WorldWideScience

Sample records for protein x-ray crystallography

  1. X-ray crystallography

    Science.gov (United States)

    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.

  2. Time-resolved protein nano-crystallography using an X-ray free-electron laser

    International Nuclear Information System (INIS)

    Aquila, Andrew; Hunter, Mark S.; Fromme, Petra; Fromme, Raimund; Grotjohann, Ingo; Doak, R. Bruce; Kirian, Richard A.; Schmidt, Kevin E.; Wang, Xiaoyu; Weierstall, Uwe; Spence, John C.H.; White, Thomas A.; Caleman, Carl; DePonte, Daniel P.; Fleckenstein, Holger; Gumprecht, Lars; Liang, Mengning; Martin, Andrew V.; Schulz, Joachim; Stellato, Francesco; Stern, Stephan; Barty, Anton; Andreasson, Jakob; Davidsson, Jan; Hajdu, Janos; Maia, Filipe R.N.C.; Seibert, M. Marvin; Timneanu, Nicusor; Arnlund, David; Johansson, Linda; Malmerberg, Erik; Neutze, Richard; Bajt, Sasa; Barthelmess, Miriam; Graafsma, Heinz; Hirsemann, Helmut; Wunderer, Cornelia; Barends, Thomas R.M.; Foucar, Lutz; Krasniqi, Faton; Lomb, Lukas; Rolles, Daniel; Schlichting, Ilme; Schmidt, Carlo; Bogan, Michael J.; Hampton, Christina Y.; Sierra, Raymond; Starodub, Dmitri; Bostedt, Christoph; Bozek, John D.; Messerschmidt, Marc; Williams, Garth J.; Bottin, Herve

    2012-01-01

    We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photo-activated states of large membrane protein complexes in the form of nano-crystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 μs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems. (authors)

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

    International Nuclear Information System (INIS)

    Šrajer, Vukica; Schmidt, Marius

    2017-01-01

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

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

    Science.gov (United States)

    Šrajer, Vukica; Schmidt, Marius

    2017-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Šrajer, Vukica; Schmidt, Marius

    2017-08-22

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

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

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    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 2 -adrenoreceptor–G 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 macromolecular

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

    Science.gov (United States)

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

    2010-01-01

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

  9. X-ray tests of microfocusing mono-capillary optic for protein crystallography

    CERN Document Server

    Bilderback, D H

    2001-01-01

    A single, borosilicate-glass capillary was drawn into a 30.5 cm long elliptical shape. The inside diameter was 0.40 mm at the large base end and 0.13 mm at the tip. With 12 keV X-rays from the CHESS D1 bending magnet, the single-bounce capillary produced a focus of better than 18 mu m in diameter (FHWM) at a 3 cm distance from the capillary tip. A flux gain of 110 in the focus position was observed along with a total flux in the spot of 4x10 sup 1 sup 0 X-rays/s (conditions: 5.3 GeV, 182 mA, 1.5% bandwidth multilayer, 12 keV X-rays). A measurement of the far field focus ring diameter yielded a divergence of 3.8 mrad, in good agreement with the 4 mrad design of the optic for protein crystallography. Using a small 25 mu m square beam, we measured the local reflectivity to be greater than 95% and the inner slope errors of the capillary to average about +-150 mu rad, both from raw and elliptically shaped tubing. Our conclusion is that more perfect starting tubing (i.e. one with lower slope errors) is needed to ma...

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Jasper J. van Thor

    2015-01-01

    Full Text Available 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.

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

    OpenAIRE

    Soichiro Tsujino; Takashi Tomizaki

    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 spinn...

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

    Science.gov (United States)

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

    2010-11-21

    Here we report the total synthesis of kaliotoxin by 'one pot' native chemical ligation of three synthetic peptides. A racemic mixture of D- and L-kaliotoxin synthetic protein molecules gave crystals in the centrosymmetric space group P1 that diffracted to atomic-resolution (0.95 Å), enabling the X-ray structure of kaliotoxin to be determined by direct methods.

  14. Watching proteins function with picosecond X-ray crystallography and molecular dynamics simulations.

    Science.gov (United States)

    Anfinrud, Philip

    2006-03-01

    Time-resolved electron density maps of myoglobin, a ligand-binding heme protein, have been stitched together into movies that unveil with molecular dynamics (MD) calculations and picosecond time-resolved X-ray structures provides single-molecule insights into mechanisms of protein function. Ensemble-averaged MD simulations of the L29F mutant of myoglobin following ligand dissociation reproduce the direction, amplitude, and timescales of crystallographically-determined structural changes. This close agreement with experiments at comparable resolution in space and time validates the individual MD trajectories, which identify and structurally characterize a conformational switch that directs dissociated ligands to one of two nearby protein cavities. This unique combination of simulation and experiment unveils functional protein motions and illustrates at an atomic level relationships among protein structure, dynamics, and function. In collaboration with Friedrich Schotte and Gerhard Hummer, NIH.

  15. CCD[charge-coupled device]-based synchrotron x-ray detector for protein crystallography: Performance projected from an experiment

    International Nuclear Information System (INIS)

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

    1986-01-01

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

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

    Science.gov (United States)

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

    2009-01-01

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

  17. Structure determination by X-ray crystallography

    CERN Document Server

    Ladd, M F C

    1977-01-01

    Crystallography may be described as the science of the structure of materi­ als, using this word in its widest sense, and its ramifications are apparent over a broad front of current scientific endeavor. It is not surprising, therefore, to find that most universities offer some aspects of crystallography in their undergraduate courses in the physical sciences. It is the principal aim of this book to present an introduction to structure determination by X-ray crystal­ lography that is appropriate mainly to both final-year undergraduate studies in crystallography, chemistry, and chemical physics, and introductory post­ graduate work in this area of crystallography. We believe that the book will be of interest in other disciplines, such as physics, metallurgy, biochemistry, and geology, where crystallography has an important part to play. In the space of one book, it is not possible either to cover all aspects of crystallography or to treat all the subject matter completely rigorously. In particular, certain ...

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

    International Nuclear Information System (INIS)

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

    1999-01-01

    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

  19. Fully convergent chemical synthesis of ester insulin: determination of the high resolution X-ray structure by racemic protein crystallography.

    Science.gov (United States)

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

    2013-02-27

    Efficient total synthesis of insulin is important to enable the application of medicinal chemistry to the optimization of the properties of this important protein molecule. Recently we described "ester insulin"--a novel form of insulin in which the function of the 35 residue C-peptide of proinsulin is replaced by a single covalent bond--as a key intermediate for the efficient total synthesis of insulin. Here we describe a fully convergent synthetic route to the ester insulin molecule from three unprotected peptide segments of approximately equal size. The synthetic ester insulin polypeptide chain folded much more rapidly than proinsulin, and at physiological pH. Both the D-protein and L-protein enantiomers of monomeric DKP ester insulin (i.e., [Asp(B10), Lys(B28), Pro(B29)]ester insulin) were prepared by total chemical synthesis. The atomic structure of the synthetic ester insulin molecule was determined by racemic protein X-ray crystallography to a resolution of 1.6 Å. Diffraction quality crystals were readily obtained from the racemic mixture of {D-DKP ester insulin + L-DKP ester insulin}, whereas crystals were not obtained from the L-ester insulin alone even after extensive trials. Both the D-protein and L-protein enantiomers of monomeric DKP ester insulin were assayed for receptor binding and in diabetic rats, before and after conversion by saponification to the corresponding DKP insulin enantiomers. L-DKP ester insulin bound weakly to the insulin receptor, while synthetic L-DKP insulin derived from the L-DKP ester insulin intermediate was fully active in binding to the insulin receptor. The D- and L-DKP ester insulins and D-DKP insulin were inactive in lowering blood glucose in diabetic rats, while synthetic L-DKP insulin was fully active in this biological assay. The structural basis of the lack of biological activity of ester insulin is discussed.

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

    International Nuclear Information System (INIS)

    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

  1. Why do We Trust X-ray Crystallography?

    Indian Academy of Sciences (India)

    IAS Admin

    crystal X-ray diffraction pattern and good chemical sense that elevates X-ray crystallography to its position as the most trusted analytical technique. Suggested Reading. [1] William Clegg, Crystal Structure Determination, Oxford Chemistry Prim-.

  2. Structure determination by X-ray crystallography

    CERN Document Server

    Ladd, M F C

    1995-01-01

    X-ray crystallography provides us with the most accurate picture we can get of atomic and molecular structures in crystals. It provides a hard bedrock of structural results in chemistry and in mineralogy. In biology, where the structures are not fully crystalline, it can still provide valuable results and, indeed, the impact here has been revolutionary. It is still an immense field for young workers, and no doubt will provide yet more striking develop­ ments of a major character. It does, however, require a wide range of intellectual application, and a considerable ability in many fields. This book will provide much help. It is a very straightforward and thorough guide to every aspect of the subject. The authors are experienced both as research workers themselves and as teachers of standing, and this is shown in their clarity of exposition. There are plenty of iliustrations and worked examples to aid the student to obtain a real grasp of the subject.

  3. Determination of the X-ray structure of the snake venom protein omwaprin by total chemical synthesis and racemic protein crystallography.

    Science.gov (United States)

    Banigan, James R; Mandal, Kalyaneswar; Sawaya, Michael R; Thammavongsa, Vilasak; Hendrickx, Antoni P A; Schneewind, Olaf; Yeates, Todd O; Kent, Stephen B H

    2010-10-01

    The 50-residue snake venom protein L-omwaprin and its enantiomer D-omwaprin were prepared by total chemical synthesis. Radial diffusion assays were performed against Bacillus megaterium and Bacillus anthracis; both L- and D-omwaprin showed antibacterial activity against B. megaterium. The native protein enantiomer, made of L-amino acids, failed to crystallize readily. However, when a racemic mixture containing equal amounts of L- and D-omwaprin was used, diffraction quality crystals were obtained. The racemic protein sample crystallized in the centrosymmetric space group P2(1)/c and its structure was determined at atomic resolution (1.33 A) by a combination of Patterson and direct methods based on the strong scattering from the sulfur atoms in the eight cysteine residues per protein. Racemic crystallography once again proved to be a valuable method for obtaining crystals of recalcitrant proteins and for determining high-resolution X-ray structures by direct methods.

  4. The 100th Anniversary of X-Ray Crystallography

    Directory of Open Access Journals (Sweden)

    Kojić-Prodić, B.

