigestible dietary protein negatively affected feed intake (P = 0.003), BW gain (P = 0.008), and feed conversion ratio (P = 0.034). This increase in dietary indigestible protein contents resulted in a decrease (P = 0.001) in total cecal volatile fatty acid concentration from 209.1 to 125.9 mmol/kg of DM digesta in broilers with increasing RSM in diets. Increase in the indigestible protein level, from RSM-0% to RSM-100%, resulted in a decrease (P = 0.042) in villus heights (1,782 vs. 1,574 µm), whereas crypt depths increased (P = 0.021; 237 vs. 274 µm). A coarse diet improved feed intake (P = 0.006), BW gain (P = 0.014), and feed conversion ratio (P = 0.009). Broilers fed coarse diets had approximately 11, 24, and 10% lower relative empty weights of the crop, proventriculus, and jejunum, respectively, whereas a 15% heavier gizzard was found compared with those fed the fine diets. Dietary coarseness resulted in approximately 16% lower gizzard pH, 21% greater villus heights, 27% lower crypt depths, 24% reduced branched-chain fatty acids, and 12% lower biogenic amines in the cecal digesta compared with broilers fed fine diets. In conclusion, feeding coarse particles improved broiler performance irrespective of digestibility of the diet. Hindgut protein fermentation can be reduced by coarse grinding of the diet.

  19. Peripartum performance and metabolism of dairy cows in response to prepartum energy and protein intake.

    PubMed

    Doepel, L; Lapierre, H; Kennelly, J J

    2002-09-01

    Twenty-six multiparous Holstein cows were used to examine the effects of prepartum energy and protein intake on periparturient metabolism and lactation performance. Two levels of energy, 1.65 Mcal/kg of net energy for lactation (NEL) and 1.30 Mcal/kg of NEL, and two levels of protein, 17.0% CP and 12.5% CP, were tested according to a factorial arrangement in a randomized block design. Dietary treatments were fed ad libitum from 21 d before expected calving date to the day of calving. After calving, all cows were fed the same diet. Increased nutrient density did not affect prepartum feed intake, but postpartum intake was higher for cows fed the high-energy diets. Treatment had no effect on cow body weight and body condition score, however, cows fed the high-energy diets were in greater energy balance throughout the study. Milk and milk component yields were unaffected by treatment. Cows fed the high-energy diets had lower plasma nonesterified fatty acid concentrations than cows fed the low energy diets (354.3 vs. 439.9 mumol/L). Hepatic triglyceride concentrations were lower for cows on the high-energy diets than for those on the low-energy diets. Liver glycogen was unaffected by treatment. Acetyl-CoA carboxylase and fatty acid synthase abundance was significantly lower at calving than pretreatment, and higher for cows on the high-energy diets relative to those on the low-energy diets. The activity of acetyl-CoA carboxylase and lipoprotein lipase was greatly decreased with the onset of lactation. Increased protein intake prepartum resulted in elevated plasma beta-hydroxybutyrate concentrations postpartum. Prepartum plasma urea nitrogen was increased and 3-methylhistidine decreased by the high protein treatments. Overall, increased energy density of prepartum diets had beneficial effects on feed intake and lipid metabolism but did not improve lactation performance. Increasing the protein content of the prepartum diet did not appear to confer any advantages to cow

  20. Supplemental dietary protein for grazing dairy cows: effect on pasture intake and lactation performance.

    PubMed

    McCormick, M E; Ward, J D; Redfearn, D D; French, D D; Blouin, D C; Chapa, A M; Fernandez, J M

    2001-04-01

    One hundred twenty-four cows (92 multiparous and 32 primiparous) were used to evaluate the effect of grain supplements containing high crude protein [(22.8% CP, 5.3% rumen undegradable protein (RUP), dry matter basis], moderate CP (16.6% CP, 6.1% RUP), and moderate CP with supplemental RUP (16.2% CP, 10.8% RUP) on lactation performance of Holstein cows rotationally grazing annual ryegrass-oat pastures. Supplemental protein was provided by solvent extracted soybean meal in the high CP and moderate CP supplements and as a corn gluten meal-blood meal mixture (2.8:1) in the moderate CP, high RUP supplement. Cows were blocked according to previous mature milk equivalent production and calving date (partum group; 0 d in milk or postpartum group; 21 to 65 d in milk) and randomly assigned to dietary treatments. Grain was individually fed, at approximately a 1:3 grain to milk ratio, before a.m. and p.m milkings. The study was replicated during two grazing seasons that averaged 199 d. Cows had ad libitum access to bermudagrass hay while on pasture (dry matter intake = 1.3 kg/d). Protein supplementation had no effect on study long pasture dry matter (12.7 +/- 1.0 kg/d) or total dry matter (23.9 +/- 1.2 kg/d) consumption. Protein concentration did not affect actual milk yield of either calving group (high CP vs. moderate CP); however, postpartum group cows receiving high CP grain supplements maintained greater milk fat concentrations (3.34 vs. 3.11%), which led to higher fat-corrected milk (FCM) yields than control cows receiving moderate CP grain diets (30.3 vs. 28.9 kg/d). Crude protein concentration in milk of high CP-supplemented, postpartum group cows was also higher than moderate CP cows (3.42 vs. 3.27%). Additional RUP did not increase FCM yield above that generated by moderate CP grain diets for partum (34.3 vs. 32.9 kg/d) or postpartum-group cows (28.9 vs. 28.2 kg/d). Increasing CP concentration of grain supplement did not affect milk yield of Holstein cows grazing

  1. Protein carbonyl levels correlate with performance in elite field hockey players.

    PubMed

    Rosa-Lima, Frederico Luis; Lannes, Luiz; Viana-Gomes, Diego; Pierucci, Anna Paola T R; Salerno, Verônica P

    2015-07-01

    Excess and incorrectly selected exercise can degrade athletic performance from an imbalance in redox homeostasis and oxidative stress, but well-planned training and nutrition can improve antioxidant capacity. The aim of the study was to investigate how nutrient intake could influence oxidative stress and cell lesion biomarkers after 5 days of training followed by a game. Blood was collected from 10 athletes at the start of training (basal), after training (pre-game), and postgame. Their acceleration capacity also was measured pre- and postgame. Blood analysis showed an increase in lactate concentration postgame (13%) and total antioxidant capacity increased both pre-game (13.1%) and postgame (12.7%), all in comparison with basal levels. An oxidative stress marker, protein carbonyl (PC), increased 3-fold over the course of the game, which correlated with a decreased acceleration (r = 0.749). For biomarkers of tissue damage, creatine kinase and aspartate transaminase (AST) increased postgame by 150% and 75%, respectively. The AST variation had a high negative correlation with energy and carbohydrate consumption and a moderate correlation with lipid and vitamin C intake. Protein intake had a positive but moderate correlation with reduced glutathione. The observed correlations suggest that nutritional monitoring can improve exercise physiological homeostasis and that PC serves as a good biomarker for oxidative stress and performance loss.

  2. Therapeutic plasma exchange performed in tandem with hemodialysis for patients with M-protein disorders.

    PubMed

    Mahmood, Aftab; Sodano, Donata; Dash, Anthony; Weinstein, Robert

    2006-07-01

    M-proteins are monoclonal immunoglobulins or immunoglobulin fragments that aberrantly accumulate in the plasma. Hemodialysis (HD) patients with M-proteins may, under certain circumstances, also need therapeutic plasma exchange (TPE). We employed a protocol for tandem TPE/HD in patients with M-protein disorders. We followed the urea reduction ratio (URR), a measure of the efficiency of HD, to compare the effect of TPE on HD efficiency during tandem procedures versus the efficiency of HD performed as a stand-alone procedure in the same patients. Three men (J.M., R.T., M.M.) underwent 23, 80, and 25 tandem TPE/HD over 3, 17, and 7 months, respectively, almost all in the outpatient setting. Mean whole blood flow rate (in ml/min) was slower during hemodialysis alone than during TPE/HD for J.M. (289 +/- 24 vs. 332 +/- 22, P < 0.0001) and R.T. (310 +/- 20 vs. 367 +/- 15, P < 0.0001) but not for M.M. (395 +/- 65 vs. 404 +/- 62, P = 0.6844). URR was equivalent during hemodialysis alone and during TPE/HD for J.M. (54 +/- 4.2 vs. 58 +/- 1.4, P = 0.3333), R.T. (69 +/- 4.9 vs. 70 +/- 2.5, P = 0.9804), and M.M. (71 +/- 2.4 vs. 67 +/- 1.5, P = 0.1143). J.M.'s renal function recovered sufficiently to permit discontinuation of hemodialysis. R.T. experienced both subjective and objective improvement of his arthritic symptoms. M.M. achieved hemostatic control but ultimately died of amyloidosis. TPE/HD is feasible using disparate pieces of equipment when the therapeutic plasma exchange circuit is connected in parallel with the low-pressure side of the hemodialysis circuit. Our experience illustrates that therapeutic plasma exchange did not adversely impact hemodialysis when the two procedures were performed in tandem.

  3. Metalloprotein structures at ambient conditions and in real-time: biological crystallography and spectroscopy using X-ray free electron lasers

    PubMed Central

    Kern, Jan; Yachandra, Vittal K; Yano, Junko

    2016-01-01

    Although the structure of enzymes and the chemistry at the catalytic sites have been studied intensively, an understanding of the atomic-scale chemistry requires a new approach beyond steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure of metallo-enzymes at ambient conditions, while overcoming the severe X-ray damage to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by the intense and ultra-short femtosecond (fs) X-ray pulses from an X-ray free electron by acquiring a signal before the sample is destroyed. This review describes the recent and pioneering uses of XFELs to study the protein structure and dynamics of metallo-enzymes using crystallography, scattering, as well as the chemical structure and dynamics of the catalytic complexes (charge, spin, and covalency) using spectroscopy during the reaction to understand the electron-transfer processes and elucidate the mechanism. PMID:26342144

  4. Cryosalts: suppression of ice formation in macromolecular crystallography.

    PubMed

    Rubinson, K A; Ladner, J E; Tordova, M; Gilliland, G L

    2000-08-01

    Quality data collection for macromolecular cryocrystallography requires suppressing the formation of crystalline or microcrystalline ice that may result from flash-freezing crystals. Described here is the use of lithium formate, lithium chloride and other highly soluble salts for forming ice-ring-free aqueous glasses upon cooling from ambient temperature to 100 K. These cryosalts are a new class of cryoprotectants that are shown to be effective with a variety of commonly used crystallization solutions and with proteins crystallized under different conditions. The influence of cryosalts on crystal mosaicity and diffraction resolution is comparable with or superior to traditional organic cryoprotectants.

  5. Analytical performances of Hemoclot Protein C Reagent on ACL TOP analyzer.

    PubMed

    Calmette, Leyla; Charpentier, Nicole; Tircot, Caroline; Bigot, Delphine; Dunois, Claire; Amiral, Jean; Tetegan, Marcelle; Sep Hieng, Sonnthida; Peltier, Jean-Yves

    2016-12-01

    Our study aimed to evaluate and validate according to standard NF EN ISO 15189 the original protocol ajustement of Hemoclot Protein C (PC) (Hyphen BioMed), clotting-based assay of PC on ACL TOP analyzer (Werfen/Instrumentation Laboratory). We evaluated the performance in terms of imprecision and we validate additional parameters in range B required by the SH GTA 04 (COFRAC): repeatability, reproducibility, detection and quantification limits, limits of linearity, stability, inter-samples and inter-reagents contamination, inaccuracy, evaluation of interferences (hemolysis, bilirubinemia and chyles). A comparison with Hemoclot PC on STA Compact analyzer (Stago) was performed. Coefficients of variation were lower than 5 %. Detection and quantification limits were respectively 8.3 % and 9.3 %. Superior limit of linearity was 140 %. The test didn't diplay any inter-samples and inter-reagents contamination. Reagent after reconstitution was stable 6 hours on ACL TOP. No interferences were observed for hemoglobin lower than 500 mg/dL, for bilirubin lower than and for chyles lower than 300 mg/dL. Comparison with Hemoclot PC on STA analyzer (Stago) was satisfactory. Hemoclot PC adjusted on ACL TOP analyzer showed satisfactory analytical performances with criteria chosen in our study. These data allow a better knowledge of the performances of this test and were useful to make a validation file in range B as recommended by SH GTA 04.