    2013-07-01

    Full Text Available The important thing in science is not so much to obtain new facts as to discover new ways of thinking about them.W. L. BraggThe 100th anniversary of X-ray crystallography dates back to the first X-ray diffraction experiment on a crystal of copper sulphate pentahydrate. Max von Laue designed the theoretical background of the experiment, which was performed by German physicists W. Friedrich and P. Knipping in 1912. At that time, the mathematical formulation of the phenomenon and the fundamental concepts of crystallography were subjects of mineralogy. Altogether, they facilitated the development of methods for determination of the structure of matter at the atomic level. In 1913, father and son Bragg started to develop X-ray structure analysis for determination of crystal structures of simple molecules. Historic examples of structure determination starting from rock salt to complex, biologically important (macromolecules, such as globular proteins haemoglobin and myoglobin, DNA, vitamin B12 and the recent discovery of ribozyme, illustrate the development of X-ray structural analysis. The determination of 3D structures of these molecules by X-ray diffraction had opened new areas of scientific research, such as molecular biophysics, molecular genetics, structural molecular biology, bioinorganic chemistry, organometallic chemistry, and many others. The discovery and development of X-ray crystallography revolutionised our understanding of natural sciences – physics, chemistry, biology, and also science of materials. The scientific community recognised these fundamental achievements (including the discovery of X-rays by awarding twenty-eight Nobel prizes to thirty-nine men and two women. The explosive growth of science and technology in the 20th and 21st centuries had been founded on the detailed knowledge of the three-dimensional structure of molecules, which was the basis for explaining and predicting the physical, chemical, biological and

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

    Directory of Open Access Journals (Sweden)

    Michael Heymann

    2014-09-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2012-09-11

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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Matthew P. Blakeley

    2015-07-01

    Full Text Available 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

  9. Colloquium: Femtosecond x-ray crystallography

    International Nuclear Information System (INIS)

    Rousse, Antoine; Rischel, Christian; Gauthier, Jean-Claude

    2001-01-01

    This article gives an overview of recent x-ray diffraction experiments with time resolutions down to 10 -13 s. The scientific motivation behind the development is outlined, using examples from solid state physics and biology. The ultrafast resolution may be provided either by fast detectors or short x-ray pulses, and the limitations of both techniques are discussed on the basis of state of the art experiments. In particular, it is shown that with present designs, high time resolution reduces the structural information attainable with high spatial resolution, thereby limiting feasible experiments on the ultrashort time-scale. The first experiment showing subpicosecond conformation changes was recently achieved with simple solids using an ultrafast laser-produced plasma x-ray source. The principles of this experiment are described in detail

  10. Conformational variability of the stationary phase survival protein E from Xylella fastidiosa revealed by X-ray crystallography, small-angle X-ray scattering studies, and normal mode analysis.

    Science.gov (United States)

    Machado, Agnes Thiane Pereira; Fonseca, Emanuella Maria Barreto; Reis, Marcelo Augusto Dos; Saraiva, Antonio Marcos; Santos, Clelton Aparecido Dos; de Toledo, Marcelo Augusto Szymanski; Polikarpov, Igor; de Souza, Anete Pereira; Aparicio, Ricardo; Iulek, Jorge

    2017-10-01

    Xylella fastidiosa is a xylem-limited bacterium that infects a wide variety of plants. Stationary phase survival protein E is classified as a nucleotidase, which is expressed when bacterial cells are in the stationary growth phase and subjected to environmental stresses. Here, we report four refined X-ray structures of this protein from X. fastidiosa in four different crystal forms in the presence and/or absence of the substrate 3'-AMP. In all chains, the conserved loop verified in family members assumes a closed conformation in either condition. Therefore, the enzymatic mechanism for the target protein might be different of its homologs. Two crystal forms exhibit two monomers whereas the other two show four monomers in the asymmetric unit. While the biological unit has been characterized as a tetramer, differences of their sizes and symmetry are remarkable. Four conformers identified by Small-Angle X-ray Scattering (SAXS) in a ligand-free solution are related to the low frequency normal modes of the crystallographic structures associated with rigid body-like protomer arrangements responsible for the longitudinal and symmetric adjustments between tetramers. When the substrate is present in solution, only two conformers are selected. The most prominent conformer for each case is associated to a normal mode able to elongate the protein by moving apart two dimers. To our knowledge, this work was the first investigation based on the normal modes that analyzed the quaternary structure variability for an enzyme of the SurE family followed by crystallography and SAXS validation. The combined results raise new directions to study allosteric features of XfSurE protein. © 2017 Wiley Periodicals, Inc.

  11. X-ray spectroscopy and X-ray crystallography of metalloenzymes at XFELs

    International Nuclear Information System (INIS)

    Yano, Junko

    2016-01-01

    The ultra-bright femtosecond X-ray pulses provided by X-ray Free Electron Lasers (XFELs) open capabilities for studying the structure and dynamics of a wide variety of biological and inorganic systems beyond what is possible at synchrotron sources. Although the structure and chemistry at the catalytic sites have been studied intensively in both biological and inorganic systems, a full understanding of the atomic-scale chemistry requires new approaches beyond the steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure at ambient conditions, while overcoming X-ray damage to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by using the intense and ultra-short femtosecond X-ray pulses from an XFEL, where sample is probed before it is damaged. We have developed methodology for simultaneously collecting crystallography data and X-ray emission spectra, using an energy dispersive spectrometer at ambient conditions. In addition, we have developed a way to collect metal L-edge data of dilute samples using soft X-rays at XFELs. The advantages and challenges of these methods will be described in this review. (author)

  12. The Beginnings of X-ray Crystallography

    Indian Academy of Sciences (India)

    IAS Admin

    significant change in his career came in 1904 when he gave a talk at Dunedin on ... In his personal reminiscences, W L Bragg talks about his school days in Australia. ... two Braggs on the occasion of the International Year of Crystallography .

  13. The founding and development of X-ray crystallography

    International Nuclear Information System (INIS)

    Mai Zhenhong

    2014-01-01

    2014 is the centennial of X-ray crystallography. Crystals have played an important role in our lives and in the development of society throughout these 100 years. In July 2012 the 66th General Assembly of the United Nations declared 2014 to be the official International Year of Crystallography (IYCr2014). The discovery of X-ray diffraction by crystals has had a profound impact on science and technology worldwide. It provides for us a distinct image of the arrangement of atoms or/and molecules in crystals. The development of X-ray spectroscopy has made it possible for us to understand the laws of atomic structure, and thus to identify the elements in all kinds of matter. In this article the greatest events in the history of X-ray crystallography, including the development of X-ray sources, detectors, experimental data analysis, and experimental methods are reviewed to commemorate the pioneers who made such important contributions to science and technology. (author)

  14. A readout system for X-ray powder crystallography

    CERN Document Server

    Loukas, D; Pavlidis, A; Karvelas, E; Psycharis, K; Misiakos, V; Mousa, J; Dre, C

    2000-01-01

    A system for capturing and processing data, from radiation detectors, in the field of X-ray crystallography has been developed. The system includes a custom-made mixed analog-digital 16-channel VLSI circuit in 50 mu m pitch. Each channel comprises a charge amplifier, a shaper, a comparator and a 21-bit counter. The circuit can be scaled in a daisy chain configuration. Data acquisition is performed with a custom made PCI card while the control software is developed with Visual C++ under the MS Windows NT environment. Performance of a fully operational system, in terms of electronic noise, statistical variations and data capture speed is presented. The noise level permits counting of X-rays down to 8 keV while the counting capability is in excess of 200 kHz. The system is intended for X-ray crystallography with silicon detectors.

  15. Polycapillary x-ray optics for macromolecular crystallography

    International Nuclear Information System (INIS)

    Owens, S.M.; Gibson, W.M.; Carter, D.C.; Sisk, R.C.; Ho, J.X.

    1996-01-01

    Polycapillary x-ray optics have found potential application in many different fields, including antiscatter and magnification in mammography, radiography, x-ray fluorescence, x-ray lithography, and x-ray diffraction techniques. In x-ray diffraction, an optic is used to collect divergent x-rays from a point source and redirect them into a quasi-parallel, or slightly focused beam. Monolithic polycapillary optics have been developed recently for macromolecular crystallography and have already shown considerable gains in diffracted beam intensity over pinhole collimation. Development is being pursued through a series of simulations and prototype optics. Many improvements have been made over the stage 1 prototype reported previously, which include better control over the manufacturing process, reducing the diameter of the output beam, and addition of a slight focusing at the output of the optic to further increase x-ray flux at the sample. The authors report the characteristics and performance of the stage 1 and stage 2 optics

  16. Novel organophosphorus compounds; synthesis, spectroscopy and X-ray crystallography

    Czech Academy of Sciences Publication Activity Database

    Shariatinia, Z.; Sohrabi, M.; Yousefi, M.; Kovaľ, Tomáš; Dušek, Michal

    2012-01-01

    Roč. 11, č. 2 (2012), s. 125-133 ISSN 1024-1221 Grant - others:AV ČR(CZ) AP0701 Program:Akademická prémie - Praemium Academiae Institutional research plan: CEZ:AV0Z10100521 Keywords : organophosphorus compounds * NMR * X-ray crystallography * hydrogen bond Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 0.686, year: 2012

  17. Metal ions-binding T4 lysozyme as an intramolecular protein purification tag compatible with X-ray crystallography

    Czech Academy of Sciences Publication Activity Database

    Bouřa, Evžen; Bäumlová, Adriana; Chalupská, Dominika; Dubánková, Anna; Klíma, Martin

    2017-01-01

    Roč. 26, č. 6 (2017), s. 1116-1123 ISSN 0961-8368 R&D Projects: GA ČR(CZ) GJ17-07058Y; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : phage T4 * lysozyme * endolysin * histidine tag * protein purification * crystal structure Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 2.523, year: 2016

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

    Science.gov (United States)

    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.

  19. The development of structural x-ray crystallography

    Science.gov (United States)

    Woolfson, M. M.

    2018-03-01

    From its birth in 1912, when only the simplest structures could be solved, x-ray structural crystallography is now able to solve macromolecular structures containing many thousands of independent non-hydrogen atoms. This progress has depended on, and been driven by, great technical advances in the development of powerful synchrotron x-ray sources, advanced automated equipment for the collection and storage of large data sets and powerful computers to deal with everything from data processing to running programmes employing complex algorithms for the automatic solution of structures. The sheer number of developments in the subject over the past century makes it impossible for this review to be exhaustive, but it will describe some major developments that will enable the reader to understand how the subject has grown from its humble beginnings to what it is today.

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

    Directory of Open Access Journals (Sweden)

    Roman Sommer

    2016-12-01

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

    Science.gov (United States)

    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.

  2. X-ray powder crystallography with vertex instrumentation

    International Nuclear Information System (INIS)

    Chatzisotiriou, V.; Christofis, I.; Dimitriou, N.; Karvelas, S.; Karydas, A.G.; Loukas, D.; Pavlidis, A.; Spirou, S.; Dre, C.; Haralabidis, N.; Misiakos, K.; Tsoi, E.; Perdikatsis, V.; Psycharis, V.; Terzis, A.; Turchetta, R.

    1998-01-01

    An X-ray Diffractometer for Powder Crystallography is described along with experimental results and future plans. This is an intermediate instrument toward a long linear array system. Three channels of a silicon microstrip detector, are the detecting elements in the present instrument. Each detector channel is followed by a VLSI readout chain, which consists of a charge preamplifier with pulse shaping circuitry, a discriminator, and a 16-bit counter. Control and data acquisition is performed with a custom made PC readout card. A motorized goniometer scans the angle range of interest. Calibration of the system is done with reference samples and data which are captured with a one-channel conventional NaI detector. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

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

    CERN Document Server

    Eikenberry, E F; Huelsen, G; Toyokawa, H; Horisberger, R P; Schmitt, B; Schulze-Briese, C; Tomizaki, T

    2003-01-01

    A large quantum-limited area X-ray detector for protein crystallography is under development at the Swiss Light Source. The final detector will be 2kx2k pixels covering 40x40 cm sup 2. A three-module prototype with 1120x157 pixels covering an active area of 24.3x3.4 cm sup 2 has been tested. X-rays above 6 keV with peak count rates exceeding 5x10 sup 5 X-ray/pixel/s could be detected in single photon counting mode. Statistics of module production and results of threshold trimming are presented. To demonstrate the potential of this new detector, protein crystal data were collected at beamline 6S of the SLS.

  4. Contribution of X-ray crystallography in energy related problems

    International Nuclear Information System (INIS)

    Majid, C.A.; Hussain, M.A.

    1995-01-01

    Crystallography is concerned with the study of the structure of matter at the atomic level in condensed state. The great practical importance of scientific knowledge of the structure of solid is self evident when consideration is given to the definition of desired physical and chemical properties. The strength of steel girders, the corrosion of alloys, the plasticity of lime, the wearing properties of case hardness steel, the dielectric capacity of materials, the lubricating properties of long chain paraffin's or of graphite, the stretching of rubber and innumerable other practical phenomena of every day life depend upon ultimate structure of these materials. To understand function to control, manipulate and best utilize their properties, and to produce materials with properties meeting a desired set of specification it is essential to understand thoroughly both the characteristics and origin of each property. Origins of materials properties lie in a combination of natural laws with the detailed structure and composition of materials, i.e. the choice, location, bonding, etc. of every atom in the material object. Therefore, to understand their various properties, it is important to explore the structure property relationship in materials. X-ray crystallography is not only helping to develop new materials having desired properties, but also in improving existing materials. Radiation effects, electrolytes, superconductors and catalysts etc. are just a few examples of many areas where crystallography is helping. With the invent of new radiation sources like synchrotron and new detectors materials and techniques, this almost 80 years old discipline continues to capture the interest of solid state physicists and chemists alike. (author)

  5. Correlation between protein sequence similarity and x-ray diffraction quality in the protein data bank.

    Science.gov (United States)

    Lu, Hui-Meng; Yin, Da-Chuan; Ye, Ya-Jing; Luo, Hui-Min; Geng, Li-Qiang; Li, Hai-Sheng; Guo, Wei-Hong; Shang, Peng

    2009-01-01

    As the most widely utilized technique to determine the 3-dimensional structure of protein molecules, X-ray crystallography can provide structure of the highest resolution among the developed techniques. The resolution obtained via X-ray crystallography is known to be influenced by many factors, such as the crystal quality, diffraction techniques, and X-ray sources, etc. In this paper, the authors found that the protein sequence could also be one of the factors. We extracted information of the resolution and the sequence of proteins from the Protein Data Bank (PDB), classified the proteins into different clusters according to the sequence similarity, and statistically analyzed the relationship between the sequence similarity and the best resolution obtained. The results showed that there was a pronounced correlation between the sequence similarity and the obtained resolution. These results indicate that protein structure itself is one variable that may affect resolution when X-ray crystallography is used.

  6. Neutron protein crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Niimura, Nobuo [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    1998-10-01

    X-ray diffraction of single crystal has enriched the knowledge of various biological molecules such as proteins, DNA, t-RNA, viruses, etc. It is difficult to make structural analysis of hydrogen atoms in a protein using X-ray crystallography, whereas neutron diffraction seems usable to directly determine the location of those hydrogen atoms. Here, neutron diffraction method was applied to structural analysis of hen egg-white lysozyme. Since the crystal size of a protein to analyze is generally small (5 mm{sup 3} at most), the neutron beam at the sample position in monochromator system was set to less than 5 x 5 mm{sup 2} and beam divergence to 0.4 degree or less. Neutron imaging plate with {sup 6}Li or Gd mixed with photostimulated luminescence material was used and about 2500 Bragg reflections were recorded in one crystal setting. A total of 38278 reflections for 2.0 A resolution were collected in less than 10 days. Thus, stereo views of Trp-111 omit map around the indol ring of Trp-111 was presented and the three-dimensional arrangement of 696H and 264D atoms in the lysozyme molecules was determined using the omit map. (M.N.)

  7. X-ray Crystallography of Biological Macromolecules -RE ...

    Indian Academy of Sciences (India)

    trons of the atoms scatter X-rays and if identical molecules are arranged in a ... Institute of Science,. Bangalore. ... The first X-ray diffraction pictures were taken and the theory .... various processes involved in biological systems in detail. We.

  8. Extending the methodology of X-ray crystallography to allow X-ray microscopy without X-ray optics

    International Nuclear Information System (INIS)

    Miao Jianwei; Kirz, Janos; Sayre, David; Charalambous, Pambos

    2000-01-01

    We demonstrate that the soft X-ray diffraction pattern from a micron-size noncrystalline specimen can be recorded and inverted to form a high-resolution image. The phase problem is overcome by oversampling the diffraction pattern. The image is obtained using an iterative algorithm. The technique provides a method for X-ray microscopy requiring no high-resolution X-ray optical elements or detectors. In the present work, a resolution of approximately 60 nm was obtained, but we believe that considerably higher resolution can be achieved

  9. Recent advances in racemic protein crystallography.

    Science.gov (United States)

    Yan, Bingjia; Ye, Linzhi; Xu, Weiliang; Liu, Lei

    2017-09-15

    Solution of the three-dimensional structures of proteins is a critical step in deciphering the molecular mechanisms of their bioactivities. Among the many approaches for obtaining protein crystals, racemic protein crystallography has been developed as a unique method to solve the structures of an increasing number of proteins. Exploiting unnatural protein enantiomers in crystallization and resolution, racemic protein crystallography manifests two major advantages that are 1) to increase the success rate of protein crystallization, and 2) to obviate the phase problem in X-ray diffraction. The requirement of unnatural protein enantiomers in racemic protein crystallography necessitates chemical protein synthesis, which is hitherto accomplished through solid phase peptide synthesis and chemical ligation reactions. This review highlights the fundamental ideas of racemic protein crystallography and surveys the harvests in the field of racemic protein crystallography over the last five years from early 2012 to late 2016. Copyright © 2017. Published by Elsevier Ltd.

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

    Science.gov (United States)

    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.…

  11. Native chemical ligation at Asx-Cys, Glx-Cys: chemical synthesis and high-resolution X-ray structure of ShK toxin by racemic protein crystallography.

    Science.gov (United States)

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

    2013-08-14

    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- site 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.

  12. History of protein crystallography in China.

    Science.gov (United States)

    Rao, Zihe

    2007-06-29

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

  13. History of protein crystallography in China

    OpenAIRE

    Rao, Zihe

    2007-01-01

    China has a strong background in X-ray crystallography dating back to the 1920s. Protein crystallography research in China was first developed following the successful synthesis of insulin in China in 1966. The subsequent determination of the three-dimensional structure of porcine insulin made China one of the few countries which could determine macromolecular structures by X-ray diffraction methods in the late 1960s and early 1970s. After a slow period during the 1970s and 1980s, protein cry...

  14. Molecular form factors in X-ray crystallography

    NARCIS (Netherlands)

    Groenewegen, P.P.M.; Feil, D.

    1969-01-01

    The calculation of molecular form factors from ab initio molecular electronic wavefunctions is discussed, and a scheme for application to X-ray diffraction structure analysis is given. The method is used to calculate the form factor of the NH+4 molecular ion from three accurate molecular

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

    International Nuclear Information System (INIS)

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

    2005-01-01

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

  16. Chemical crystallography with pulsed neutrons and synchrotron x-rays

    International Nuclear Information System (INIS)

    Carrondo, M.A.; Jeffrey, G.A.

    1988-01-01

    Solid-state chemists and physicists, crystallographers and molecular biologists who are using or who plan to use the special properties of pulsed neutron spallation and synchrotron X-ray sources will find this book invaluable. Those scientists who have not yet gained experience in working with such sources will find the basic physics of the radiations, their production and their scattering properties explained, together with descriptions of the different types of diffraction experiments which use them

  17. Phosphoramidates: synthesis, spectroscopy, and X-ray crystallography

    Czech Academy of Sciences Publication Activity Database

    Shariatinia, Z.; Sohrabi, M.; Yousefi, M.; Kovaľ, Tomáš; Dušek, Michal

    2012-01-01

    Roč. 23, č. 5 (2012), s. 478-485 ISSN 1042-7163 Grant - others:AV ČR(CZ) AP0701 Program:Akademická prémie - Praemium Academiae Institutional research plan: CEZ:AV0Z10100521 Keywords : phosphoramidates * X-ray diffraction * spectroscopy Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 1.577, year: 2012

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

    Science.gov (United States)

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

    2017-09-01

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

  19. Structural investigation of bistrifluron using x-ray crystallography, NMR spectroscopy, and molecular modeling

    CERN Document Server

    Moon, J K; Rhee, S K; Kim, G B; Yun, H S; Chung, B J; Lee, S S; Lim, Y H

    2002-01-01

    A new insecticide, bistrifluron acts as an inhibitor of insect development and interferes with the cuticle formation of insects. Since it shows low acute oral and dermal toxicities, it can be one of potent insecticides. Based on X-ray crystallography, NMR spectroscopy and molecular modeling, the structural studies of bistrifluron have been carried out.

  20. Synthesis, spectroscopy, X-ray crystallography, and DFT computations of nanosized phosphazenes

    Czech Academy of Sciences Publication Activity Database

    Shariatinia, Z.; Moghadam, E.J.; Maghsoudi, N.; Mousavi, H.S.M.; Dušek, Michal; Eigner, Václav

    2015-01-01

    Roč. 641, č. 5 (2015), s. 967-978 ISSN 0044-2313 Grant - others:AV ČR(CZ) Praemium Academiae Institutional support: RVO:68378271 Keywords : phosphazene * ultrasonic * nanoparticle * x-ray crystallography * DFT calculation Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 1.261, year: 2015

  1. Synthesis of new nano Schiff base complexes: X-ray crystallography ...

    African Journals Online (AJOL)

    This study presents synthesis and characterization of new nano uranyl Schiff base complexes. Electrochemistry of these complexes showed a quasireversible redox reaction without any successive reactions. Furthermore, X-ray crystallography exhibited that beside the coordination of tetradentate Schiff base, one solvent ...

  2. 100 Years later: Celebrating the contributions of x-ray crystallography to allergy and clinical immunology.

    Science.gov (United States)

    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. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. All rights reserved.

  3. A convolutional neural network-based screening tool for X-ray serial crystallography.

    Science.gov (United States)

    Ke, Tsung Wei; Brewster, Aaron S; Yu, Stella X; Ushizima, Daniela; Yang, Chao; Sauter, Nicholas K

    2018-05-01

    A new tool is introduced for screening macromolecular X-ray crystallography diffraction images produced at an X-ray free-electron laser light source. Based on a data-driven deep learning approach, the proposed tool executes a convolutional neural network to detect Bragg spots. Automatic image processing algorithms described can enable the classification of large data sets, acquired under realistic conditions consisting of noisy data with experimental artifacts. Outcomes are compared for different data regimes, including samples from multiple instruments and differing amounts of training data for neural network optimization. open access.

  4. Semi-empirical atom-atom interaction models and X-ray crystallography

    International Nuclear Information System (INIS)

    Braam, A.W.M.

    1981-01-01

    Several aspects of semi-empirical energy calculations in crystallography are considered. Solid modifications of ethane have been studied using energy calculations and a fast summation technique has been evaluated. The structure of tetramethylpyrazine has been determined at room temperature and at 100K and accurate structure factors have been derived from measured Bragg intensities. Finally electrostatic properties have been deduced from X-ray structure factors. (C.F.)

  5. Synthesis, X-ray crystallography, spectroscopy, electrochemistry, thermal and kinetic study of uranyl Schiff base complexes

    Czech Academy of Sciences Publication Activity Database

    Asadi, Z.; Golzard, F.; Eigner, Václav; Dušek, Michal

    2013-01-01

    Roč. 66, č. 20 (2013), s. 3629-3646 ISSN 0095-8972 R&D Projects: GA ČR(CZ) GAP204/11/0809 Institutional support: RVO:68378271 Keywords : X-ray crystallography * uranyl Schiff base complex * kinetics of thermal decomposition * cyclic voltammetry * kinetics and mechanism Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 2.224, year: 2013

  6. FreeDam - A webtool for free-electron laser-induced damage in femtosecond X-ray crystallography

    Science.gov (United States)

    Jönsson, H. Olof; Östlin, Christofer; Scott, Howard A.; Chapman, Henry N.; Aplin, Steve J.; Tîmneanu, Nicuşor; Caleman, Carl

    2018-03-01

    Over the last decade X-ray free-electron laser (XFEL) sources have been made available to the scientific community. One of the most successful uses of these new machines has been protein crystallography. When samples are exposed to the intense short X-ray pulses provided by the XFELs, the sample quickly becomes highly ionized and the atomic structure is affected. Here we present a webtool dubbed FreeDam based on non-thermal plasma simulations, for estimation of radiation damage in free-electron laser experiments in terms of ionization, temperatures and atomic displacements. The aim is to make this tool easily accessible to scientists who are planning and performing experiments at XFELs.