  6. Protein-Pacing from Food or Supplementation Improves Physical Performance in Overweight Men and Women: The PRISE 2 Study

    PubMed Central

    Arciero, Paul J.; Edmonds, Rohan C.; Bunsawat, Kanokwan; Gentile, Christopher L.; Ketcham, Caitlin; Darin, Christopher; Renna, Mariale; Zheng, Qian; Zhang, Jun Zhu; Ormsbee, Michael J.

    2016-01-01

    We recently reported that protein-pacing (P; six meals/day @ 1.4 g/kg body weight (BW), three of which included whey protein (WP) supplementation) combined with a multi-mode fitness program consisting of resistance, interval sprint, stretching, and endurance exercise training (RISE) improves body composition in overweight individuals. The purpose of this study was to extend these findings and determine whether protein-pacing with only food protein (FP) is comparable to WP supplementation during RISE training on physical performance outcomes in overweight/obese individuals. Thirty weight-matched volunteers were prescribed RISE training and a P diet derived from either whey protein supplementation (WP, n = 15) or food protein sources (FP, n = 15) for 16 weeks. Twenty-one participants completed the intervention (WP, n = 9; FP, n = 12). Measures of body composition and physical performance were significantly improved in both groups (p < 0.05), with no effect of protein source. Likewise, markers of cardiometabolic disease risk (e.g., LDL (low-density lipoprotein) cholesterol, glucose, insulin, adiponectin, systolic blood pressure) were significantly improved (p < 0.05) to a similar extent in both groups. These results demonstrate that both whey protein and food protein sources combined with multimodal RISE training are equally effective at improving physical performance and cardiometabolic health in obese individuals. PMID:27187451

  7. X-ray crystallography, an essential tool for the determination of thermodynamic relationships between crystalline polymorphs.

    PubMed

    Céolin, R; Rietveld, I-B

    2016-01-01

    After a short review of the controversies surrounding the discovery of crystalline polymorphism in relation to our present day understanding, the methods of how to solve the stability hierarchy of different polymorphs will be briefly discussed. They involve either theoretical calculations, or, more commonly, experimental methods based on classical thermodynamics. The experimental approach is mainly carried out using heat-exchange data associated to the transition of one form into another. It will be demonstrated that work-related data associated to the phase transition should be taken into account and the role of X-ray crystallography therein will be discussed. X-ray crystallography has become increasingly precise and can nowadays provide specific volumes and their differences as a function of temperature, and also as a function of pressure, humidity, and time.

  8. Structural isomserism in gold nanoparticles revealed by X-ray crystallography

    PubMed Central

    Tian, Shubo; Li, Yi-Zhi; Li, Man-Bo; Yuan, Jinyun; Yang, Jinlong; Wu, Zhikun; Jin, Rongchao

    2015-01-01

    Revealing structural isomerism in nanoparticles using single-crystal X-ray crystallography remains a largely unresolved task, although it has been theoretically predicted with some experimental clues. Here we report a pair of structural isomers, Au38T and Au38Q, as evidenced using electrospray ionization mass spectrometry, X-ray photoelectron spectroscopy, thermogravimetric analysis and indisputable single-crystal X-ray crystallography. The two isomers show different optical and catalytic properties, and differences in stability. In addition, the less stable Au38T can be irreversibly transformed to the more stable Au38Q at 50 °C in toluene. This work may represent an important advance in revealing structural isomerism at the nanoscale. PMID:26482704

  9. Beyond crystallography: Diffractive imaging using coherent x-ray light sources

    SciTech Connect

    Miao, J.; Ishikawa, T.; Robinson, I. K.; Murnane, M. M.

    2015-04-30

    X-ray crystallography has been central to the development of many fields of science over the past century. It has now matured to a point that as long as good-quality crystals are available, their atomic structure can be routinely determined in three dimensions. However, many samples in physics, chemistry, materials science, nanoscience, geology, and biology are noncrystalline, and thus their three-dimensional structures are not accessible by traditional x-ray crystallography. Overcoming this hurdle has required the development of new coherent imaging methods to harness new coherent x-ray light sources. Here we review the revolutionary advances that are transforming x-ray sources and imaging in the 21st century.

  10. Mapping the continuous reciprocal space intensity distribution of X-ray serial crystallography.

    PubMed

    Yefanov, Oleksandr; Gati, Cornelius; Bourenkov, Gleb; Kirian, Richard A; White, Thomas A; Spence, John C H; Chapman, Henry N; Barty, Anton

    2014-07-17

    Serial crystallography using X-ray free-electron lasers enables the collection of tens of thousands of measurements from an equal number of individual crystals, each of which can be smaller than 1 µm in size. This manuscript describes an alternative way of handling diffraction data recorded by serial femtosecond crystallography, by mapping the diffracted intensities into three-dimensional reciprocal space rather than integrating each image in two dimensions as in the classical approach. We call this procedure 'three-dimensional merging'. This procedure retains information about asymmetry in Bragg peaks and diffracted intensities between Bragg spots. This intensity distribution can be used to extract reflection intensities for structure determination and opens up novel avenues for post-refinement, while observed intensity between Bragg peaks and peak asymmetry are of potential use in novel direct phasing strategies.

  11. Engineering fluorescent proteins towards ultimate performances: lessons from the newly developed cyan variants

    NASA Astrophysics Data System (ADS)

    Mérola, Fabienne; Erard, Marie; Fredj, Asma; Pasquier, Hélène

    2016-03-01

    New fluorescent proteins (FPs) are constantly discovered from natural sources, and submitted to intensive engineering based on random mutagenesis and directed evolution. However, most of these newly developed FPs fail to achieve all the performances required for their bioimaging applications. The design of highly optimised FP-based reporters, simultaneously displaying appropriate colour, multimeric state, chromophore maturation, brightness, photostability and environmental sensitivity will require a better understanding of the structural and dynamic determinants of FP photophysics. The recent development of cyan fluorescent proteins (CFPs) like mCerulean3, mTurquoise2 and Aquamarine brings a different view on these questions, as in this particular case, a step by step evaluation of critical mutations has been performed within a family of spectrally identical and evolutionary close variants. These efforts have led to CFPs with quantum yields close to unity, near single exponential emission decays, high photostability and complete insensitivity to pH, making them ideal choices as energy transfer donors in FRET and FLIM imaging applications. During this process, it was found that a proper amino-acid choice at only two positions (148 and 65) is sufficient to transform the performances of CFPs: with the help of structural and theoretical investigations, we rationalise here how these two positions critically control the CFP photophysics, in the context of FPs derived from the Aequorea victoria species. Today, these results provide a useful toolbox for upgrading the different CFP donors carried by FRET biosensors. They also trace the route towards the de novo design of FP-based optogenetic devices that will be perfectly tailored to dedicated imaging and sensing applications.

  12. Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II.

    PubMed

    Sierra, Raymond G; Gati, Cornelius; Laksmono, Hartawan; Dao, E Han; Gul, Sheraz; Fuller, Franklin; Kern, Jan; Chatterjee, Ruchira; Ibrahim, Mohamed; Brewster, Aaron S; Young, Iris D; Michels-Clark, Tara; Aquila, Andrew; Liang, Mengning; Hunter, Mark S; Koglin, Jason E; Boutet, Sébastien; Junco, Elia A; Hayes, Brandon; Bogan, Michael J; Hampton, Christina Y; Puglisi, Elisabetta V; Sauter, Nicholas K; Stan, Claudiu A; Zouni, Athina; Yano, Junko; Yachandra, Vittal K; Soltis, S Michael; Puglisi, Joseph D; DeMirci, Hasan

    2016-01-01

    We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).

  13. Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II

    SciTech Connect

    Sierra, Raymond G.; Gati, Cornelius; Laksmono, Hartawan; Dao, E. Han; Gul, Sheraz; Fuller, Franklin; Kern, Jan; Chatterjee, Ruchira; Ibrahim, Mohamed; Brewster, Aaron S.; Young, Iris D.; Michels-Clark, Tara; Aquila, Andrew; Liang, Mengning; Hunter, Mark S.; Koglin, Jason E.; Boutet, Sébastien; Junco, Elia A.; Hayes, Brandon; Bogan, Michael J.; Hampton, Christina Y.; Puglisi, Elisabetta V.; Sauter, Nicholas K.; Stan, Claudiu A.; Zouni, Athina; Yano, Junko; Yachandra, Vittal K.; Soltis, S. Michael; Puglisi, Joseph D.; DeMirci, Hasan

    2015-11-30

    We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).

  14. Stress Corrosion Cracking Facet Crystallography of Ti-8Al-1Mo-1V (Preprint)

    DTIC Science & Technology

    2011-05-01

    both specimens, the regions not characterized by faceted fracture generally consisted of features like flutes [33;34] and dimples consistent with...received specimen studied here switches from ductile dimple / fluted growth [34] to faceted growth with increasing crack length. Since crack formation...Crystallography of fluted fracture in near-α titanium alloys, Metall. Mater. Trans. A. 41 (2010) 22-25. [35] J.C. Chesnutt, J.C. Williams, Comments on the

  15. SPINE-compatible ‘carboloops’: a new microshaped vitreous carbon sample mount for X-ray and neutron crystallography

    PubMed Central

    Romoli, Filippo; Mossou, Estelle; Cuypers, Maxime; van der Linden, Peter; Carpentier, Philippe; Mason, Sax A.; Forsyth, V. Trevor; McSweeney, Sean

    2014-01-01

    A novel vitreous carbon mount for macromolecular crystallography, suitable for neutron and X-ray crystallographic studies, has been developed. The technology described here is compatible both with X-ray and neutron cryo-crystallography. The mounts have low density and low background scattering for both neutrons and X-rays. They are prepared by laser cutting, allowing high standards of production quality, the ability to custom-design the mount to specific crystal sizes and large-scale production. PMID:24817737

  16. Effect of dietary protein sources on production performance, egg quality, and plasma parameters of laying hens

    PubMed Central

    Wang, Xiaocui; Zhang, Haijun; Wang, Hao; Wang, Jing; Wu, Shugeng; Qi, Guanghai

    2017-01-01

    Objective This study was conducted to evaluate the effects of dietary protein sources (soybean meal, SBM; low-gossypol cottonseed meal, LCSM; double-zero rapeseed meal, DRM) on laying performance, egg quality, and plasma parameters of laying hens. Methods A total of 432 32-wk-old laying hens were randomly divided into 6 treatments with 6 replicates of 12 birds each. The birds were fed diets containing SBM, LCSM100, or DRM100 individually or in combination with an equal amount of crude protein (CP) (LCSM50, DRM50, and LCSM50-DRM50). The experimental diets, which were isocaloric (metabolizable energy, 11.11 MJ/kg) and isonitrogenous (CP, 16.5%), had similar digestible amino acid profile. The feeding trial lasted 12 weeks. Results The daily egg mass was decreased in the LCSM100 and LCSM50-DRM50 groups (p<0.05) in weeks 41 to 44. The LCSM50 group did not affect egg production compared to the SBM group in weeks 41 to 44 (p>0.05) and showed increased yolk color at the end of the trial (p<0.05). Compared to the SBM group, the LCSM100 and LCSM50-DRM50 groups showed decreased albumen weight (p<0.05), CP weight in the albumen (p<0.05) and CP weight in the whole egg (p<0.05) at 44 weeks. Plasma total protein (TP) levels were lower in the LCSM100 group than in the SBM group at 44 weeks (p<0.05); however, TP, albumin, and globulin levels were not significantly different between the LCSM50 group and the SBM group or between the DRM50 group and the SBM group (p>0.05). Conclusion Together, our results suggest that the LCSM100 or DRM100 diets may produce the adverse effects on laying performance and egg quality after feeding for 8 more weeks. The 100.0 g/kg LCSM diet or the148.7 g/kg DRM diet has no adverse effects on laying performance and egg quality. PMID:27608634

  17. X-ray crystallography, mass spectrometry and single crystal microspectrophotometry: a multidisciplinary characterization of catechol 1,2 dioxygenase.