  7. Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging

    OpenAIRE

    Warren, Anna J.; Armour, Wes; Axford, Danny; Basham, Mark; Connolley, Thomas; Hall, David R.; Horrell, Sam; McAuley, Katherine E.; Mykhaylyk, Vitaliy; Wagner, Armin; Evans, Gwyndaf

    2013-01-01

    The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolec...

  8. Sources, instrumentation and detectors for protein crystallography

    CERN Document Server

    Nave, C

    2001-01-01

    Some of the requirements for protein crystallography experiments on a synchrotron are described. Although data from different types of crystal are often collected without changing the X-ray beam properties, there are benefits if the incident beam is matched to a particular crystal and its diffraction pattern. These benefits are described with some examples. Radiation damage and other effects impose limits on the dose and dose rate on a protein crystal if the maximum amount of data is to be obtained. These limitations have possible consequences for the X-ray source required. Presently available commercial detector systems provide excellent data for protein crystallography but do not quite reach the specifications of the 'ideal' detector. In order to collect the most accurate data (e.g. for very weak anomalous scattering applications) detectors that produce near photon counting statistics over a wide dynamic range are required. It is possible that developments in 'pixel' detectors will allow these demanding exp...

  9. Coded diffraction system in X-ray crystallography using a boolean phase coded aperture approximation

    Science.gov (United States)

    Pinilla, Samuel; Poveda, Juan; Arguello, Henry

    2018-03-01

    Phase retrieval is a problem present in many applications such as optics, astronomical imaging, computational biology and X-ray crystallography. Recent work has shown that the phase can be better recovered when the acquisition architecture includes a coded aperture, which modulates the signal before diffraction, such that the underlying signal is recovered from coded diffraction patterns. Moreover, this type of modulation effect, before the diffraction operation, can be obtained using a phase coded aperture, just after the sample under study. However, a practical implementation of a phase coded aperture in an X-ray application is not feasible, because it is computationally modeled as a matrix with complex entries which requires changing the phase of the diffracted beams. In fact, changing the phase implies finding a material that allows to deviate the direction of an X-ray beam, which can considerably increase the implementation costs. Hence, this paper describes a low cost coded X-ray diffraction system based on block-unblock coded apertures that enables phase reconstruction. The proposed system approximates the phase coded aperture with a block-unblock coded aperture by using the detour-phase method. Moreover, the SAXS/WAXS X-ray crystallography software was used to simulate the diffraction patterns of a real crystal structure called Rhombic Dodecahedron. Additionally, several simulations were carried out to analyze the performance of block-unblock approximations in recovering the phase, using the simulated diffraction patterns. Furthermore, the quality of the reconstructions was measured in terms of the Peak Signal to Noise Ratio (PSNR). Results show that the performance of the block-unblock phase coded apertures approximation decreases at most 12.5% compared with the phase coded apertures. Moreover, the quality of the reconstructions using the boolean approximations is up to 2.5 dB of PSNR less with respect to the phase coded aperture reconstructions.

  10. Racemic protein crystallography.

    Science.gov (United States)

    Yeates, Todd O; Kent, Stephen B H

    2012-01-01

    Although natural proteins are chiral and are all of one "handedness," their mirror image forms can be prepared by chemical synthesis. This opens up new opportunities for protein crystallography. A racemic mixture of the enantiomeric forms of a protein molecule can crystallize in ways that natural proteins cannot. Recent experimental data support a theoretical prediction that this should make racemic protein mixtures highly amenable to crystallization. Crystals obtained from racemic mixtures also offer advantages in structure determination strategies. The relevance of these potential advantages is heightened by advances in synthetic methods, which are extending the size limit for proteins that can be prepared by chemical synthesis. Recent ideas and results in the area of racemic protein crystallography are reviewed.

  11. One-dimensional curved wire chamber for powder x-ray crystallography

    International Nuclear Information System (INIS)

    Ortendahl, D.; Perez-Mendez, V.; Stoker, J.; Beyermann, W.

    1978-01-01

    A xenon filled single anode wire chamber with delay line readout has been constructed for use in powder x-ray crystallography using 8 to 20 keV x-rays. The entire chamber including the anode wire and the delay line which forms part of the cathode plane is a section of a circular arc whose center is the powder specimen. The anode wire--38 μm gold-plated tungsten--is suspended in a circular arc by the interaction of a current flowing through it and magnetic field provided by two permanent magnets, above and below the wire, extending along the active length of the chamber. When filled with xenon to 3 atmospheres the chamber has uniform sensitivity in excess of 80% at 8 keV and a spatial resolution better than 0.3 mm

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

    Science.gov (United States)

    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.

  13. From electron microscopy to X-ray crystallography: molecular-replacement case studies

    International Nuclear Information System (INIS)

    Xiong, Yong

    2008-01-01

    Test studies have been conducted on five crystal structures of large molecular assemblies, in which EM maps are used as models for structure solution by molecular replacement using various standard MR packages such as AMoRe, MOLREP and Phaser. Multi-component molecular complexes are increasingly being tackled by structural biology, bringing X-ray crystallography into the purview of electron-microscopy (EM) studies. X-ray crystallography can utilize a low-resolution EM map for structure determination followed by phase extension to high resolution. Test studies have been conducted on five crystal structures of large molecular assemblies, in which EM maps are used as models for structure solution by molecular replacement (MR) using various standard MR packages such as AMoRe, MOLREP and Phaser. The results demonstrate that EM maps are viable models for molecular replacement. Possible difficulties in data analysis, such as the effects of the EM magnification error, and the effect of MR positional/rotational errors on phase extension are discussed

  14. Mapping the topographic epitope landscape on the urokinase plasminogen activator receptor (uPAR) by surface plasmon resonance and X-ray crystallography

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Gandhi, Sonu; Yuan, Cai

    2015-01-01

    The urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycolipid-anchored membrane protein often expressed in the microenvironment of invasive solid cancers and high levels are generally associated with poor patient prognosis (Kriegbaum et al., 2011 [1]). uPAR is organized as a dy...... of these mAbs by X-ray crystallography alone and in complex with uPAR [deposited in the PDB database as 4QTH and 4QTI, respectively]....

  15. Native State Mass Spectrometry, Surface Plasmon Resonance, and X-ray Crystallography Correlate Strongly as a Fragment Screening Combination.

    Science.gov (United States)

    Woods, Lucy A; Dolezal, Olan; Ren, Bin; Ryan, John H; Peat, Thomas S; Poulsen, Sally-Ann

    2016-03-10

    Fragment-based drug discovery (FBDD) is contingent on the development of analytical methods to identify weak protein-fragment noncovalent interactions. Herein we have combined an underutilized fragment screening method, native state mass spectrometry, together with two proven and popular fragment screening methods, surface plasmon resonance and X-ray crystallography, in a fragment screening campaign against human carbonic anhydrase II (CA II). In an initial fragment screen against a 720-member fragment library (the "CSIRO Fragment Library") seven CA II binding fragments, including a selection of nonclassical CA II binding chemotypes, were identified. A further 70 compounds that comprised the initial hit chemotypes were subsequently sourced from the full CSIRO compound collection and screened. The fragment results were extremely well correlated across the three methods. Our findings demonstrate that there is a tremendous opportunity to apply native state mass spectrometry as a complementary fragment screening method to accelerate drug discovery.

  16. Synthesis, x-ray crystallography and leishmanicidal activity of benzimidazolinyl piperidine derivative

    International Nuclear Information System (INIS)

    Saify, Z.S.; Begum, N.; Yousuf, S.; Ashraf, S.

    2014-01-01

    Protozoan parasites of the Leishmania genus are the main cause of vector-borne disease leishmaniasis throughout the world. It is caused by at least 17 different species of protozoan Leishmania and transmitted by the bite of infected sand flies. Leishmaniasis could be fatal. Present drugs have limitations to cure it due to the development of drug resistance. Hence, to design an effective leishmanicidal agent would be of great interest. Benzimidazolinyl piperidine has served as potential target due to a vast range of biological activities. In the present study a new 4-(2-keto-1-benzimidazolinyl)piperidine derivative, 1-(2-(4-fluorophenyl)-2-oxoethyl)-4-(2-oxo-2,3-dihydro-1H-benzo(d)imidazol) piperidinium bromide has been synthesized and characterized by X-ray crystallography, 1D and 2D NMR spectroscopy. Evaluation by in vitro leishmanicidal assay showed good activity. (author)

  17. Ultra-high resolution protein crystallography

    International Nuclear Information System (INIS)

    Takeda, Kazuki; Hirano, Yu; Miki, Kunio

    2010-01-01

    Many protein structures have been determined by X-ray crystallography and deposited with the Protein Data Bank. However, these structures at usual resolution (1.5< d<3.0 A) are insufficient in their precision and quantity for elucidating the molecular mechanism of protein functions directly from structural information. Several studies at ultra-high resolution (d<0.8 A) have been performed with synchrotron radiation in the last decade. The highest resolution of the protein crystals was achieved at 0.54 A resolution for a small protein, crambin. In such high resolution crystals, almost all of hydrogen atoms of proteins and some hydrogen atoms of bound water molecules are experimentally observed. In addition, outer-shell electrons of proteins can be analyzed by the multipole refinement procedure. However, the influence of X-rays should be precisely estimated in order to derive meaningful information from the crystallographic results. In this review, we summarize refinement procedures, current status and perspectives for ultra high resolution protein crystallography. (author)

  18. Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging

    International Nuclear Information System (INIS)

    Warren, Anna J.; Armour, Wes; Axford, Danny; Basham, Mark; Connolley, Thomas; Hall, David R.; Horrell, Sam; McAuley, Katherine E.; Mykhaylyk, Vitaliy; Wagner, Armin; Evans, Gwyndaf

    2013-01-01

    A comparison of X-ray diffraction and radiographic techniques for the location and characterization of protein crystals is demonstrated on membrane protein crystals mounted within lipid cubic phase material. The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required

  19. Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging

    Energy Technology Data Exchange (ETDEWEB)

    Warren, Anna J. [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Armour, Wes [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Oxford e-Research Centre, 7 Keble Road, Oxford OX1 3QG (United Kingdom); Axford, Danny; Basham, Mark; Connolley, Thomas; Hall, David R. [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Horrell, Sam [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); University of Liverpool, Liverpool L69 3BX (United Kingdom); McAuley, Katherine E.; Mykhaylyk, Vitaliy; Wagner, Armin; Evans, Gwyndaf, E-mail: gwyndaf.evans@diamond.ac.uk [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom)

    2013-07-01

    A comparison of X-ray diffraction and radiographic techniques for the location and characterization of protein crystals is demonstrated on membrane protein crystals mounted within lipid cubic phase material. The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required.

  20. NATO Advanced Study Institute on Chemical Crystallography with Pulsed Neutrons and Synchrotron X-Rays

    CERN Document Server

    Jeffrey, George

    1988-01-01

    X-ray and neutron crystallography have played an increasingly impor­ tant role in the chemical and biochemical sciences over the past fifty years. The principal obstacles in this methodology, the phase problem and com­ puting, have been overcome. The former by the methods developed in the 1960's and just recognised by the 1985 Chemistry Nobel Prize award to Karle and Hauptman, the latter by the dramatic advances that have taken place in computer technology in the past twenty years. Within the last decade, two new radiation sources have been added to the crystallographer's tools. One is synchrotron X-rays and the other is spallation neutrons. Both have much more powerful fluxes than the pre­ vious sources and they are pulsed rather than continuos. New techniques are necessary to fully exploit the intense continuos radiation spectrum and its pulsed property. Both radiations are only available from particular National Laboratories on a guest-user basis for scientists outside these Na­ tional Laboratories. Hi...

  1. Serial Millisecond Crystallography of Membrane Proteins.

    Science.gov (United States)

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

    2016-01-01

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

  2. Structural elucidation of dendritic host-guest complexes by X-ray crystallography and molecular dynamics simulations

    NARCIS (Netherlands)

    Chang, T.; Pieterse, K.; Broeren, M.A.C.; Kooijman, H.; Spek, A.L.; Hilbers, P.A.J.; Meijer, E.W.

    2007-01-01

    The multiple monovalent binding of adamantyl-urea poly(propyleneimine) dendrimers with carboxylic acid-urea guests was investigated using molecular dynamics simulations and X-ray crystallography to better understand the structure and behavior of the dynamic multivalent complex in solution. The

  3. NATURAL CYCLOPENTANOID CYANOHYDRIN GLYCOSIDES .13. STRUCTURE DETERMINATION OF NATURAL EPOXYCYCLOPENTANES BY X-RAY CRYSTALLOGRAPHY AND NMR-SPECTROSCOPY

    DEFF Research Database (Denmark)

    Olafsdottir, E. S.; Sorensen, A. M.; Cornett, Claus

    1991-01-01

    nonannellated cyclopentane derivatives. The new glucosides were shown, by NMR spectroscopy (including NOE measurements), X-ray crystallography, and enzymatic hydrolysis to the corresponding cyanohydrins, to be (1R,2R,3R,4R)- and (1S,2S,3S,4S)-1-(beta-D-glucopyranosyloxy)-2,3-epoxy-4-hydroxycyclopenta ne-1...

  4. Hydrogen atoms in protein structures: high-resolution X-ray diffraction structure of the DFPase

    Science.gov (United States)

    2013-01-01

    Background Hydrogen atoms represent about half of the total number of atoms in proteins and are often involved in substrate recognition and catalysis. Unfortunately, X-ray protein crystallography at usual resolution fails to access directly their positioning, mainly because light atoms display weak contributions to diffraction. However, sub-Ångstrom diffraction data, careful modeling and a proper refinement strategy can allow the positioning of a significant part of hydrogen atoms. Results A comprehensive study on the X-ray structure of the diisopropyl-fluorophosphatase (DFPase) was performed, and the hydrogen atoms were modeled, including those of solvent molecules. This model was compared to the available neutron structure of DFPase, and differences in the protein and the active site solvation were noticed. Conclusions A further examination of the DFPase X-ray structure provides substantial evidence about the presence of an activated water molecule that may constitute an interesting piece of information as regard to the enzymatic hydrolysis mechanism. PMID:23915572

  5. Measurement and Interpretation of Diffuse Scattering in X-Ray Diffraction for Macromolecular Crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Wall, Michael E. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2017-10-16

    X-ray diffraction from macromolecular crystals includes both sharply peaked Bragg reflections and diffuse intensity between the peaks. The information in Bragg scattering reflects the mean electron density in the unit cells of the crystal. The diffuse scattering arises from correlations in the variations of electron density that may occur from one unit cell to another, and therefore contains information about collective motions in proteins.

  6. Kissing G domains of MnmE monitored by X-ray crystallography and pulse electron paramagnetic resonance spectroscopy.

    Directory of Open Access Journals (Sweden)

    Simon Meyer

    2009-10-01

    Full Text Available MnmE, which is involved in the modification of the wobble position of certain tRNAs, belongs to the expanding class of G proteins activated by nucleotide-dependent dimerization (GADs. Previous models suggested the protein to be a multidomain protein whose G domains contact each other in a nucleotide dependent manner. Here we employ a combined approach of X-ray crystallography and pulse electron paramagnetic resonance (EPR spectroscopy to show that large domain movements are coupled to the G protein cycle of MnmE. The X-ray structures show MnmE to be a constitutive homodimer where the highly mobile G domains face each other in various orientations but are not in close contact as suggested by the GDP-AlF(x structure of the isolated domains. Distance measurements by pulse double electron-electron resonance (DEER spectroscopy show that the G domains adopt an open conformation in the nucleotide free/GDP-bound and an open/closed two-state equilibrium in the GTP-bound state, with maximal distance variations of 18 A. With GDP and AlF(x, which mimic the transition state of the phosphoryl transfer reaction, only the closed conformation is observed. Dimerization of the active sites with GDP-AlF(x requires the presence of specific monovalent cations, thus reflecting the requirements for the GTPase reaction of MnmE. Our results directly demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle. They show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein, and they are of crucial importance for understanding the mechanistic principles of this GAD.

  7. Quantum Crystallography: Density Matrix-Density Functional Theory and the X-Ray Diffraction Experiment

    Science.gov (United States)

    Soirat, Arnaud J. A.

    Density Matrix Theory is a Quantum Mechanical formalism in which the wavefunction is eliminated and its role taken over by reduced density matrices. The interest of this is that, it allows one, in principle, to calculate any electronic property of a physical system, without having to solve the Schrodinger equation, using only two entities much simpler than an N-body wavefunction: first and second -order reduced density matrices. In practice, though, this very promising possibility faces the tremendous theoretical problem of N-representability, which has been solved for the former, but, until now, voids any hope of theoretically determining the latter. However, it has been shown that single determinant reduced density matrices of any order may be recovered from coherent X-ray diffraction data, if one provides a proper Quantum Mechanical description of the Crystallography experiment. A deeper investigation of this method is the purpose of this work, where we, first, further study the calculation of X-ray reduced density matrices N-representable by a single Slater determinant. In this context, we independently derive necessary and sufficient conditions for the uniqueness of the method. We then show how to account for electron correlation in this model. For the first time, indeed, we derive highly accurate, yet practical, density matrices approximately N-representable by correlated-determinant wavefunctions. The interest of such a result lies in the Quantum Mechanical validity of these density matrices, their property of being entirely obtainable from X-ray coherent diffraction data, their very high accuracy conferred by this known property of the N-representing wavefunction, as well as their definition as explicit functionals of the density. All of these properties are finally used in both a theoretical and a numerical application: in the former, we show that these density matrices may be used in the context of Density Functional Theory to highly accurately determine

  8. Bioactive Formylated Flavonoids from Eugenia rigida: Isolation, Synthesis, and X-ray Crystallography.

    Science.gov (United States)

    Zaki, Mohamed A; Nanayakkara, N P Dhammika; Hetta, Mona H; Jacob, Melissa R; Khan, Shabana I; Mohammed, Rabab; Ibrahim, Mohamed A; Samoylenko, Volodymyr; Coleman, Christina; Fronczek, Frank R; Ferreira, Daneel; Muhammad, Ilias

    2016-09-23

    Two new flavonoids, rac-6-formyl-5,7-dihydroxyflavanone (1) and 2',6'-dihydroxy-4'-methoxy-3'-methylchalcone (2), together with five known derivatives, rac-8-formyl-5,7-dihydroxyflavanone (3), 4',6'-dihydroxy-2'-methoxy-3'-methyldihydrochalcone (4), rac-7-hydroxy-5-methoxy-6-methylflavanone (5), 3'-formyl-2',4',6'-trihydroxy-5'-methyldihydrochalcone (6), and 3'-formyl-2',4',6'-trihydroxydihydrochalcone (7), were isolated from the leaves of Eugenia rigida. The individual (S)- and (R)-enantiomers of 1 and 3, together with the corresponding formylated flavones 8 (6-formyl-5,7-dihydroxyflavone) and 9 (8-formyl-5,7-dihydroxyflavone), as well as 2',4',6'-trihydroxychalcone (10), 3'-formyl-2',4',6'-trihydroxychalcone (11), and the corresponding 3'-formyl-2',4',6'-trihydroxydihydrochalcone (7) and 2',4',6'-trihydroxydihydrochalcone (12), were synthesized. The structures of the isolated and synthetic compounds were established via NMR, HRESIMS, and electronic circular dichroism data. In addition, the structures of 3, 5, and 8 were confirmed by single-crystal X-ray diffraction crystallography. The isolated and synthetic flavonoids were evaluated for their antimicrobial and cytotoxic activities against a panel of microorganisms and solid tumor cell lines.

  9. Synthesis, X-ray crystallography, and computational analysis of 1-azafenestranes.

    Science.gov (United States)

    Denmark, Scott E; Montgomery, Justin I; Kramps, Laurenz A

    2006-09-06

    The tandem [4+2]/[3+2] cycloaddition of nitroalkenes has been employed in the synthesis of 1-azafenestranes, molecules of theoretical interest because of planarizing distortion of their central carbon atoms. The synthesis of c,c,c,c-[5.5.5.5]-1-azafenestrane was completed in good yield from a substituted nitrocyclopentene, and its borane adduct was analyzed through X-ray crystallography, which showed a moderate distortion from ideal tetrahedral geometry. The syntheses of two members of the [4.5.5.5] family of 1-azafenestranes are also reported, including one with a trans fusion at a bicyclic ring junction which brings about considerable planarization of one of the central angles (16.8 degrees deviation from tetrahedral geometry). While investigating the [4.5.5.5]-1-azafenestranes, a novel dyotropic rearrangement that converts nitroso acetals into tetracyclic aminals was discovered. Through conformational analysis, a means to prevent this molecular reorganization was formulated and realized experimentally with the use of a bulky vinyl ether in the key [4+2] cycloaddition reaction. Finally, DFT calculations on relative strain energy for the 1-azafenestranes, as well as their predicted central angles, are disclosed.

  10. Selenium Derivatization of Nucleic Acids for Phase and Structure Determination in Nucleic Acid X-ray Crystallography

    Directory of Open Access Journals (Sweden)

    Zhen Huang

    2008-03-01

    Full Text Available Selenium derivatization (via selenomethionine of proteins for crystal structure determination via MAD phasing has revolutionized protein X-ray crystallography. It is estimated that over two thirds of all new crystal structures of proteins have been determined via Se-Met derivatization. Similarly, selenium functionalities have also been successfully incorporated into nucleic acids to facilitate their structure studies and it has been proved that this Se-derivatization has advantages over halogen strategy, which was usually used as a traditional method in this field. This review reports the development of site-specific selenium derivatization of nucleic acids for phase determination since the year of 2001 (mainly focus on the 2’-position of the ribose. All the synthesis of 2’-SeMe modified phosphoramidite building blocks (U, C, T, A, G and the according oligonucleotides are included. In addition, several structures of selenium contained nucleic acid are also described in this paper.

  11. X-ray crystallography, electrochemistry, spectral and thermal analysis of some tetradentate schiff base complexes and formation constant measurements

    Czech Academy of Sciences Publication Activity Database

    Asadi, Z.; Savarypour, N.; Dušek, Michal; Eigner, Václav

    2017-01-01

    Roč. 47, č. 11 (2017), s. 1501-1508 ISSN 2470-1556 R&D Projects: GA ČR(CZ) GA15-12653S Institutional support: RVO:68378271 Keywords : X-ray crystallography * transition metal Schiff base complexes * thermogravimetry * electrochemistry * formation constant measurements Subject RIV: BM - Solid Matter Physics ; Magnetism OBOR OECD: Condensed matter physics (including formerly solid state physics, supercond.)

  12. Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging.