    PubMed

    Micalella, Chiara; Martignon, Sara; Bruno, Stefano; Pioselli, Barbara; Caglio, Raffaella; Valetti, Francesca; Pessione, Enrica; Giunta, Carlo; Rizzi, Menico

    2011-06-01

    Intradiol-cleaving catechol 1,2 dioxygenases are Fe(III) dependent enzymes that act on catechol and substituted catechols, including chlorocatechols pollutants, by inserting molecular oxygen in the aromatic ring. Members of this class are the object of intense biochemical investigations aimed at the understanding of their catalytic mechanism, particularly for designing mutants with selected catalytic properties. We report here an in depth investigation of catechol 1,2 dioxygenase IsoB from Acinetobacter radioresistens LMG S13 and its A72G and L69A mutants. By applying a multidisciplinary approach that includes high resolution X-rays crystallography, mass spectrometry and single crystal microspectrophotometry, we characterised the phospholipid bound to the enzyme and provided a structural framework to understand the inversion of substrate specificity showed by the mutants. Our results might be of help for the rational design of enzyme mutants showing a biotechnologically relevant substrate specificity, particularly to be used in bioremediation. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

  18. Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals

    PubMed Central

    Uervirojnangkoorn, Monarin; Zeldin, Oliver B; Lyubimov, Artem Y; Hattne, Johan; Brewster, Aaron S; Sauter, Nicholas K; Brunger, Axel T; Weis, William I

    2015-01-01

    There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as the resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited. DOI: http://dx.doi.org/10.7554/eLife.05421.001 PMID:25781634

  19. Effects of Forage Sources on Rumen Fermentation Characteristics, Performance, and Microbial Protein Synthesis in Midlactation Cows

    PubMed Central

    Xu, Jun; Hou, Yujie; Yang, Hongbo; Shi, Renhuang; Wu, Caixia; Huo, Yongjiu; Zhao, Guoqi

    2014-01-01

    Eight multiparous Holstein cows (632±12 kg BW; 135±16 DIM) were used in a replicated 4×4 Latin square design to evaluate the effects of forage sources on rumen fermentation characteristics, performance, and microbial protein (MCP) synthesis. The forage portion of the diets contained alfalfa hay (AH), oat hay (OH), Leymus chinensis (LC), or rice straw (RS) as the primary source of fiber. Diets were isonitrogenous and isocaloric, and cows were fed four corn silages based total mixed rations with equivalent nonfiber carbohydrate (NFC) and forage neutral detergent fiber (NDF). Dry matter intake was not affected by the source of dietary forages, ranging from 18.83 to 19.20 kg/d, consequently, milk yield was similar among diets. Because of the numerical differences in milk fat and milk protein concentrations, 4% FCM and ECM yields were unchanged (p>0.05). Mean rumen pH, NH3-N content, and concentrations of volatile fatty acids in the rumen fluid were not affected by the treatments (p>0.05). Dietary treatments did not affect the total tract apparent digestibility of dry matter, organic matter, and crude protein (p>0.05); however, digestibility of NDF and acid detergent fiber in RS diet was higher compared with AH, OH, and LC diets (p<0.05). Total purine derivative excretion was higher in cows fed AH, OH, and LC diets compared with those fed RS diet (p<0.05), consequently, estimated MCP synthesis was 124.35 g/d higher in cows fed AH diet compared with those fed RS diet (p<0.05). The results indicated that cows fed AH, OH, LC, and RS diets with an equivalent forage NDF and NFC have no unfavourable effect on the ruminal fermentation and productive parameters. PMID:25050001

  20. Visible vs near-infrared optical fiber plasmonics: performance comparison for protein sensing

    NASA Astrophysics Data System (ADS)

    Caucheteur, Christophe; Ribaut, Clotilde; Wattiez, Ruddy

    2016-04-01

    In this work, two plasmonic optical fiber sensor configurations are used for protein sensing and their relative performances in terms of limit of detection and sensitivity are compared. The first configuration consists in unclad 200 μm optical fibers that produce a broadband resonance in the visible wavelength range around 650 nm while the second configuration makes use of multiple narrowband resonances produced in the C+L bands with weakly tilted fiber Bragg gratings photo-inscribed in telecommunication-grade single-mode optical fibers. In both cases, the sensitive regions are surrounded by a ~50 nm gold layer so that the evanescent wave can excite a surface plasmon polariton at the metalsurrounding medium interface. Both configurations are used to sense green fluorescent proteins. Our experimental results demonstrate that the two sensor configurations present a complementary measurement dynamics as a function of the investigated concentration in the range 10-12 - 10-7 g/ml. We attribute this difference of sensitivity to the difference of penetration depth of the evanescent wave in the surrounding medium, which is proportional to the light wavelength.

  1. Performance of Leishmania braziliensis enolase protein for the serodiagnosis of canine and human visceral leishmaniosis.

    PubMed

    Duarte, Mariana Costa; Lage, Daniela Pagliara; Martins, Vívian Tamietti; Costa, Lourena Emanuele; Salles, Beatriz Cristina Silveira; Carvalho, Ana Maria Ravena Severino; de Oliveira Santos, Thaís Teodoro; Dias, Daniel Silva; Ribeiro, Patrícia Aparecida Fernandes; Chávez-Fumagalli, Miguel Angel; Machado-de-Ávila, Ricardo Andrez; Roatt, Bruno Mendes; Menezes-Souza, Daniel; de Magalhães-Soares, Danielle Ferreira; Ferraz Coelho, Eduardo Antonio

    2017-03-30

    In the present study, Leishmania braziliensis enolase was cloned and the recombinant protein (rEnolase) was evaluated for the serodiagnosis of canine and human visceral leishmaniosis (VL). For the canine VL diagnosis, this study examined serum samples of Leishmania infantum-infected dogs, from non-infected animals living in endemic or non-endemic areas of leishmaniosis, as well as those from Leish-Tec(®)-vaccinated dogs and Trypanosoma cruzi or Ehrlichia canis experimentally infected animals. For the human VL diagnosis, this study analyzed serum samples from VL patients, from non-infected subjects living in endemic or non-endemic areas of leishmaniosis, as well as those from T. cruzi-infected patients. In the results, an indirect ELISA method using rEnolase showed diagnostic sensitivity and specificity values of 100% and 98.57%, respectively, for canine VL serodiagnosis, and of 100% and 97.87%, respectively, for human VL diagnosis. These results showed rEnolase with an improved diagnostic performance when compared to the recombinant A2 protein, the crude soluble Leishmania antigenic preparation, and the recombinant K39-based immunochromatographic test. In conclusion, preliminary results suggest that the detection of antibodies against rEnolase improves the serodiagnosis of human and canine visceral leishmaniosis.

  2. Conceptual design of novel IP-conveyor-belt Weissenberg-mode data-collection system with multi-readers for macromolecular crystallography. A comparison between Galaxy and Super Galaxy.

    PubMed

    Sakabe, N; Sakabe, K; Sasaki, K

    2004-01-01

    Galaxy is a Weissenberg-type high-speed high-resolution and highly accurate fully automatic data-collection system using two cylindrical IP-cassettes each with a radius of 400 mm and a width of 450 mm. It was originally developed for static three-dimensional analysis using X-ray diffraction and was installed on bending-magnet beamline BL6C at the Photon Factory. It was found, however, that Galaxy was also very useful for time-resolved protein crystallography on a time scale of minutes. This has prompted us to design a new IP-conveyor-belt Weissenberg-mode data-collection system called Super Galaxy for time-resolved crystallography with improved time and crystallographic resolution over that achievable with Galaxy. Super Galaxy was designed with a half-cylinder-shaped cassette with a radius of 420 mm and a width of 690 mm. Using 1.0 A incident X-rays, these dimensions correspond to a maximum resolutions of 0.71 A in the vertical direction and 1.58 A in the horizontal. Upper and lower screens can be used to set the frame size of the recorded image. This function is useful not only to reduce the frame exchange time but also to save disk space on the data server. The use of an IP-conveyor-belt and many IP-readers make Super Galaxy well suited for time-resolved, monochromatic X-ray crystallography at a very intense third-generation SR beamline. Here, Galaxy and a conceptual design for Super Galaxy are described, and their suitability for use as data-collection systems for macromolecular time-resolved monochromatic X-ray crystallography are compared.

  3. High-pressure protein crystallography of hen egg-white lysozyme

    PubMed Central

    Yamada, Hiroyuki; Nagae, Takayuki; Watanabe, Nobuhisa

    2015-01-01

    Crystal structures of hen egg-white lysozyme (HEWL) determined under pressures ranging from ambient pressure to 950 MPa are presented. From 0.1 to 710 MPa, the molecular and internal cavity volumes are monotonically compressed. However, from 710 to 890 MPa the internal cavity volume remains almost constant. Moreover, as the pressure increases to 950 MPa, the tetragonal crystal of HEWL undergoes a phase transition from P43212 to P43. Under high pressure, the crystal structure of the enzyme undergoes several local and global changes accompanied by changes in hydration structure. For example, water molecules penetrate into an internal cavity neighbouring the active site and induce an alternate conformation of one of the catalytic residues, Glu35. These phenomena have not been detected by conventional X-ray crystal structure analysis and might play an important role in the catalytic activity of HEWL. PMID:25849385

  4. High-pressure protein crystallography of hen egg-white lysozyme

    SciTech Connect

    Yamada, Hiroyuki; Nagae, Takayuki; Watanabe, Nobuhisa

    2015-04-01

    The crystal structure of hen egg-white lysozyme (HEWL) was analyzed under pressures of up to 950 MPa. The high pressure modified the conformation of the molecule and induced a novel phase transition in the tetragonal crystal of HEWL. Crystal structures of hen egg-white lysozyme (HEWL) determined under pressures ranging from ambient pressure to 950 MPa are presented. From 0.1 to 710 MPa, the molecular and internal cavity volumes are monotonically compressed. However, from 710 to 890 MPa the internal cavity volume remains almost constant. Moreover, as the pressure increases to 950 MPa, the tetragonal crystal of HEWL undergoes a phase transition from P4{sub 3}2{sub 1}2 to P4{sub 3}. Under high pressure, the crystal structure of the enzyme undergoes several local and global changes accompanied by changes in hydration structure. For example, water molecules penetrate into an internal cavity neighbouring the active site and induce an alternate conformation of one of the catalytic residues, Glu35. These phenomena have not been detected by conventional X-ray crystal structure analysis and might play an important role in the catalytic activity of HEWL.

  5. Comparing Chemistry to Outcome: The Development of a Chemical Distance Metric, Coupled with Clustering and Hierarchal Visualization Applied to Macromolecular Crystallography

    PubMed Central

    Bruno, Andrew E.; Ruby, Amanda M.; Luft, Joseph R.; Grant, Thomas D.; Seetharaman, Jayaraman; Montelione, Gaetano T.; Hunt, John F.; Snell, Edward H.

    2014-01-01

    Many bioscience fields employ high-throughput methods to screen multiple biochemical conditions. The analysis of these becomes tedious without a degree of automation. Crystallization, a rate limiting step in biological X-ray crystallography, is one of these fields. Screening of multiple potential crystallization conditions (cocktails) is the most effective method of probing a proteins phase diagram and guiding crystallization but the interpretation of results can be time-consuming. To aid this empirical approach a cocktail distance coefficient was developed to quantitatively compare macromolecule crystallization conditions and outcome. These coefficients were evaluated against an existing similarity metric developed for crystallization, the C6 metric, using both virtual crystallization screens and by comparison of two related 1,536-cocktail high-throughput crystallization screens. Hierarchical clustering was employed to visualize one of these screens and the crystallization results from an exopolyphosphatase-related protein from Bacteroides fragilis, (BfR192) overlaid on this clustering. This demonstrated a strong correlation between certain chemically related clusters and crystal lead conditions. While this analysis was not used to guide the initial crystallization optimization, it led to the re-evaluation of unexplained peaks in the electron density map of the protein and to the insertion and correct placement of sodium, potassium and phosphate atoms in the structure. With these in place, the resulting structure of the putative active site demonstrated features consistent with active sites of other phosphatases which are involved in binding the phosphoryl moieties of nucleotide triphosphates. The new distance coefficient, CDcoeff, appears to be robust in this application, and coupled with hierarchical clustering and the overlay of crystallization outcome, reveals information of biological relevance. While tested with a single example the potential applications

  6. In crystallo optical spectroscopy (icOS) as a complementary tool on the macromolecular crystallography beamlines of the ESRF

    SciTech Connect

    Stetten, David von; Giraud, Thierry; Carpentier, Philippe; Sever, Franc; Terrien, Maxime; Dobias, Fabien; Juers, Douglas H.; Flot, David; Mueller-Dieckmann, Christoph; Leonard, Gordon A.; Sanctis, Daniele de; Royant, Antoine

    2015-01-01

    The current version of the Cryobench in crystallo optical spectroscopy facility of the ESRF is presented. The diverse experiments that can be performed at the Cryobench are also reviewed. The analysis of structural data obtained by X-ray crystallography benefits from information obtained from complementary techniques, especially as applied to the crystals themselves. As a consequence, optical spectroscopies in structural biology have become instrumental in assessing the relevance and context of many crystallographic results. Since the year 2000, it has been possible to record such data adjacent to, or directly on, the Structural Biology Group beamlines of the ESRF. A core laboratory featuring various spectrometers, named the Cryobench, is now in its third version and houses portable devices that can be directly mounted on beamlines. This paper reports the current status of the Cryobench, which is now located on the MAD beamline ID29 and is thus called the ID29S-Cryobench (where S stands for ‘spectroscopy’). It also reviews the diverse experiments that can be performed at the Cryobench, highlighting the various scientific questions that can be addressed.