    Science.gov (United States)

    Warren, Anna J; Armour, Wes; Axford, Danny; Basham, Mark; Connolley, Thomas; Hall, David R; Horrell, Sam; McAuley, Katherine E; Mykhaylyk, Vitaliy; Wagner, Armin; Evans, Gwyndaf

    2013-07-01

    The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required.

  13. A new fixed-target approach for serial crystallography at synchrotron light sources and X-ray free electron lasers

    Energy Technology Data Exchange (ETDEWEB)

    Roedig, Philip

    2017-07-15

    In the framework of this thesis, a new method for high-speed fixed-target serial crystallography experiments and its applicability to biomacromolecular crystallography at both synchrotron light sources and X-ray free electron lasers (XFELs) is presented. The method is based on a sample holder, which can carry up to 20,000 microcrystals and which is made of single-crystalline silicon. Using synchrotron radiation, the structure of Operophtera brumata cytoplasmic polyhedrosis virus type 18 polyhedrin, lysozyme and cubic insulin was determined by collecting X-ray diffraction data from multiple microcrystals. Data collection was shown to be possible at both cryogenic and ambient conditions. For room-temperature measurements, both global and specific indications of radiation damage were investigated and characterized. Due to the sieve-like structure of the chip, the microcrystals tend to arrange themselves according to the micropore pattern, which allows for efficient sampling of the sample material. In combination with a high-speed scanning stage, the sample holder was furthermore shown to be highly suitable for serial femtosecond crystallography experiments. By fast raster scanning of the chip through the pulsed X-ray beam of an XFEL, structure determination of a virus, using the example of bovine enterovirus type 2, has been demonstrated at an XFEL for the first time. Hit rates of up to 100% were obtained by the presented method, which refers to a reduction in sample consumption by at least three orders of magnitude with respect to common liquid-jet injection methods used for sample delivery. In this way, the typical time needed for data collection in serial femtosecond crystallography is significantly decreased. The presented technique for sample loading of the chip is easy to learn and results in efficient removal of the surrounding mother liquor, thereby reducing the generated background signal. Since the chip is made of single-crystalline silicon, in principle no

  14. A new fixed-target approach for serial crystallography at synchrotron light sources and X-ray free electron lasers

    International Nuclear Information System (INIS)

    Roedig, Philip

    2017-07-01

    In the framework of this thesis, a new method for high-speed fixed-target serial crystallography experiments and its applicability to biomacromolecular crystallography at both synchrotron light sources and X-ray free electron lasers (XFELs) is presented. The method is based on a sample holder, which can carry up to 20,000 microcrystals and which is made of single-crystalline silicon. Using synchrotron radiation, the structure of Operophtera brumata cytoplasmic polyhedrosis virus type 18 polyhedrin, lysozyme and cubic insulin was determined by collecting X-ray diffraction data from multiple microcrystals. Data collection was shown to be possible at both cryogenic and ambient conditions. For room-temperature measurements, both global and specific indications of radiation damage were investigated and characterized. Due to the sieve-like structure of the chip, the microcrystals tend to arrange themselves according to the micropore pattern, which allows for efficient sampling of the sample material. In combination with a high-speed scanning stage, the sample holder was furthermore shown to be highly suitable for serial femtosecond crystallography experiments. By fast raster scanning of the chip through the pulsed X-ray beam of an XFEL, structure determination of a virus, using the example of bovine enterovirus type 2, has been demonstrated at an XFEL for the first time. Hit rates of up to 100% were obtained by the presented method, which refers to a reduction in sample consumption by at least three orders of magnitude with respect to common liquid-jet injection methods used for sample delivery. In this way, the typical time needed for data collection in serial femtosecond crystallography is significantly decreased. The presented technique for sample loading of the chip is easy to learn and results in efficient removal of the surrounding mother liquor, thereby reducing the generated background signal. Since the chip is made of single-crystalline silicon, in principle no

  15. Optics for protein microcrystallography using synchrotron and laboratory X-ray sources

    International Nuclear Information System (INIS)

    Varghese, J.N.; Van Donkelaar, A.; Balaic, D.X.; Barnea, Z.

    2000-01-01

    Full text: For protein crystallography, a highly-intense focused beam overcomes a serious constraint in current biological research: the inability of many protein molecules to form crystals larger than a few tens of microns in size. High structure-resolution X-ray diffraction analysis of microcrystals is currently only being studied at synchrotron X-ray sources. We shall examine how this is being carried out, and also report the development of a novel tapered glass monocapillary toroidal-mirror optic, which achieves a high-intensity, low-divergence focused beam from a rotating-anode Xray generator. We have used this optic, which demonstrates an ∼28x intensity gain at the beam focus to solve the structure of a plant exoglucanse/inhibtor complex microcrystal to 2.8 Angstroms, with volume equivalent to a 30-micron-edge cube

  16. The O2-Evolving Complex of Photosystem II: Recent Insights from Quantum Mechanics/Molecular Mechanics (QM/MM), Extended X-ray Absorption Fine Structure (EXAFS), and Femtosecond X-ray Crystallography Data.

    Science.gov (United States)

    Askerka, Mikhail; Brudvig, Gary W; Batista, Victor S

    2017-01-17

    Efficient photoelectrochemical water oxidation may open a way to produce energy from renewable solar power. In biology, generation of fuel due to water oxidation happens efficiently on an immense scale during the light reactions of photosynthesis. To oxidize water, photosynthetic organisms have evolved a highly conserved protein complex, Photosystem II. Within that complex, water oxidation happens at the CaMn 4 O 5 inorganic catalytic cluster, the so-called oxygen-evolving complex (OEC), which cycles through storage "S" states as it accumulates oxidizing equivalents and produces molecular oxygen. In recent years, there has been significant progress in understanding the OEC as it evolves through the catalytic cycle. Studies have combined conventional and femtosecond X-ray crystallography with extended X-ray absorption fine structure (EXAFS) and quantum mechanics/molecular mechanics (QM/MM) methods and have addressed changes in protonation states of μ-oxo bridges and the coordination of substrate water through the analysis of ammonia binding as a chemical analog of water. These advances are thought to be critical to understanding the catalytic cycle since protonation states regulate the relative stability of different redox states and the geometry of the OEC. Therefore, establishing the mechanism for substrate water binding and the nature of protonation/redox state transitions in the OEC is essential for understanding the catalytic cycle of O 2 evolution. The structure of the dark-stable S 1 state has been a target for X-ray crystallography for the past 15 years. However, traditional X-ray crystallography has been hampered by radiation-induced reduction of the OEC. Very recently, a revolutionary X-ray free electron laser (XFEL) technique was applied to PSII to reveal atomic positions at 1.95 Å without radiation damage, which brought us closer than ever to establishing the ultimate structure of the OEC in the S 1 state. However, the atom positions in this crystal

  17. Small angle X-ray scattering from protein in solution

    International Nuclear Information System (INIS)

    Souza, C.F. de; Torriani, I.L.

    1988-01-01

    In this work we report experiments performed with giant respiratory proteins from annelids. X-ray scattering data were obtained both by the use of conventional rotating anod source and synchotron radiation. Data from solutions with several protein concentrations were analyzed. (A.C.A.S.) [pt

  18. Structural Data on the Periplasmic Aldehyde Oxidoreductase PaoABC from Escherichia coli: SAXS and Preliminary X-ray Crystallography Analysis

    Directory of Open Access Journals (Sweden)

    Ana Rita Otrelo-Cardoso

    2014-01-01

    Full Text Available The periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli is a molybdenum enzyme involved in detoxification of aldehydes in the cell. It is an example of an αβγ heterotrimeric enzyme of the xanthine oxidase family of enzymes which does not dimerize via its molybdenum cofactor binding domain. In order to structurally characterize PaoABC, X-ray crystallography and small angle X-ray scattering (SAXS have been carried out. The protein crystallizes in the presence of 20% (w/v polyethylene glycol 3350 using the hanging-drop vapour diffusion method. Although crystals were initially twinned, several experiments were done to overcome twinning and lowering the crystallization temperature (293 K to 277 K was the solution to the problem. The non-twinned crystals used to solve the structure diffract X-rays to beyond 1.80 Å and belong to the C2 space group, with cell parameters a = 109.42 Å, b = 78.08 Å, c = 151.77 Å, β = 99.77°, and one molecule in the asymmetric unit. A molecular replacement solution was found for each subunit separately, using several proteins as search models. SAXS data of PaoABC were also collected showing that, in solution, the protein is also an αβγ heterotrimer.

  19. Combined analysis of 1,3-benzodioxoles by crystalline sponge X-ray crystallography and laser desorption ionization mass spectrometry.

    Science.gov (United States)

    Hayashi, Yukako; Ohara, Kazuaki; Taki, Rika; Saeki, Tomomi; Yamaguchi, Kentaro

    2018-03-12

    The crystalline sponge (CS) method, which employs single-crystal X-ray diffraction to determine the structure of an analyte present as a liquid or an oil and having a low melting point, was used in combination with laser desorption ionization mass spectrometry (LDI-MS). 1,3-Benzodioxole derivatives were encapsulated in CS and their structures were determined by combining X-ray crystallography and MS. After the X-ray analysis, the CS was subjected to imaging mass spectrometry (IMS) with an LDI spiral-time-of-flight mass spectrometer (TOF-MS). The ion detection area matched the microscopic image of the encapsulated CS. In addition, the accumulated 1D mass spectra showed that fragmentation of the guest molecule (hereafter, guest) can be easily visualized without any interference from the fragment ions of CS except for two strong ion peaks derived from the tridentate ligand TPT (2,4,6-tris(4-pyridyl)-1,3,5-triazine) of the CS and its fragment. X-ray analysis clearly showed the presence of the guest as well as the π-π, CH-halogen, and CH-O interactions between the guest and the CS framework. However, some guests remained randomly diffused in the nanopores of CS. In addition, the detection limit was less than sub-pmol order based on the weight and density of CS determined by X-ray analysis. Spectroscopic data, such as UV-vis and NMR, also supported the encapsulation of the guest through the interaction between the guest and CS components. The results denote that the CS-LDI-MS method, which combines CS, X-ray analysis and LDI-MS, is effective for structure determination.

  20. Langmuir-Blodgett nanotemplates for protein crystallography.

    Science.gov (United States)

    Pechkova, Eugenia; Nicolini, Claudio

    2017-12-01

    The new generation of synchrotrons and microfocused beamlines has enabled great progress in X-ray protein crystallography, resulting in new 3D atomic structures for proteins of high interest to the pharmaceutical industry and life sciences. It is, however, often still challenging to produce protein crystals of sufficient size and quality (order, intensity of diffraction, radiation stability). In this protocol, we provide instructions for performing the Langmuir-Blodgett (LB) nanotemplate method, a crystallization approach that can be used for any protein (including membrane proteins). We describe how to produce highly ordered 2D LB protein monolayers at the air-water interface and deposit them on glass slides. LB-film formation can be observed by surface-pressure measurements and Brewster angle microscopy (BAM), although its quality can be characterized by atomic force microscopy (AFM) and nanogravimetry. Such films are then used as a 2D template for triggering 3D protein crystal formation by hanging-drop vapor diffusion. The procedure for forming the 2D template takes a few minutes. Structural information about the protein reorganization in the LB film during the crystallization process on the nano level can be obtained using an in situ submicron GISAXS (grazing-incidence small-angle X-ray scattering) method. MicroGISAXS spectra, measured directly at the interface of the LB films and protein solution in real time, as described in this protocol, can be interpreted in terms of the buildup of layers, islands, or holes. In our experience, the obtained LB crystals take 1-10 d to prepare and they are more ordered and radiation stable as compared with those produced using other crystallization methods.

  1. X-ray spectromicroscopy studies of protein-polymer Interactions

    International Nuclear Information System (INIS)

    Hitchcock, A.P.; Tyliszczak, T.; Heng, Y.M.; Cornelius, R.; Brash, J.L.; Ade, H.; Anders, S.; Scholl, A.; Nolting, F.