  7. Growth performance of calves fed microbially enhanced soy protein in pelleted starters.

    PubMed

    Senevirathne, N D; Anderson, J L; Gibbons, W R; Clapper, J A

    2017-01-01

    interaction. Treatments had similar total-tract dry matter digestibility, but calves fed MSPC had greater crude protein digestibility than SBMA, with MSPA similar to both. Results demonstrated calves fed pelleted starters with MSP had maintained growth performance with less starter intake compared with SBM.

  8. Combined Protein A and size exclusion high performance liquid chromatography for the single-step measurement of mAb, aggregates and host cell proteins.

    PubMed

    Gjoka, Xhorxhi; Schofield, Mark; Cvetkovic, Aleksandar; Gantier, Rene

    2014-12-01

    Quantification of monoclonal antibody (mAb) monomer, mAb aggregates, and host cell proteins (HCPs) is critical for the optimization of the mAb production process. The present work describes a single high throughput analytical tool capable of tracking the concentration of mAb, mAb aggregate and HCPs in a growing cell culture batch. By combining two analytical HPLC methods, Protein A affinity and size-exclusion chromatography (SEC), it is possible to detect a relative increase or decrease in the concentration of all three entities simultaneously. A comparison of the combined Protein A-SEC assay to SEC alone was performed, demonstrating that it can be useful tool for the quantification of mAb monomer along with trending data for mAb aggregate and HCP. Furthermore, the study shows that the Protein A-SEC method is at least as accurate as other commonly used analytical methods such as ELISA and Bradford.

  9. Timing influence of carbohydrate-protein ingestion on muscle soreness and next-day running performance.

    PubMed

    Greer, Beau Kjerulf; Price, Anna; Jones, Brett

    2014-06-01

    The present study investigates timing effects of a carbohydrate-protein (CHO-PROT) beverage on indicators of muscle damage and next day running performance. Nine trained subjects completed three trials of a 30 min downhill run, followed by a 1.5 mile treadmill running time trial 24 hr later in a blinded, crossover design. Either a CHO-PROT or noncaloric placebo beverage was given 30 and 5 min prior to, at the 15 min mark during, immediately after, and 30 min after the downhill running protocol. In the first treatment (T1), a total of 360 kilocalories were given 30 and 5 min prior to downhill running, as well as at the 15 min mark, with placebos used at other time points. In the second treatment (T2), an isocaloric amount was given but only immediately after and 30 min after downhill running, with placebos used at other time points. In the placebo treatment, a placebo was given at all time points. There were no significant differences in the 1.5 mile time trial or soreness between trials (p > .05). Regardless of timing, the ingestion of a CHO-PROT beverage had no effect on next day running performance or muscular soreness versus a placebo.

  10. Textural performance of crosslinked or reduced-calcium milk protein ingredients in model high-protein nutrition bars.

    PubMed

    Banach, J C; Clark, S; Metzger, L E; Lamsal, B P

    2016-08-01

    Transglutaminase (Tgase) crosslinking and calcium reduction were investigated as ways to improve the texture and storage stability of high-protein nutrition (HPN) bars formulated with milk protein concentrate (MPC) and micellar casein concentrate (MCC). The MPC and MCC crosslinked at none, low, and high levels, and a reduced-calcium MPC (RCMPC) were each formulated into model HPN bars. Hardness, crumbliness, moisture content, pH, color, and water activity of the HPN bars were measured during accelerated storage. The HPN bars prepared with MPC were harder and more cohesive than those prepared with MCC. Higher levels of Tgase crosslinking improved HPN bar cohesiveness and decreased hardening during storage. The RCMPC produced softer, yet crumblier HPN bars. Small textural differences were observed for the HPN bars formulated with the transglutaminase crosslinked proteins or RCMPC when compared with their respective controls. However, modification only slightly improved protein ingredient ability to slow hardening while balancing cohesion and likely requires further improvement for increased applicability in soft-texture HPN bars.

  11. Effect of dietary protein content on live and carcase performance of heterozygous naked neck and normally feathered broilers.

    PubMed

    Yalçin, S; Ozkan, S; Açikgöz, Z; Ozkan, K

    1996-12-01

    1. An experiment was conducted to determine the effect of different dietary protein contents on the performance of naked neck (Na/na) and normally feathered (na/na) broilers. 2. Chicks from the two genotypes were reared in wire-floored cages and divided at random into 3 groups. Birds were fed on high protein (HP, 12.99 MJ ME, 238 g crude protein/kg and 12.94 MJ ME, 216 g crude protein/kg from 0 to 3 and 3 to 7 weeks, respectively), medium protein (MP, 12.99 MJ ME, 219 g crude protein/kg and 12.87 MJ ME, 201 g crude protein/kg from 0 to 3 and 3 to 7 weeks), and low protein (LP, 12.94 MJ ME, 205 g crude protein/kg and 12.75 MJ ME, 184 g protein/kg from 0 to 3 and 3 to 7 weeks) diets. 3. The LP diets resulted in a significantly lower daily body weight gain of males from 0 to 3 weeks. Dietary protein content had no effect on body weight gain from 3 to 7 weeks, body weight at 7 weeks, and the food intake of birds. Carcase composition of birds from both genotypes was unaffected by dietary protein. 4. Naked neck birds had significantly higher body weights at 7 weeks. Yields of carcase and breast of Na/na males were significantly higher than those of na/na males. There were no significant differences between females from the two genotypes as regards carcase yield. 5. It was concluded that the dietary protein requirements of naked neck birds were similar to those for normally feathered birds.

  12. Combined carbohydrate-protein supplementation improves competitive endurance exercise performance in the heat.

    PubMed

    Cathcart, Andrew J; Murgatroyd, Scott R; McNab, Alison; Whyte, Laura J; Easton, Chris

    2011-09-01

    Laboratory-based studies have demonstrated that adding protein (PRO) to a carbohydrate (CHO) supplement can improve thermoregulatory capacity, exercise performance and recovery. However, no study has investigated these effects in a competitive sporting context. This study assessed the effects of combined CHO-PRO supplementation on physiological responses and exercise performance during 8 days of strenuous competition in a hot environment. Twenty-eight cyclists participating in the TransAlp mountain bike race were randomly assigned to fitness-matched placebo (PLA 76 g L(-1) CHO) or CHO-PRO (18 g L(-1) PRO, 72 g L(-1) CHO) groups. Participants were given enough supplements to allow ad libitum consumption. Physiological and anthropometric variables were recorded pre- and post-exercise. Body mass decreased significantly from race stage 1 to 8 in the PLA group (-0.75 ± 0.22 kg, P = 0.01) but did not change in the CHO-PRO group (0.42 ± 0.42 kg, P = 0.35). Creatine kinase concentration and muscle soreness were substantially elevated during the race, but were not different between groups (P = 0.82, P = 0.44, respectively). Urine osmolality was significantly higher in the CHO-PRO versus the PLA group (P = 0.04) and the rise in tympanic temperature from pre- to post-exercise was significantly less in CHO-PRO versus PLA (P = 0.01). The CHO-PRO group also completed the 8 stages significantly quicker than the PLA group (2,277 ± 127 vs. 2,592 ± 68 min, respectively, P = 0.02). CHO-PRO supplementation therefore appears to prevent body mass loss, enhance thermoregulatory capacity and improve competitive exercise performance despite no effect on muscle damage.

  13. Sake Protein Supplementation Affects Exercise Performance and Biochemical Profiles in Power-Exercise-Trained Mice

    PubMed Central

    Chen, Yi-Ming; Lin, Che-Li; Wei, Li; Hsu, Yi-Ju; Chen, Kuan-Neng; Huang, Chi-Chang; Kao, Chin-Hsung

    2016-01-01

    Exercise and fitness training programs have attracted the public’s attention in recent years. Sports nutrition supplementation is an important issue in the global sports market. Purpose: In this study, we designed a power exercise training (PET) program with a mouse model based on a strength and conditional training protocol for humans. We tested the effect of supplementation with functional branched-chain amino acid (BCAA)-rich sake protein (SP) to determine whether the supplement had a synergistic effect during PET and enhanced athletic performance and resistance to fatigue. Methods: Male ICR mice were divided into three groups (n = 8 per group) for four-week treatment: sedentary controls with vehicle (SC), and PET and PET groups with SP supplementation (3.8 g/kg, PET + SP). Exercise performance was evaluated by forelimb grip strength and exhaustive swimming time as well as changes in body composition and anti-fatigue activity levels of serum lactate, ammonia, glucose, and creatine kinase (CK) after a 15-min swimming exercise. The biochemical parameters were measured at the end of the experiment. Results: four-week PET significantly increased grip strength and exhaustive swimming time and decreased epididymal fat pad (EFP) weight and area. Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, and uric acid (UA) were significantly increased. PET + SP supplementation significantly decreased serum lactate, ammonia and CK levels after the 15-min swimming exercise. The resting serum levels of AST, ALT, CREA and UA were all significantly decreased with PET + SP. Conclusion: The PET program could increase the exercise performance and modulate the body composition of mice. PET with SP conferred better anti-fatigue activity, improved biochemical profiles, and may be an effective ergogenic aid in strength training. PMID:26907336

  14. Structure of ATP synthase from Paracoccus denitrificans determined by X-ray crystallography at 4.0 Å resolution

    PubMed Central

    Morales-Rios, Edgar; Montgomery, Martin G.; Leslie, Andrew G. W.; Walker, John E.

    2015-01-01

    The structure of the intact ATP synthase from the α-proteobacterium Paracoccus denitrificans, inhibited by its natural regulatory ζ-protein, has been solved by X-ray crystallography at 4.0 Å resolution. The ζ-protein is bound via its N-terminal α-helix in a catalytic interface in the F1 domain. The bacterial F1 domain is attached to the membrane domain by peripheral and central stalks. The δ-subunit component of the peripheral stalk binds to the N-terminal regions of two α-subunits. The stalk extends via two parallel long α-helices, one in each of the related b and b′ subunits, down a noncatalytic interface of the F1 domain and interacts in an unspecified way with the a-subunit in the membrane domain. The a-subunit lies close to a ring of 12 c-subunits attached to the central stalk in the F1 domain, and, together, the central stalk and c-ring form the enzyme’s rotor. Rotation is driven by the transmembrane proton-motive force, by a mechanism where protons pass through the interface between the a-subunit and c-ring via two half-channels in the a-subunit. These half-channels are probably located in a bundle of four α-helices in the a-subunit that are tilted at ∼30° to the plane of the membrane. Conserved polar residues in the two α-helices closest to the c-ring probably line the proton inlet path to an essential carboxyl group in the c-subunit in the proton uptake site and a proton exit path from the proton release site. The structure has provided deep insights into the workings of this extraordinary molecular machine. PMID:26460036

  15. NMR Structures of Membrane Proteins in Phospholipid Bilayers

    PubMed Central

    Radoicic, Jasmina; Lu, George J.; Opella, Stanley J.

    2014-01-01

    Membrane proteins have always presented technical challenges for structural studies because of their requirement for a lipid environment. Multiple approaches exist including X-ray crystallography and electron microscopy that can give significant insights into their structure and function. However, nuclear magnetic resonance (NMR) is unique in that it offers the possibility of determining the structures of unmodified membrane proteins in their native environment of phospholipid bilayers under physiological conditions. Furthermore, NMR enables the characterization of the structure and dynamics of backbone and side chain sites of the proteins alone and in complexes with both small molecules and other biopolymers. The learning curve has been steep for the field as most initial studies were performed under non-native environments using modified proteins until ultimately progress in both techniques and instrumentation led to the possibility of examining unmodified membrane proteins in phospholipid bilayers under physiological conditions. This review aims to provide an overview of the development and application of NMR to membrane proteins. It highlights some of the most significant structural milestones that have been reached by NMR spectroscopy of membrane proteins; especially those accomplished with the proteins in phospholipid bilayer environments where they function. PMID:25032938

  16. Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

    PubMed Central

    Wan, Qun; Parks, Jerry M.; Hanson, B. Leif; Fisher, Suzanne Zoe; Ostermann, Andreas; Schrader, Tobias E.; Graham, David E.; Coates, Leighton; Langan, Paul; Kovalevsky, Andrey

    2015-01-01

    Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen. PMID:26392527

  17. How reliable is immunohistochemical staining for DNA mismatch repair proteins performed after neoadjuvant chemoradiation?

    PubMed

    Vilkin, Alex; Halpern, Marisa; Morgenstern, Sara; Brazovski, Eli; Gingold-Belfer, Rachel; Boltin, Doron; Purim, Ofer; Kundel, Yulia; Welinsky, Sara; Brenner, Baruch; Niv, Yaron; Levi, Zohar

    2014-10-01

    Immunohistochemistry (IHC) testing for mismatch repair proteins (MMRP) is currently being used primarily in colorectal cancer resection specimens. We aimed to compare the results of IHC staining performed on biopsy specimens obtained at endoscopy with that performed on surgical specimens after neoadjuvant therapy. Thirty-two rectal cancer subjects had paired preneoadjuvant and postneoadjuvant tissue available for IHC staining (MLH1, MSH2, MSH6, and PMS2), whereas 39 rectosigmoid cancer patients who did not receive neoadjuvant treatment served as controls. Each slide received a qualitative (absent, focal, and strong) and quantitative score (immunoreactivity [0-3] × percent positivity [0-4]). The quantitative scores of MMRP from the operative material were significantly lower in the neoadjuvant group than in the control (P < .05 for all).The scores of all MMRP from endoscopic biopsies were not significantly different between the neoadjuvant and the control groups. Disagreement between the endoscopic biopsy and the operative material was evident in 23 of 128 stains (18.5%) in the neoadjuvant group and in 12 of 156 stains (7.7%) in the control group (P = .009). In the neoadjuvant group, a disagreement pattern of "endoscopic strong operative focal" was observed in 28.1% for PMS2, 12.5% for MSH6, 12.5% for MLH1, and 6.3% for MSH2, and in the control group, this same disagreement pattern was found in 12.8% for PMS2, 7.7% for MSH6, 7.7% for MLH1, and 0% for MSH2. Based on our findings, we suggest that for rectal cancer, the endoscopic material rather than the operative material should serve as the primary material for IHC staining.

  18. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  19. Intake of Protein Plus Carbohydrate during the First Two Hours after Exhaustive Cycling Improves Performance the following Day.

    PubMed

    Rustad, Per I; Sailer, Manuela; Cumming, Kristoffer T; Jeppesen, Per B; Kolnes, Kristoffer J; Sollie, Ove; Franch, Jesper; Ivy, John L; Daniel, Hannelore; Jensen, Jørgen

    2016-01-01

    Intake of protein immediately after exercise stimulates protein synthesis but improved recovery of performance is not consistently observed. The primary aim of the present study was to compare performance 18 h after exhaustive cycling in a randomized diet-controlled study (175 kJ·kg(-1) during 18 h) when subjects were supplemented with protein plus carbohydrate or carbohydrate only in a 2-h window starting immediately after exhaustive cycling. The second aim was to investigate the effect of no nutrition during the first 2 h and low total energy intake (113 kJ·kg(-1) during 18 h) on performance when protein intake was similar. Eight endurance-trained subjects cycled at 237±6 Watt (~72% VO2max) until exhaustion (TTE) on three occasions, and supplemented with 1.2 g carbohydrate·kg(-1)·h(-1) (CHO), 0.8 g carbohydrate + 0.4 g protein·kg(-1)·h(-1) (CHO+PRO) or placebo without energy (PLA). Intake of CHO+PROT increased plasma glucose, insulin, and branch chained amino acids, whereas CHO only increased glucose and insulin. Eighteen hours later, subjects performed another TTE at 237±6 Watt. TTE was increased after intake of CHO+PROT compared to CHO (63.5±4.4 vs 49.8±5.4 min; p<0.05). PLA reduced TTE to 42.8±5.1 min (p<0.05 vs CHO). Nitrogen balance was positive in CHO+PROT, and negative in CHO and PLA. In conclusion, performance was higher 18 h after exhaustive cycling with intake of CHO+PROT compared to an isocaloric amount of carbohydrate during the first 2 h post exercise. Intake of a similar amount of protein but less carbohydrate during the 18 h recovery period reduced performance.

  20. Intake of Protein Plus Carbohydrate during the First Two Hours after Exhaustive Cycling Improves Performance the following Day

    PubMed Central

    Rustad, Per I.; Kolnes, Kristoffer J.; Sollie, Ove; Franch, Jesper; Ivy, John L.; Daniel, Hannelore; Jensen, Jørgen

    2016-01-01

    Intake of protein immediately after exercise stimulates protein synthesis but improved recovery of performance is not consistently observed. The primary aim of the present study was to compare performance 18 h after exhaustive cycling in a randomized diet-controlled study (175 kJ·kg-1 during 18 h) when subjects were supplemented with protein plus carbohydrate or carbohydrate only in a 2-h window starting immediately after exhaustive cycling. The second aim was to investigate the effect of no nutrition during the first 2 h and low total energy intake (113 kJ·kg-1 during 18 h) on performance when protein intake was similar. Eight endurance-trained subjects cycled at 237±6 Watt (~72% VO2max) until exhaustion (TTE) on three occasions, and supplemented with 1.2 g carbohydrate·kg-1·h-1 (CHO), 0.8 g carbohydrate + 0.4 g protein·kg-1·h-1 (CHO+PRO) or placebo without energy (PLA). Intake of CHO+PROT increased plasma glucose, insulin, and branch chained amino acids, whereas CHO only increased glucose and insulin. Eighteen hours later, subjects performed another TTE at 237±6 Watt. TTE was increased after intake of CHO+PROT compared to CHO (63.5±4.4 vs 49.8±5.4 min; p<0.05). PLA reduced TTE to 42.8±5.1 min (p<0.05 vs CHO). Nitrogen balance was positive in CHO+PROT, and negative in CHO and PLA. In conclusion, performance was higher 18 h after exhaustive cycling with intake of CHO+PROT compared to an isocaloric amount of carbohydrate during the first 2 h post exercise. Intake of a similar amount of protein but less carbohydrate during the 18 h recovery period reduced performance. PMID:27078151

  1. Accounting for partiality in serial crystallography using ray-tracing principles

    SciTech Connect

    Kroon-Batenburg, Loes M. J. Schreurs, Antoine M. M.; Ravelli, Raimond B. G.; Gros, Piet

    2015-08-25

    Serial crystallography generates partial reflections from still diffraction images. Partialities are estimated with EVAL ray-tracing simulations, thereby improving merged reflection data to a similar quality as conventional rotation data. Serial crystallography generates ‘still’ diffraction data sets that are composed of single diffraction images obtained from a large number of crystals arbitrarily oriented in the X-ray beam. Estimation of the reflection partialities, which accounts for the expected observed fractions of diffraction intensities, has so far been problematic. In this paper, a method is derived for modelling the partialities by making use of the ray-tracing diffraction-integration method EVAL. The method estimates partialities based on crystal mosaicity, beam divergence, wavelength dispersion, crystal size and the interference function, accounting for crystallite size. It is shown that modelling of each reflection by a distribution of interference-function weighted rays yields a ‘still’ Lorentz factor. Still data are compared with a conventional rotation data set collected from a single lysozyme crystal. Overall, the presented still integration method improves the data quality markedly. The R factor of the still data compared with the rotation data decreases from 26% using a Monte Carlo approach to 12% after applying the Lorentz correction, to 5.3% when estimating partialities by EVAL and finally to 4.7% after post-refinement. The merging R{sub int} factor of the still data improves from 105 to 56% but remains high. This suggests that the accuracy of the model parameters could be further improved. However, with a multiplicity of around 40 and an R{sub int} of ∼50% the merged still data approximate the quality of the rotation data. The presented integration method suitably accounts for the partiality of the observed intensities in still diffraction data, which is a critical step to improve data quality in serial crystallography.

  2. The Stanford Automated Mounter: Pushing the limits of sample exchange at the SSRL macromolecular crystallography beamlines

    SciTech Connect

    Russi, Silvia; Song, Jinhu; McPhillips, Scott E.; Cohen, Aina E.

    2016-02-24

    The Stanford Automated Mounter System, a system for mounting and dismounting cryo-cooled crystals, has been upgraded to increase the throughput of samples on the macromolecular crystallography beamlines at the Stanford Synchrotron Radiation Lightsource. This upgrade speeds up robot maneuvers, reduces the heating/drying cycles, pre-fetches samples and adds an air-knife to remove frost from the gripper arms. As a result, sample pin exchange during automated crystal quality screening now takes about 25 s, five times faster than before this upgrade.

  3. The Stanford Automated Mounter: Pushing the limits of sample exchange at the SSRL macromolecular crystallography beamlines

    DOE PAGES

    Russi, Silvia; Song, Jinhu; McPhillips, Scott E.; ...

    2016-02-24

    The Stanford Automated Mounter System, a system for mounting and dismounting cryo-cooled crystals, has been upgraded to increase the throughput of samples on the macromolecular crystallography beamlines at the Stanford Synchrotron Radiation Lightsource. This upgrade speeds up robot maneuvers, reduces the heating/drying cycles, pre-fetches samples and adds an air-knife to remove frost from the gripper arms. As a result, sample pin exchange during automated crystal quality screening now takes about 25 s, five times faster than before this upgrade.

  4. The Stanford Automated Mounter: pushing the limits of sample exchange at the SSRL macromolecular crystallography beamlines

    PubMed Central

    Russi, Silvia; Song, Jinhu; McPhillips, Scott E.; Cohen, Aina E.

    2016-01-01

    The Stanford Automated Mounter System, a system for mounting and dismounting cryo-cooled crystals, has been upgraded to increase the throughput of samples on the macromolecular crystallography beamlines at the Stanford Synchrotron Radiation Lightsource. This upgrade speeds up robot maneuvers, reduces the heating/drying cycles, pre-fetches samples and adds an air-knife to remove frost from the gripper arms. Sample pin exchange during automated crystal quality screening now takes about 25 s, five times faster than before this upgrade. PMID:27047309

  5. Preparation, X-ray crystallography, and thermal decomposition of some transition metal perchlorate complexes of hexamethylenetetramine.

    PubMed

    Singh, Gurdip; Baranwal, B P; Kapoor, I P S; Kumar, Dinesh; Fröhlich, Roland

    2007-12-20

    The perchlorate complexes of manganese, nickel, and zinc with hexamethylenetetramine (HMTA) of the general formula [M(H2O-HMTA-H2O)2(H2O-ClO4)2(H2O)2] (where M=Mn, Ni, and Zn) have been prepared and characterized by X-ray crystallography. Thermal studies were undertaken using thermogravimetry (TG), differential thermal analysis (DTA), and explosion delay (DE) measurements. The kinetics of thermal decomposition of these complexes was investigated using isothermal TG data by applying isoconversional method. The decomposition pathways of the complexes have also been proposed. These were found to explode when subjected to higher temperatures.

  6. Influence of acid-induced conformational variability on protein separation in reversed phase high performance liquid chromatography.

    PubMed

    Bobály, Balázs; Tóth, Eszter; Drahos, László; Zsila, Ferenc; Visy, Júlia; Fekete, Jenő; Vékey, Károly

    2014-01-17

    Influence of acid concentration in the mobile phase on protein separation was studied in a wide concentration range using trifluoroacetic acid (TFA) and formic acid (FA). At low, 0.001-0.01 (v/v%) TFA concentration and appropriate solvent strength proteins elute before the column's dead time. This is explained by the proteins having a structured, but relatively extended conformation in the eluent; and are excluded from the pores of the stationary phase. Above ca. 0.01-0.05 (v/v%) TFA concentration proteins undergo further conformational change, leading to a compact, molten globule-like structure, likely stabilized by ion pairing. Proteins in this conformation enter the pores and are retained on the column. The results suggest a pore exclusion induced separation related to protein conformation. This effect is influenced by the pH and type of acid used, and is likely to involve ion-pair formation. The TFA concentration needed to result in protein folding (and therefore to observe retention on the column) depends on the protein; and therefore can be utilized to improve chromatographic performance. Conformation change was monitored by circular dichroism spectroscopy and mass spectrometry; and it was shown that not only TFA but FA can also induce molten globule formation.