    2000-01-01

    Identification of the sites of protein binding on laterally heterogeneous polymer surfaces can help understand mechanisms of bio-passivation, which in turn has practical impact on developing improved polymers for medical uses, such as blood contact applications. We have used scanning X-ray transmission microscopy and photoelectron emission microscopy to explore the sensitivity of these X-ray microscopies to detection of human serum albumin adsorbed onto the surface of two different polyurethane polymers. Both techniques appear capable of the requisite monolayer sensitivity

  2. Review: Radiation Chemists Look at Damage in Redox Proteins Induced by X-rays.

    Science.gov (United States)

    Wherland, Scot; Pecht, Israel

    2018-04-30

    The three-dimensional structure of proteins, especially as determined by X-ray crystallography, is critical to the understanding of their function. However, the X-ray exposure may lead to damage that must be recognized and understood to interpret the crystallographic results. This is especially relevant for proteins with transition metal ions that can be oxidized or reduced. The detailed study of proteins in aqueous solution by the technique of pulse radiolysis has provided a wealth of information on the production and fate of radicals that are the same as those produced by X-ray exposure. The results reviewed here illustrate how the products of the interaction of radiation with water or with solutes added to the crystallization medium, and with proteins themselves, are formed, and about their fate. Of particular focus is how electrons are produced and transferred through the polypeptide matrix to redox centers such as metal ions or to specific amino acid residues, e.g. disulfides, and how the hydroxyl radicals formed may be converted to reducing equivalents or scavenged. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

  3. X-ray transparent Microfluidics for Protein Crystallization and Biomineralization

    Science.gov (United States)

    Opathalage, Achini

    Protein crystallization demands the fundamental understanding of nucleation and applying techniques to find the optimal conditions to achieve the kinetic pathway for a large and defect free crystal. Classical nucleation theory predicts that the nucleation occurs at high supersaturation conditions. In this dissertation we sought out to develop techniques to attain optimal supersaturation profile to a large defect free crystal and subject it to in-situ X-ray diffraction using microfluidics. We have developed an emulsion-based serial crystallographic technology in nanolitre-sized droplets of protein solution encapsulated in to nucleate 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 un-oriented crystals. As proof of concept, the structure of Glucose Isomerase was solved to 2.1 A. We have developed a suite of X-ray semi-transparent micrfluidic devices which enables; controlled evaporation as a method of increasing supersaturation and manipulating the phase space of proteins and small molecules. We exploited the inherently high water permeability of the thin X-ray semi-transparent devices as a mean of increasing the supersaturation by controlling the evaporation. We fabricated the X-ray semi-transparent version of the PhaseChip with a thin PDMS membrane by which the storage and the reservoir layers are separated, and studies the phase transition of amorphous CaCO3.

  4. A CCD-based area detector for X-ray crystallography using synchrotron and laboratory sources

    International Nuclear Information System (INIS)

    Phillips, W.C.; Li Youli; Stanton, M.; Xie Yuanhui; O'Mara, D.; Kalata, K.

    1993-01-01

    The design and characteristics of a CCD-based area detector suitable for X-ray crystallographic studies using both synchrotron and laboratory sources are described. The active area is 75 mm in diameter, the FWHM of the point response function is 0.20 mm, and for Bragg peaks the dynamic range is 900 and the DQE ∼0.3. The 1320x1035-pixel Kodak CCD is read out into an 8 Mbyte memory system in 0.14 s and digitized to 12 bits. X-ray crystallographic data collected at the NSLS synchrotron from cubic insulin crystals are presented. (orig.)

  5. Diffracted X-ray tracking for monitoring intramolecular motion in individual protein molecules using broad band X-ray

    Energy Technology Data Exchange (ETDEWEB)

    Ichiyanagi, Kouhei; Sasaki, Yuji C. [Department of Advanced Materials Science, Graduate School of Frontier Sciences, The University of Tokyo, 609 Kiban Building 5-1-5 Kashiwanoha, Kahiwashi, Chiba 277-8561 (Japan); Japan Science and Technology Agency, CREST, CREST, Sasaki-Team, 609 Kiban Building, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8561 (Japan); Sekiguchi, Hiroshi; Hoshino, Masato; Kajiwara, Kentaro; Senba, Yasunori; Ohashi, Haruhiko; Ohta, Noboru [Japan Synchrotron Radiation Research Institute, SPring-8, 1-1-1 Kouto, Sayo, Hyogo 679-5198 (Japan); Hoshisashi, Kentaro; Jae-won, Chang; Tokue, Maki; Matsushita, Yufuku [Department of Advanced Materials Science, Graduate School of Frontier Sciences, The University of Tokyo, 609 Kiban Building 5-1-5 Kashiwanoha, Kahiwashi, Chiba 277-8561 (Japan); Nishijima, Masaki; Inoue, Yoshihisa [Department of Applied Chemistry and Office for University-Industry Collaboration, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Yagi, Naoto [Japan Science and Technology Agency, CREST, CREST, Sasaki-Team, 609 Kiban Building, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8561 (Japan); Japan Synchrotron Radiation Research Institute, SPring-8, 1-1-1 Kouto, Sayo, Hyogo 679-5198 (Japan)

    2013-10-15

    Diffracted X-ray tracking (DXT) enables the tilting and twisting motions of single protein molecules to be monitored with micro- to milliradian resolution using a highly brilliant X-ray source with a wide energy bandwidth. We have developed a technique to monitor single molecules using gold nanocrystals attached to individual protein molecules using the BL28B2 beamline at SPring-8. In this paper we present the installation of a single toroidal X-ray mirror at BL28B2 to focus X-rays in an energy range of 10–20 keV (△E/E = 82% for an X-ray with a wide energy bandwidth). With this beamline we tracked diffraction spots from gold nanocrystals over a wide angle range than that using quasi-monochromatic X-rays. Application of the wide angle DXT technique to biological systems enabled us to observe the on-site motions of single protein molecules that have been functionalized in vivo. We further extend the capability of DXT by observing the fractional tilting and twisting motions of inner proteins under various conditions. As a proof of this methodology and to determine instrumental performance the intramolecular motions of a human serum albumin complex with 2-anthracenecarboxylic acid was investigated using the BL28B2 beamline. The random tilting and twisting intramolecular motions are shown to be directly linked to the movement of individual protein molecules in the buffer solution.

  6. X-ray-excited optical luminescence of protein crystals: a new tool for studying radiation damage during diffraction data collection.

    Science.gov (United States)

    Owen, Robin L; Yorke, Briony A; Pearson, Arwen R

    2012-05-01

    During X-ray irradiation protein crystals radiate energy in the form of small amounts of visible light. This is known as X-ray-excited optical luminescence (XEOL). The XEOL of several proteins and their constituent amino acids has been characterized using the microspectrophotometers at the Swiss Light Source and Diamond Light Source. XEOL arises primarily from aromatic amino acids, but the effects of local environment and quenching within a crystal mean that the XEOL spectrum of a crystal is not the simple sum of the spectra of its constituent parts. Upon repeated exposure to X-rays XEOL spectra decay non-uniformly, suggesting that XEOL is sensitive to site-specific radiation damage. However, rates of XEOL decay were found not to correlate to decays in diffracting power, making XEOL of limited use as a metric for radiation damage to protein crystals. © 2012 International Union of Crystallography

  7. Synthesis, X-ray crystallography, and DFT calculations of a novel phosphoramide

    Czech Academy of Sciences Publication Activity Database

    Shariatinia, Z.; Dušek, Michal; Eigner, Václav

    2014-01-01

    Roč. 640, č. 14 (2014), 2945-2955 ISSN 0044-2313 R&D Projects: GA ČR(CZ) GA14-03276S Institutional support: RVO:68378271 Keywords : phosphoramide * x-ray structure * DFT Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 1.160, year: 2014

  8. Weak activity of haloalkane dehalogenase LinB with 1,2,3-trichloropropane revealed by X-Ray crystallography and microcalorimetry.

    Science.gov (United States)

    Monincová, Marta; Prokop, Zbynek; Vévodová, Jitka; Nagata, Yuji; Damborsky, Jirí

    2007-03-01

    1,2,3-Trichloropropane (TCP) is a highly toxic and recalcitrant compound. Haloalkane dehalogenases are bacterial enzymes that catalyze the cleavage of a carbon-halogen bond in a wide range of organic halogenated compounds. Haloalkane dehalogenase LinB from Sphingobium japonicum UT26 has, for a long time, been considered inactive with TCP, since the reaction cannot be easily detected by conventional analytical methods. Here we demonstrate detection of the weak activity (k(cat) = 0.005 s(-1)) of LinB with TCP using X-ray crystallography and microcalorimetry. This observation makes LinB a useful starting material for the development of a new biocatalyst toward TCP by protein engineering. Microcalorimetry is proposed to be a universal method for the detection of weak enzymatic activities. Detection of these activities is becoming increasingly important for engineering novel biocatalysts using the scaffolds of proteins with promiscuous activities.

  9. Weak Activity of Haloalkane Dehalogenase LinB with 1,2,3-Trichloropropane Revealed by X-Ray Crystallography and Microcalorimetry▿

    Science.gov (United States)

    Monincová, Marta; Prokop, Zbyněk; Vévodová, Jitka; Nagata, Yuji; Damborský, Jiří

    2007-01-01

    1,2,3-Trichloropropane (TCP) is a highly toxic and recalcitrant compound. Haloalkane dehalogenases are bacterial enzymes that catalyze the cleavage of a carbon-halogen bond in a wide range of organic halogenated compounds. Haloalkane dehalogenase LinB from Sphingobium japonicum UT26 has, for a long time, been considered inactive with TCP, since the reaction cannot be easily detected by conventional analytical methods. Here we demonstrate detection of the weak activity (kcat = 0.005 s−1) of LinB with TCP using X-ray crystallography and microcalorimetry. This observation makes LinB a useful starting material for the development of a new biocatalyst toward TCP by protein engineering. Microcalorimetry is proposed to be a universal method for the detection of weak enzymatic activities. Detection of these activities is becoming increasingly important for engineering novel biocatalysts using the scaffolds of proteins with promiscuous activities. PMID:17259360

  10. Synthesis and structure elucidation of a series of pyranochromene chalcones and flavanones using 1D and 2D NMR spectroscopy and X-ray crystallography.

    Science.gov (United States)

    Pawar, Sunayna S; Koorbanally, Neil A

    2014-06-01

    A series of novel pyranochromene chalcones and corresponding flavanones were synthesized. This is the first report on the confirmation of the absolute configuration of chromene-based flavanones using X-ray crystallography. These compounds were characterized by 2D NMR spectroscopy, and their assignments are reported herein. The 3D structure of the chalcone 3b and flavanone 4g was determined by X-ray crystallography, and the structure of the flavanone was confirmed to be in the S configuration at C-2. Copyright © 2014 John Wiley & Sons, Ltd.