  7. Integrated crystal mounting and alignment system for high-throughput biological crystallography

    DOEpatents

    Nordmeyer, Robert A.; Snell, Gyorgy P.; Cornell, Earl W.; Kolbe, William; Yegian, Derek; Earnest, Thomas N.; Jaklevic, Joseph M.; Cork, Carl W.; Santarsiero, Bernard D.; Stevens, Raymond C.

    2005-07-19

    A method and apparatus for the transportation, remote and unattended mounting, and visual alignment and monitoring of protein crystals for synchrotron generated x-ray diffraction analysis. The protein samples are maintained at liquid nitrogen temperatures at all times: during shipment, before mounting, mounting, alignment, data acquisition and following removal. The samples must additionally be stably aligned to within a few microns at a point in space. The ability to accurately perform these tasks remotely and automatically leads to a significant increase in sample throughput and reliability for high-volume protein characterization efforts. Since the protein samples are placed in a shipping-compatible layered stack of sample cassettes each holding many samples, a large number of samples can be shipped in a single cryogenic shipping container.

  8. Integrated crystal mounting and alignment system for high-throughput biological crystallography

    DOEpatents

    Nordmeyer, Robert A.; Snell, Gyorgy P.; Cornell, Earl W.; Kolbe, William F.; Yegian, Derek T.; Earnest, Thomas N.; Jaklevich, Joseph M.; Cork, Carl W.; Santarsiero, Bernard D.; Stevens, Raymond C.

    2007-09-25

    A method and apparatus for the transportation, remote and unattended mounting, and visual alignment and monitoring of protein crystals for synchrotron generated x-ray diffraction analysis. The protein samples are maintained at liquid nitrogen temperatures at all times: during shipment, before mounting, mounting, alignment, data acquisition and following removal. The samples must additionally be stably aligned to within a few microns at a point in space. The ability to accurately perform these tasks remotely and automatically leads to a significant increase in sample throughput and reliability for high-volume protein characterization efforts. Since the protein samples are placed in a shipping-compatible layered stack of sample cassettes each holding many samples, a large number of samples can be shipped in a single cryogenic shipping container.

  9. [EFFECTS OF INGESTING CARBOHYDRATE-PROTEIN SUPPLEMENTS DURING EXERCISE ON ENDURANCE PERFORMANCE: A SYSTEMATIC REVIEW].

    PubMed

    Espino-González, Ever; Muñoz-Daw, María de Jesús; Candia-Lujan, Ramon

    2015-11-01

    Introducción: las bebidas deportivas ayudan a mejorar el rendimiento físico de forma significativa debido a su aporte de carbohidratos, electrolitos y agua. Sin embargo, en las últimas décadas se ha encontrado que ingerir una bebida deportiva con proteína durante el ejercicio mejora el rendimiento físico, produce menores pérdidas de peso corporal inducidas por la deshidratación y ayuda a disminuir el daño muscular postejercicio en comparación con una bebida únicamente con carbohidratos y electrolitos. Objetivo: analizar los principales estudios sobre la efectividad de la ingesta de un suplemento con carbohidratos, proteína y electrolitos durante el ejercicio. Método: se realizó una búsqueda automatizada en Google académico, EBSCO, PubMEd y Scopus, utilizando las palabras clave: Carbohydrate-protein and performance y Added protein and sports drink. Se evaluó la calidad metodológica de los ensayos y se tomó en cuenta que la ingesta del suplemento fuera durante el ejercicio. Resultados: de los veinte artículos que se incluyeron, trece obtuvieron resultados en los que la ingesta de una bebida deportiva con proteína generó mejoras significativas en el rendimiento físico en comparación con una bebida únicamente con carbohidratos y electrolitos, o un placebo. Discusión: aumentar el contenido calórico de las bebidas deportivas al agregar proteína es probablemente una estrategia más efectiva en comparación con disminuir el contenido de carbohidratos para igualar la cantidad de energía. Conclusiones: el consumo de proteína durante el ejercicio posiblemente sirva como una ayuda ergogénica, retardando el tiempo hasta llegar al agotamiento. Sin embargo, hace falta más evidencia que así lo demuestre.

  10. Protein

    MedlinePlus

    ... Search for: Harvard T.H. Chan School of Public Health Email People Departments Calendar Careers Give my.harvard ... Nutrition Source Harvard T.H. Chan School of Public Health > The Nutrition Source > What Should I Eat? > Protein ...

  11. Protein

    MedlinePlus

    ... Go lean with protein. • Choose lean meats and poultry. Lean beef cuts include round steaks (top loin, ... main dishes. • Use nuts to replace meat or poultry, not in addition to meat or poultry (i. ...

  12. High Pressure X-Ray Crystallography With the Diamond Cell at NIST/NBS

    PubMed Central

    Piermarini, Gasper J.

    2001-01-01

    Scientists in the Crystallography Section at NIST/NBS made several outstanding contributions which greatly promoted the development and advancement of high pressure x-ray crystallography during the second-half of the 20th century. These milestone achievements or “firsts” included: (1) the invention of the lever-arm type diamond anvil cell (DAC) in 1958; (2) the development of DAC technology for powder x-ray diffraction at high pressure in 1960; (3) the introduction of DAC methodology for single crystal x-ray diffraction at high pressure in 1964; (4) the invention of the optical fluorescence ruby method of pressure measurement in 1971; and (5) the discovery of hydrostatic pressure-transmitting media useful to unprecedented pressures for that time. These achievements provided the spark that ignited the explosion of activity in high pressure research that occurred in laboratories throughout the world during the latter part of the 20th century. It is still going on, unabated, today. An estimated 5000 DACs were built during the last 40 years. PMID:27500054

  13. The use of a mini-κ goniometer head in macromolecular crystallography diffraction experiments

    SciTech Connect

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

    2013-07-01

    Hardware and software solutions for MX data-collection strategies using the EMBL/ESRF miniaturized multi-axis goniometer head are presented. Most macromolecular crystallography (MX) diffraction experiments at synchrotrons use a single-axis goniometer. This markedly contrasts with small-molecule crystallography, in which the majority of the diffraction data are collected using multi-axis goniometers. A novel miniaturized κ-goniometer head, the MK3, has been developed to allow macromolecular crystals to be aligned. It is available on the majority of the structural biology beamlines at the ESRF, as well as elsewhere. In addition, the Strategy for the Alignment of Crystals (STAC) software package has been developed to facilitate the use of the MK3 and other similar devices. Use of the MK3 and STAC is streamlined by their incorporation into online analysis tools such as EDNA. The current use of STAC and MK3 on the MX beamlines at the ESRF is discussed. It is shown that the alignment of macromolecular crystals can result in improved diffraction data quality compared with data obtained from randomly aligned crystals.

  14. Redox-coupled structural changes in nitrite reductase revealed by serial femtosecond and microfocus crystallography

    PubMed Central

    Fukuda, Yohta; Suzuki, Mamoru; Matsumura, Hiroyoshi; Inoue, Tsuyoshi; Iwata, So; Mizohata, Eiichi

    2016-01-01

    Serial femtosecond crystallography (SFX) has enabled the damage-free structural determination of metalloenzymes and filled the gaps of our knowledge between crystallographic and spectroscopic data. Crystallographers, however, scarcely know whether the rising technique provides truly new structural insights into mechanisms of metalloenzymes partly because of limited resolutions. Copper nitrite reductase (CuNiR), which converts nitrite to nitric oxide in denitrification, has been extensively studied by synchrotron radiation crystallography (SRX). Although catalytic Cu (Type 2 copper (T2Cu)) of CuNiR had been suspected to tolerate X-ray photoreduction, we here showed that T2Cu in the form free of nitrite is reduced and changes its coordination structure in SRX. Moreover, we determined the completely oxidized CuNiR structure at 1.43 Å resolution with SFX. Comparison between the high-resolution SFX and SRX data revealed the subtle structural change of a catalytic His residue by X-ray photoreduction. This finding, which SRX has failed to uncover, provides new insight into the reaction mechanism of CuNiR. PMID:26769972

  15. Structure determination of a partially ordered layered silicate material with an NMR crystallography approach.

    PubMed

    Brouwer, Darren Henry; Cadars, Sylvian; Hotke, Kathryn; Van Huizen, Jared; Van Huizen, Nicholas

    2017-03-01

    Structure determination of layered materials can present challenges for conventional diffraction methods due to the fact that such materials often lack full three-dimensional periodicity since adjacent layers may not stack in an orderly and regular fashion. In such cases, NMR crystallography strategies involving a combination of solid-state NMR spectroscopy, powder X-ray diffraction, and computational chemistry methods can often reveal structural details that cannot be acquired from diffraction alone. We present here the structure determination of a surfactant-templated layered silicate material that lacks full three-dimensional crystallinity using such an NMR crystallography approach. Through a combination of powder X-ray diffraction and advanced (29)Si solid-state NMR spectroscopy, it is revealed that the structure of the silicate layer of this layered silicate material templated with cetyltrimethylammonium surfactant cations is isostructural with the silicate layer of a previously reported material referred to as ilerite, octosilicate, or RUB-18. High-field (1)H NMR spectroscopy reveals differences between the materials in terms of the ordering of silanol groups on the surfaces of the layers, as well as the contents of the inter-layer space.

  16. Back-exchange of deuterium in neutron crystallography: characterization by IR spectroscopy

    PubMed Central

    Yee, Ai Woon; Moulin, Martine; Haertlein, Michael; Mitchell, Edward; Forsyth, V. Trevor

    2017-01-01

    The application of IR spectroscopy to the characterization and quality control of samples used in neutron crystallography is described. While neutron crystallography is a growing field, the limited availability of neutron beamtime means that there may be a delay between crystallogenesis and data collection. Since essentially all neutron crystallographic work is carried out using D2O-based solvent buffers, a particular concern for these experiments is the possibility of H2O back-exchange across reservoir or capillary sealants. This may limit the quality of neutron scattering length density maps and of the associated analysis. Given the expense of central facility beamtime and the effort that goes into the production of suitably sized (usually perdeuterated) crystals, a systematic method of exploiting IR spectroscopy for the analysis of back-exchange phenomena in the reservoirs used for crystal growth is valuable. Examples are given in which the characterization of D2O/H2O back-exchange in transthyretin crystals is described. PMID:28381984

  17. Effect of dietary supplementation of omega-3 fatty acids and high levels of dietary protein on performance of sows.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was conducted to determine the effect of dietary supplementation of omega-3 fatty acids (O3FA), with or without high levels of dietary protein supplementation, on the performance of sows and their litters during first and subsequent parities. Sixty-four pregnant gilts with body weight (BW...

  18. Effect of dietary supplementation of omega-3 fatty acids and high levels of dietary protein on performance of sows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A study was conducted to determine the effect of dietary supplementation of omega-3 fatty acids (O3FA), with or without high levels of protein, on the performance of sows during first and subsequent parity. Sixty-four pregnant gilts with BW of 195.0 ± 2.1 kg and backfat (BF) thickness of 12.9 ± 0.2 ...

  19. The influences of ambient temperature and crude protein levels on performance and serum biochemical parameters in broilers.

    PubMed

    Liu, Q W; Feng, J H; Chao, Z; Chen, Y; Wei, L M; Wang, F; Sun, R P; Zhang, M H

    2016-04-01

    This study was undertaken to investigate the effects of ambient temperature, crude protein levels and their interaction on performance and serum biochemical parameters of broiler chickens. A total of 216 Arbor Acre broiler chickens (108 males and 108 females) were used in a 2 × 3 factorial arrangement and randomly reared at two temperatures (normal temperature: 23 °C; daily cyclic high temperature: 28-32 °C) and fed on three diets with different crude protein levels (153.3, 183.3 or 213.3 g/kg, with constant essential amino acids) from 28 to 42 days of age. Daily cyclic high ambient temperature decreased final body weight, average daily weight gain, average daily feed intake and serum total protein contents (p < 0.001, p < 0.001, p < 0.001, p = 0.008 respectively), but increased feed/gain, mortality, respiratory rate, rectal temperature, serum uric acid contents and serum creatine kinase activity (p = 0.008, p = 0.003, p < 0.0001, p < 0.0001, p < 0.0001, p = 0.003 respectively), irrespective of crude protein levels. At the ambient temperature, reducing crude protein levels resulted in an increase in feed/gain (p < 0.001), but a decrease in serum total protein and uric acid contents. Only serum creatine kinase activity in broiler chickens was interacted by daily cyclic high ambient temperature and dietary crude protein levels (p = 0.003). These results indicated that daily cyclic high ambient temperature had a great effect on performance and serum biochemical parameters in broiler chickens, whereas dietary crude protein levels affected them partially.

  20. Protein losses in ion-exchange and hydrophobic interaction high-performance liquid chromatography

    SciTech Connect

    Goheen, Steven C.; Gibbins, Betty M.

    2000-01-01

    Protein losses in ion-exchange and hydrophobic interaction HPLC were examined. The supports were allnon-porous, packed in columns of identical dimensions. Two ion-exchange chromatography (IEC), anion and cation, as well as a hydrophobic interaction chromatography (HIC) columns were tested. Proteins included cytochrome c, bovine serum albumin (BSA), immunoglobulin G and fibrinogen. Temperature effects on HIC supports were studied for cytochrome c and BSA. Both retention times and recoveries of the proteins were measured. The influence of column residence time on the recovery of proteins were also investigated. We found a linear relationship between the amount of protein recovered and the log of the molecular mass. Retention times also generally increased with temperature for both HIC and IEC. Other trends in retention behavior and recoveries are discussed.

  1. Effects of winter military training on energy balance, whole-body protein balance, muscle damage, soreness, and physical performance.

    PubMed

    Margolis, Lee M; Murphy, Nancy E; Martini, Svein; Spitz, Marissa G; Thrane, Ingjerd; McGraw, Susan M; Blatny, Janet-Martha; Castellani, John W; Rood, Jennifer C; Young, Andrew J; Montain, Scott J; Gundersen, Yngvar; Pasiakos, Stefan M

    2014-12-01

    Physiological consequences of winter military operations are not well described. This study examined Norwegian soldiers (n = 21 males) participating in a physically demanding winter training program to evaluate whether short-term military training alters energy and whole-body protein balance, muscle damage, soreness, and performance. Energy expenditure (D2(18)O) and intake were measured daily, and postabsorptive whole-body protein turnover ([(15)N]-glycine), muscle damage, soreness, and performance (vertical jump) were assessed at baseline, following a 4-day, military task training phase (MTT) and after a 3-day, 54-km ski march (SKI). Energy intake (kcal·day(-1)) increased (P < 0.01) from (mean ± SD (95% confidence interval)) 3098 ± 236 (2985, 3212) during MTT to 3461 ± 586 (3178, 3743) during SKI, while protein (g·kg(-1)·day(-1)) intake remained constant (MTT, 1.59 ± 0.33 (1.51, 1.66); and SKI, 1.71 ± 0.55 (1.58, 1.85)). Energy expenditure increased (P < 0.05) during SKI (6851 ± 562 (6580, 7122)) compared with MTT (5480 ± 389 (5293, 5668)) and exceeded energy intake. Protein flux, synthesis, and breakdown were all increased (P < 0.05) 24%, 18%, and 27%, respectively, during SKI compared with baseline and MTT. Whole-body protein balance was lower (P < 0.05) during SKI (-1.41 ± 1.11 (-1.98, -0.84) g·kg(-1)·10 h) than MTT and baseline. Muscle damage and soreness increased and performance decreased progressively (P < 0.05). The physiological consequences observed during short-term winter military training provide the basis for future studies to evaluate nutritional strategies that attenuate protein loss and sustain performance during severe energy deficits.

  2. Study of the inclusion of the (R)- and (S)-camphor enantiomers in alpha-cyclodextrin by X-ray crystallography and molecular dynamics.

    PubMed

    Kokkinou, Areti; Tsorteki, Frantzeska; Karpusas, Michael; Papakyriakou, Athanasios; Bethanis, Kostas; Mentzafos, Dimitris

    2010-05-27

    The inclusion of (R)- and (S)-camphor compounds in alpha-cyclodextrin has been studied by X-ray crystallography. The crystal structures of the complexes reveal that one guest molecule is accommodated inside the cavity formed by a head-to-head cyclodextrin dimer. In the crystal lattice, the dimers form layers which are successively shifted by half a dimer. In both (R)- and (S)-cases, the camphor molecule exhibits disorder and occupies three major sites with orientations that can be described as either 'polar' or 'equatorial'. Molecular dynamics simulations performed for the observed complexes indicate that although the carbonyl oxygen of both (R)- and (S)-camphor switches between different hydrogen bonding partners, it maintains the observed mode of 'polar' or 'equatorial' alignment.

  3. The application of tailor-made force fields and molecular dynamics for NMR crystallography: a case study of free base cocaine

    PubMed Central

    Neumann, Marcus A.

    2017-01-01

    Motional averaging has been proven to be significant in predicting the chemical shifts in ab initio solid-state NMR calculations, and the applicability of motional averaging with molecular dynamics has been shown to depend on the accuracy of the molecular mechanical force field. The performance of a fully automatically generated tailor-made force field (TMFF) for the dynamic aspects of NMR crystallography is evaluated and compared with existing benchmarks, including static dispersion-corrected density functional theory calculations and the COMPASS force field. The crystal structure of free base cocaine is used as an example. The results reveal that, even though the TMFF outperforms the COMPASS force field for representing the energies and conformations of predicted structures, it does not give significant improvement in the accuracy of NMR calculations. Further studies should direct more attention to anisotropic chemical shifts and development of the method of solid-state NMR calculations. PMID:28250956

  4. Soft Computing Techniques for the Protein Folding Problem on High Performance Computing Architectures.

    PubMed

    Llanes, Antonio; Muñoz, Andrés; Bueno-Crespo, Andrés; García-Valverde, Teresa; Sánchez, Antonia; Arcas-Túnez, Francisco; Pérez-Sánchez, Horacio; Cecilia, José M

    2016-01-01

    The protein-folding problem has been extensively studied during the last fifty years. The understanding of the dynamics of global shape of a protein and the influence on its biological function can help us to discover new and more effective drugs to deal with diseases of pharmacological relevance. Different computational approaches have been developed by different researchers in order to foresee the threedimensional arrangement of atoms of proteins from their sequences. However, the computational complexity of this problem makes mandatory the search for new models, novel algorithmic strategies and hardware platforms that provide solutions in a reasonable time frame. We present in this revision work the past and last tendencies regarding protein folding simulations from both perspectives; hardware and software. Of particular interest to us are both the use of inexact solutions to this computationally hard problem as well as which hardware platforms have been used for running this kind of Soft Computing techniques.

  5. Using antibody arrays to measure protein abundance and glycosylation: considerations for optimal performance

    PubMed Central

    Haab, Brian B.; Partyka, Katie; Cao, Zheng

    2013-01-01

    Antibody arrays provide a valuable method of obtaining multiple protein measurements from low volumes of biological samples. Antibody arrays can be designed to target not only core protein abundances (relative or absolute abundances, depending on the availability of standards for calibration), but also protein post-translational modifications, provided antibodies or other affinity reagents are available to detect the modifications. Glycosylation is a common modification that has important and diverse functions, both in normal and disease biology. The methods for measuring glycan levels on multiple, specific proteins using antibody arrays and glycan-binding reagents have made significant progress. Here we describe practical approaches to develop, optimize and use antibody array assays to determine both protein abundances and glycosylation states. We cover the use of low-volume arrays to reduce sample consumption and a new way to improve the binding strength of particular glycan-binding reagents through multimerization. These methods could be useful for a wide range of biological studies in which glycosylation may be changing or affect protein function. PMID:24510592

  6. Supplemental Protein during Heavy Cycling Training and Recovery Impacts Skeletal Muscle and Heart Rate Responses but Not Performance.

    PubMed

    D'Lugos, Andrew C; Luden, Nicholas D; Faller, Justin M; Akers, Jeremy D; McKenzie, Alec I; Saunders, Michael J

    2016-09-07

    The effects of protein supplementation on cycling performance, skeletal muscle function, and heart rate responses to exercise were examined following intensified (ICT) and reduced-volume training (RVT). Seven cyclists performed consecutive periods of normal training (NT), ICT (10 days; average training duration 220% of NT), and RVT (10 days; training duration 66% of NT). In a crossover design, subjects consumed supplemental carbohydrate (CHO) or an equal amount of carbohydrate with added protein (CP) during and following each exercise session (CP = +0.94 g/kg/day protein during ICT; +0.39 g/kg/day during RVT). A 30-kilometer time trial performance (following 120 min at 50% Wmax) was modestly impaired following ICT (+2.4 ± 6.4% versus NT) and returned to baseline levels following RVT (-0.7 ± 4.5% versus NT), with similar responses between CHO and CP. Skeletal muscle torque at 120 deg/s benefited from CP, compared to CHO, following ICT. However, this effect was no longer present at RVT. Following ICT, muscle fiber cross-sectional area was increased with CP, while there were no clear changes with CHO. Reductions in constant-load heart rates (at 50% Wmax) following RVT were likely greater with CP than CHO (-9 ± 9 bpm). Overall it appears that CP supplementation impacted skeletal muscle and heart rate responses during a period of heavy training and recovery, but this did not result in meaningful changes in time trial performance.

  7. Supplemental Protein during Heavy Cycling Training and Recovery Impacts Skeletal Muscle and Heart Rate Responses but Not Performance

    PubMed Central

    D’Lugos, Andrew C.; Luden, Nicholas D.; Faller, Justin M.; Akers, Jeremy D.; McKenzie, Alec I.; Saunders, Michael J.

    2016-01-01

    The effects of protein supplementation on cycling performance, skeletal muscle function, and heart rate responses to exercise were examined following intensified (ICT) and reduced-volume training (RVT). Seven cyclists performed consecutive periods of normal training (NT), ICT (10 days; average training duration 220% of NT), and RVT (10 days; training duration 66% of NT). In a crossover design, subjects consumed supplemental carbohydrate (CHO) or an equal amount of carbohydrate with added protein (CP) during and following each exercise session (CP = +0.94 g/kg/day protein during ICT; +0.39 g/kg/day during RVT). A 30-kilometer time trial performance (following 120 min at 50% Wmax) was modestly impaired following ICT (+2.4 ± 6.4% versus NT) and returned to baseline levels following RVT (−0.7 ± 4.5% versus NT), with similar responses between CHO and CP. Skeletal muscle torque at 120 deg/s benefited from CP, compared to CHO, following ICT. However, this effect was no longer present at RVT. Following ICT, muscle fiber cross-sectional area was increased with CP, while there were no clear changes with CHO. Reductions in constant-load heart rates (at 50% Wmax) following RVT were likely greater with CP than CHO (−9 ± 9 bpm). Overall it appears that CP supplementation impacted skeletal muscle and heart rate responses during a period of heavy training and recovery, but this did not result in meaningful changes in time trial performance. PMID:27618091

  8. The influence of 8 weeks of whey-protein and leucine supplementation on physical and cognitive performance.

    PubMed

    Walker, Thomas B; Smith, Jessica; Herrera, Monica; Lebegue, Breck; Pinchak, Andrea; Fischer, Joseph

    2010-10-01

    The purpose of this study was to investigate the ability of whey-protein and leucine supplementation to enhance physical and cognitive performance and body composition. Thirty moderately fit participants completed a modified Air Force fitness test, a computer-based cognition test, and a dual-energy X-ray-absorptiometry scan for body composition before and after supplementing their daily diet for 8 wk with either 19.7 g of whey protein and 6.2 g leucine (WPL) or a calorie-equivalent placebo (P). Bench-press performance increased significantly from Week 1 to Week 8 in the WPL group, whereas the increase in the P group was not significant. Push-up performance increased significantly for WPL, and P showed a nonsignificant increase. Total mass, fat-free mass, and lean body mass all increased significantly in the WPL group but showed no change in the P group. No differences were observed within or between groups for crunches, chin-ups, 3-mile-run time, or cognition. The authors conclude that supplementing with whey protein and leucine may provide an advantage to people whose performance benefits from increased upper body strength and/or lean body mass.

  9. The Molecular Mechanisms Underlying the Hijack of Host Proteins by the 1918 Spanish Influenza Virus.

    PubMed

    Shen, Qingliang; Zeng, Danyun; Zhao, Baoyu; Bhatt, Veer S; Li, Pingwei; Cho, Jae-Hyun

    2017-04-05

    The 1918 Spanish influenza A virus (IAV) caused one of the most serious pandemics in history. The nonstructural protein 1 (NS1) of the 1918 IAV hijacks the interaction between human CrkII and JNK1. Little is, however, known about its molecular mechanism. Here, we performed X-ray crystallography, NMR relaxation dispersion experiment, and fluorescence spectroscopy to determine the structural, kinetic, and thermodynamic mechanisms underlying the hijacking of CrkII by 1918 IAV NS1. We observed that the interaction between a proline-rich motif in NS1 and the N-terminal SH3 domain of CrkII displays strikingly rapid kinetics and exceptionally high affinity with 100-fold faster kon and 3300-fold lower Kd compared to those for the CrkII-JNK1 interaction. These results provide molecular insight into the mechanism by which 1918 IAV NS1 hijacks CrkII and disrupts its interactions with critical cellular signaling proteins.

  10. Effect on digestion and performance of dietary protein content and of increased substitution of lucerne hay with soya-bean protein concentrate in starter diets for young rabbits.

    PubMed

    Chamorro, S; Gómez-Conde, M S; Pérez de Rozas, A M; Badiola, I; Carabaño, R; De Blas, J C

    2007-06-01

    The aim of this work was to study the effect of protein source / availability on the intestinal microbiota, digestive traits and nutritional performance of early-weaned rabbits. The effects of supplemental antibiotics in the drinking water were also evaluated. Four isoenergetic and isofibrous diets were formulated: a control diet with a high protein (207 g/kg dry matter (DM)) and lucerne hay content (HPHL), a diet with low crude protein (CP) (179 g/kg DM) and high lucerne hay content (LPHL) and low protein diets in which the lucerne hay in diet LPHL was replaced partially (LPML) or totally (LPLL) with soya-bean protein concentrate. Rabbits, weaned at 25 days (52 per diet), were fed the experimental diets for a 2-week period and thereafter received a commercial diet until 56 days of age. The incidence of mortality was investigated using 70 animals per diet without supplemental medication. The profile of the ileal microbiota was studied at 35 days of age in rabbits treated (18 per diet) or not (12 per diet) with antibiotic. As expected, supplementation with antibiotics effectively reduced fattening mortality rate and microbial biodiversity. However, lowering of also the dietary CP content led to a reduction in the mortality rate ( P < 0.05), both in animals treated with (by 80%) or without (by 39%) antibiotics. In addition, there was a reduction ( P < 0.05) in the frequency of Clostridium perfringens in non-medicated animals. Neither jejunal morphology nor growth performance, over the whole fattening period, was affected by dietary CP content of the experimental diets. However, with HPHL, feed efficiency was higher (by 4.8%; P < 0.01) than with LPHL diets. Substitution of lucerne hay with soya-bean meal in low protein diets did not affect apparent faecal or ileal digestibility of DM and CP. However, the ileal digestibility of cystine, alanine, aspartic acid, and proline was lowered ( P < 0.05) with increasing substitution by soya bean. Nevertheless

  11. Using antibody arrays to measure protein abundance and glycosylation: considerations for optimal performance.

    PubMed

    Haab, Brian B; Partyka, Katie; Cao, Zheng

    2013-09-24

    Antibody arrays provide a valuable method for obtaining multiple protein measurements from small volumes of biological samples. Antibody arrays can be designed to target not only core protein abundances (relative or absolute abundances, depending on the availability of standards for calibration), but also posttranslational modifications, provided antibodies or other affinity reagents are available to detect them. Glycosylation is a common modification that has important and diverse functions in both normal and disease biology. Significant progress has been made in developing methods for measuring glycan levels on multiple specific proteins using antibody arrays and glycan-binding reagents. This unit describes practical approaches for developing, optimizing, and using antibody array assays to determine both protein abundance and glycosylation state. Low-volume arrays can be used to reduce sample consumption, and a new way to improve the binding strength of particular glycan-binding reagents through multimerization is discussed. These methods can be useful for a wide range of biological studies in which glycosylation may change and/or affect protein function.

  12. Breakthrough performance of large proteins on ion-exchange membrane columns.

    PubMed

    Montesinos-Cisneros, Rosa Maria; Lucero-Acuña, Armando; Ortega, Jaime; Guzmán, Roberto; Tejeda-Mansir, Armando

    2007-10-01

    Protein adsorption of large proteins on ion-exchange membrane columns was theoretically and experimentally investigated using batch and fixed-bed systems. Thyroglobulin was used as the model protein. The study strongly suggests that part of the protein is physically retained inside the column during frontal mode operation. These experimental results were used to obtain a filtration function of the chromatographic system. In the theoretical analysis of the frontal protein adsorption, a model was integrated by the serial coupling of the membrane-transport model, the filtration model and the system-dispersion model. Two different techniques were employed in the estimation of the maximum adsorption capacity, the equilibrium desorption constant and the forward interaction rate constant, which are the parameters of the membrane-transport model. The fit of the model to the experimental data was not possible using the equilibrium parameters obtained in the batch experiments. The parameter estimation using a simplex optimization routine coupled to the solution of the partial differential model equations yields full prediction of the adsorption phenomena.

  13. Effect of additives on the tensile performance and protein solubility of industrial oilseed residual based plastics.

    PubMed

    Newson, William R; Kuktaite, Ramune; Hedenqvist, Mikael S; Gällstedt, Mikael; Johansson, Eva

    2014-07-16

    Ten chemical additives were selected from the literature for their proposed modifying activity in protein-protein interactions. These consisted of acids, bases, reducing agents, and denaturants and were added to residual deoiled meals of Crambe abyssinica (crambe) and Brassica carinata (carinata) to modify the properties of plastics produced through hot compression molding at 130 °C. The films produced were examined for tensile properties, protein solubility, molecular weight distribution, and water absorption. Of the additives tested, NaOH had the greatest positive effect on tensile properties, with increases of 105% in maximum stress and 200% in strain at maximum stress for crambe and a 70% increase in strain at maximum stress for carinata. Stiffness was not increased by any of the applied additives. Changes in tensile strength and elongation for crambe and elongation for carinata were related to changes in protein solubility. Increased pH was the most successful in improving the protein aggregation and mechanical properties within the complex chemistry of residual oilseed meals.

  14. Probing the binding behavior and conformational states of globular proteins in reversed-phase high-performance liquid chromatography.

    PubMed

    Purcell, A W; Aguilar, M I; Hearn, M T

    1999-07-01

    Reversed-phase high-performance liquid chromatography (RP-HPLC) is a widely used technique for the separation of proteins under low pH aquo-organic solvent gradient elution conditions, typically carried out at ambient temperatures. These conditions can however induce conformational effects with proteins as evident from changes in their biological or immunological activities. By monitoring the influence of temperature on the retention and band-broadening characteristics of proteins, the role of conformational processes in these lipophilic environments can be examined. These processes can then be interpreted in terms of a two-state model involving a native (N) and a fully unfolded species (U) or more complex folding/unfolding models. In the present study, the gradient elution RP-HPLC behavior of sperm whale myoglobin (SWMYO) and hen egg white lysozyme (HEWL) has been investigated at temperatures between 5 and 85 degrees C with n-octadecyl (C18)- and n-butyl (C4)-silica reversed-phase sorbents. The interaction of these proteins with these reversed-phase sorbents has also been examined in terms of the contributions that the heme prosthetic group of SWMYO and the disulfide bonds in HEWL make to the stabilization of the native conformation of these proteins in these hydrophobic environments. The observed interconversions of multiple peak zones of SWMYO and HEWL in the presence of C18 and C4 ligands have been subsequently analyzed in terms of the unfolding processes that these proteins can undergo at low pH and at elevated temperatures. The ability of hydrocarbonaceous ligands to trap ensemblies of partially unfolded conformational intermediates of proteins in these perturbing environments has been examined. Pseudo-first-order rate constants have been derived for these processes from analysis of the dependencies on time of the concentration of the different protein species at specified temperatures. The relationship of these processes to the conformational transitions

  15. Community proteogenomics highlights microbial strain-variant protein expression within activated sludge performing enhanced biological phosphorus removal.

    SciTech Connect

    Wilmes, P; Andersson, Anders F.; Lefsrud, Mark G; Wexler, Margaret; Shah, Manesh B; Zhang, B; Hettich, Robert {Bob} L; Bond, P. L.; Verberkmoes, Nathan C; Banfield, Jillian F.

    2008-01-01

    Enhanced biological phosphorus removal (EBPR) selects for polyphosphate accumulating organisms to achieve phosphate removal from wastewater. We used highresolution community proteomics to identify key metabolic pathways in "Candidatus Accumulibacter phosphatis"-mediated EBPR and to evaluate the contributions of co- 5 existing strains within the dominant population. Results highlight the importance of denitrification, fatty acid cycling and the glyoxylate bypass in EBPR. Despite overall strong similarity in protein profiles under anaerobic and aerobic conditions, fatty acid degradation proteins were more abundant during the anaerobic phase. By comprehensive genome-wide alignment of orthologous proteins, we uncovered strong 10 functional partitioning for enzyme variants involved in both core-metabolism and EBPR-specific pathways among the dominant strains. These findings emphasize the importance of genetic diversity in maintaining the stable performance of EBPR systems and demonstrate the power of integrated cultivation-independent genomics and proteomics for analysis of complex biotechnological systems.

  16. Serum protein profile study of normal and cervical cancer subjects by high performance liquid chromatography with laser-induced fluorescence.

    PubMed

    Sujatha; Rai, Lavanya; Kumar, Pratap; Mahato, Krishna K; Kartha, Vasudevan B; Santhosh, Chidangil

    2008-01-01

    High performance liquid chromatography with high sensitivity laser-induced fluorescence detection is used to study the protein profiles of serum samples from healthy volunteers and cervical cancer subjects. The protein profiles are subjected to principal component analysis (PCA). PCA shows that the large number of chromatograms of a given class of serum samples--say normal/malignant--can be expressed in terms of a small number of factors (principal components). Three parameters--scores of the factors, squared residuals, and Mahalanobis distance--are derived from PCA. The parameters are observed to have a narrow range for protein profiles of standard calibration sets formed from groups of clinically confirmed normal/malignant classes. Limit tests using match/no match of the parameters of any test sample with parameters derived for the standard calibration sets give very good discrimination between malignant and normal samples with high sensitivity (approximately 100%) aand specificity (approximately 94%).

  17. In-plate protein crystallization, in situ ligand soaking and X-ray diffraction.

    PubMed

    le Maire, Albane; Gelin, Muriel; Pochet, Sylvie; Hoh, François; Pirocchi, Michel; Guichou, Jean François; Ferrer, Jean Luc; Labesse, Gilles

    2011-09-01

    X-ray crystallography is now a recognized technique for ligand screening, especially for fragment-based drug design. However, protein crystal handling is still tedious and limits further automation. An alternative method for the solution of crystal structures of proteins in complex with small ligands is proposed. Crystallization drops are directly exposed to an X-ray beam after cocrystallization or soaking with the desired ligands. The use of dedicated plates in connection with an optimal parametrization of the G-rob robot allows efficient data collection. Three proteins currently under study in our laboratory for ligand screening by X-ray crystallography were used as validation test cases. The protein crystals belonged to different space groups, including a challenging monoclinic case. The resulting diffraction data can lead to clear ligand recognition, including indication of alternating conformations. These results demonstrate a possible method for automation of ligand screening by X-ray crystallography.

  18. Teaching with the Case Study Method to Promote Active Learning in a Small Molecule Crystallography Course for Chemistry Students

    ERIC Educational Resources Information Center

    Campbell, Michael G.; Powers, Tamara M.; Zheng, Shao-Liang

    2016-01-01

    Implementing the case study method in a practical X-ray crystallography course designed for graduate or upper-level undergraduate chemistry students is described. Compared with a traditional lecture format, assigning small groups of students to examine literature case studies encourages more active engagement with the course material and…

  19. Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

    SciTech Connect

    Wan, Qun; Parks, Jerry M.; Hanson, B. Leif; Fisher, Suzanne Zoe; Ostermann, Andreas; Schrader, Tobias E.; Graham, David E.; Coates, Leighton; Langan, Paul; Kovalevsky, Andrey

    2015-09-21

    Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. Lastly, these findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.

  20. Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

    DOE PAGES

    Wan, Qun; Parks, Jerry M.; Hanson, B. Leif; ...

    2015-09-21

    Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The generalmore » acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. Lastly, these findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.« less