  11. Hydrogen atoms can be located accurately and precisely by x-ray crystallography.

    Science.gov (United States)

    Woińska, Magdalena; Grabowsky, Simon; Dominiak, Paulina M; Woźniak, Krzysztof; Jayatilaka, Dylan

    2016-05-01

    Precise and accurate structural information on hydrogen atoms is crucial to the study of energies of interactions important for crystal engineering, materials science, medicine, and pharmacy, and to the estimation of physical and chemical properties in solids. However, hydrogen atoms only scatter x-radiation weakly, so x-rays have not been used routinely to locate them accurately. Textbooks and teaching classes still emphasize that hydrogen atoms cannot be located with x-rays close to heavy elements; instead, neutron diffraction is needed. We show that, contrary to widespread expectation, hydrogen atoms can be located very accurately using x-ray diffraction, yielding bond lengths involving hydrogen atoms (A-H) that are in agreement with results from neutron diffraction mostly within a single standard deviation. The precision of the determination is also comparable between x-ray and neutron diffraction results. This has been achieved at resolutions as low as 0.8 Å using Hirshfeld atom refinement (HAR). We have applied HAR to 81 crystal structures of organic molecules and compared the A-H bond lengths with those from neutron measurements for A-H bonds sorted into bonds of the same class. We further show in a selection of inorganic compounds that hydrogen atoms can be located in bridging positions and close to heavy transition metals accurately and precisely. We anticipate that, in the future, conventional x-radiation sources at in-house diffractometers can be used routinely for locating hydrogen atoms in small molecules accurately instead of large-scale facilities such as spallation sources or nuclear reactors.

  12. Protein crystal growth studies at the Center for Macromolecular Crystallography

    International Nuclear Information System (INIS)

    DeLucas, Lawrence J.; Long, Marianna M.; Moore, Karen M.; Harrington, Michael; McDonald, William T.; Smith, Craig D.; Bray, Terry; Lewis, Johanna; Crysel, William B.; Weise, Lance D.

    2000-01-01

    The Center for Macromolecular Crystallography (CMC) has been involved in fundamental studies of protein crystal growth (PCG) in microgravity and in our earth-based laboratories. A large group of co-investigators from academia and industry participated in these experiments by providing protein samples and by performing the x-ray crystallographic analysis. These studies have clearly demonstrated the usefulness of a microgravity environment for enhancing the quality and size of protein crystals. Review of the vapor diffusion (VDA) PCG results from nineteen space shuttle missions is given in this paper

  13. FIST - a suite of X-ray powder crystallography programs for use with a HP-65 calculator

    International Nuclear Information System (INIS)

    Ferguson, I.F.; Turek, M.

    1977-12-01

    Programs for X-ray powder crystallography are defined for use with a Hewlett Packard HP-65 (programmable) pocket calculator. These include the prediction of all Bragg reflections for defined P-, F-, I-cubic, tetragonal, hexagonal and orthorhombic cells; the calculation of the position of a specific Bragg reflection from defined unit cells with all symmetries except triclinic; interconversion of theta, 2theta, sin 2 theta and d, as well as the calculation of the Nelson-Riley function; the computation of crystal densities; the interconversion of rhombohedral and hexagonal unit cells, lsub(c) determinations for graphite, the calculation of a and c for boron carbide; and Miller index transformations between various unit cells. (author)

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

    OpenAIRE

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

    2010-01-01

    The Protein Crystallography Station user facility at Los Alamos National Laboratory not only offers open access to a high-performance neutron beamline, but also actively supports and develops new methods in protein expression, deuteration, purification, robotic crystallization and the synthesis of substrates with stable isotopes and provides assistance with data-reduction and structure-refinement software and comprehensive neutron structure analysis.

  15. Unambiguous determination of H-atom positions: comparing results from neutron and high-resolution X-ray crystallography.

    Science.gov (United States)

    Gardberg, Anna S; Del Castillo, Alexis Rae; Weiss, Kevin L; Meilleur, Flora; Blakeley, Matthew P; Myles, Dean A A

    2010-05-01

    The locations of H atoms in biological structures can be difficult to determine using X-ray diffraction methods. Neutron diffraction offers a relatively greater scattering magnitude from H and D atoms. Here, 1.65 A resolution neutron diffraction studies of fully perdeuterated and selectively CH(3)-protonated perdeuterated crystals of Pyrococcus furiosus rubredoxin (D-rubredoxin and HD-rubredoxin, respectively) at room temperature (RT) are described, as well as 1.1 A resolution X-ray diffraction studies of the same protein at both RT and 100 K. The two techniques are quantitatively compared in terms of their power to directly provide atomic positions for D atoms and analyze the role played by atomic thermal motion by computing the sigma level at the D-atom coordinate in simulated-annealing composite D-OMIT maps. It is shown that 1.65 A resolution RT neutron data for perdeuterated rubredoxin are approximately 8 times more likely overall to provide high-confidence positions for D atoms than 1.1 A resolution X-ray data at 100 K or RT. At or above the 1.0sigma level, the joint X-ray/neutron (XN) structures define 342/378 (90%) and 291/365 (80%) of the D-atom positions for D-rubredoxin and HD-rubredoxin, respectively. The X-ray-only 1.1 A resolution 100 K structures determine only 19/388 (5%) and 8/388 (2%) of the D-atom positions above the 1.0sigma level for D-rubredoxin and HD-rubredoxin, respectively. Furthermore, the improved model obtained from joint XN refinement yielded improved electron-density maps, permitting the location of more D atoms than electron-density maps from models refined against X-ray data only.

  16. Small angle x-ray scattering from proteins in solution

    International Nuclear Information System (INIS)

    de Souza, C.F.; Torriani, I.L.; Bonafe, C.F.S.; Merrelles, N.C.; Vachette, P.

    1989-01-01

    In this work the authors report experiments performed with giant respiratory proteins from annelids (erythrocruorins), known to have a molecular weight in the order of four million Daltons. Preliminary x-ray scattering data was obtained using a conventional rotating anode source. High resolution small angle scattering curves were obtained with synchrotron radiation from the DCI storage ring at LURE. Data from solutions with several protein concentrations were analyzed in order to determine low resolution dimensional parameters, using Guinier plots from the smeared scattering curves and the inverse transformation method

  17. Automatic protein structure solution from weak X-ray data

    Science.gov (United States)

    Skubák, Pavol; Pannu, Navraj S.

    2013-11-01

    Determining new protein structures from X-ray diffraction data at low resolution or with a weak anomalous signal is a difficult and often an impossible task. Here we propose a multivariate algorithm that simultaneously combines the structure determination steps. In tests on over 140 real data sets from the protein data bank, we show that this combined approach can automatically build models where current algorithms fail, including an anisotropically diffracting 3.88 Å RNA polymerase II data set. The method seamlessly automates the process, is ideal for non-specialists and provides a mathematical framework for successfully combining various sources of information in image processing.

  18. Serial Femtosecond Crystallography

    OpenAIRE

    Chapman, Henry N.

    2015-01-01

    X-ray free-electron lasers produce brief flashes of X-rays that are of about a billion times higher peak brightness than achievable from storage ring sources. Such a tremendous jump in X-ray source capabilities, which came in 2009 when the Linac Coherent Light Source began operations, was unprecedented in the history of X-ray science. Protein structure determination through the method of macromolecular crystallography has consistently benefited from the many increases in source performance fr...

  19. Simulated x-ray scattering of protein solutions using explicit-solvent models

    International Nuclear Information System (INIS)

    Park, Sanghyun; Bardhan, Jaydeep P.; Makowski, Lee; Roux, Benoit

    2009-01-01

    X-ray solution scattering shows new promise for the study of protein structures, complementing crystallography and nuclear magnetic resonance. In order to realize the full potential of solution scattering, it is necessary to not only improve experimental techniques but also develop accurate and efficient computational schemes to relate atomistic models to measurements. Previous computational methods, based on continuum models of water, have been unable to calculate scattering patterns accurately, especially in the wide-angle regime which contains most of the information on the secondary, tertiary, and quaternary structures. Here we present a novel formulation based on the atomistic description of water, in which scattering patterns are calculated from atomic coordinates of protein and water. Without any empirical adjustments, this method produces scattering patterns of unprecedented accuracy in the length scale between 5 and 100 A, as we demonstrate by comparing simulated and observed scattering patterns for myoglobin and lysozyme.

  20. Synthesis, X-ray crystallography, thermal studies, spectroscopic and electrochemistry investigations of uranyl Schiff base complexes.

    Science.gov (United States)

    Asadi, Zahra; Shorkaei, Mohammad Ranjkesh

    2013-03-15

    Some tetradentate salen type Schiff bases and their uranyl complexes were synthesized and characterized by UV-Vis, NMR, IR, TG, C.H.N. and X-ray crystallographic studies. From these investigations it is confirmed that a solvent molecule occupied the fifth position of the equatorial plane of the distorted pentagonal bipyramidal structure. Also, the kinetics of complex decomposition by using thermo gravimetric methods (TG) was studied. The thermal decomposition reactions are first order for the studied complexes. To examine the properties of uranyl complexes according to the substitutional groups, we have carried out the electrochemical studies. The electrochemical reactions of uranyl Schiff base complexes in acetonitrile were reversible. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. X-ray-mediated cross linking of protein and DNA

    International Nuclear Information System (INIS)

    Minsky, B.D.; Braun, A.

    1977-01-01

    Using a simple filter assay for the binding of BSA or lysozyme to DNA, two mechanisms of x-ray-mediated cross linking are shown to occur. One, a fast reaction, appears to involve a radical intermediate, is inhibited by high pH and salt, and seems to be enhanced by deoxygenation. The second mechanism, a slow time-dependent component, differs from the fast reaction in its stimulation by histidine, its inhibition by catalase, and the lack of an oxygen effect. Separate irradiation of DNA or water does not lead to cross linking. However, separate irradiation of protein leads to cross linking which proceeds with slow-component kinetics

  2. Protein energy landscapes determined by five-dimensional crystallography

    International Nuclear Information System (INIS)

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

    2013-01-01

    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

  3. The Barium Site in a Potassium Channel by X-Ray Crystallography

    Science.gov (United States)

    Jiang, Youxing; MacKinnon, Roderick

    2000-01-01

    X-ray diffraction data were collected from frozen crystals (100°K) of the KcsA K+ channel equilibrated with solutions containing barium chloride. Difference electron density maps (Fbarium − Fnative, 5.0 Å resolution) show that Ba2+ resides at a single location within the selectivity filter. The Ba2+ blocking site corresponds to the internal aspect (adjacent to the central cavity) of the “inner ion” position where an alkali metal cation is found in the absence of the blocking Ba2+ ion. The location of Ba2+ with respect to Rb+ ions in the pore is in good agreement with the findings on the functional interaction of Ba2+ with K+ (and Rb+) in Ca2+-activated K+ channels (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549–567). Taken together, these structural and functional data imply that at physiological ion concentrations a third ion may interact with two ions in the selectivity filter, perhaps by entering from one side and displacing an ion on the opposite side. PMID:10694255

  4. High-resolution X-ray crystal structure of bovine H-protein using the high-pressure cryocooling method

    International Nuclear Information System (INIS)

    Higashiura, Akifumi; Ohta, Kazunori; Masaki, Mika; Sato, Masaru; Inaka, Koji; Tanaka, Hiroaki; Nakagawa, Atsushi

    2013-01-01

    Using the high-pressure cryocooling method, the high-resolution X-ray crystal structure of bovine H-protein was determined at 0.86 Å resolution. This is the first ultra-high-resolution structure obtained from a high-pressure cryocooled crystal. Recently, many technical improvements in macromolecular X-ray crystallography have increased the number of structures deposited in the Protein Data Bank and improved the resolution limit of protein structures. Almost all high-resolution structures have been determined using a synchrotron radiation source in conjunction with cryocooling techniques, which are required in order to minimize radiation damage. However, optimization of cryoprotectant conditions is a time-consuming and difficult step. To overcome this problem, the high-pressure cryocooling method was developed (Kim et al., 2005 ▶) and successfully applied to many protein-structure analyses. In this report, using the high-pressure cryocooling method, the X-ray crystal structure of bovine H-protein was determined at 0.86 Å resolution. Structural comparisons between high- and ambient-pressure cryocooled crystals at ultra-high resolution illustrate the versatility of this technique. This is the first ultra-high-resolution X-ray structure obtained using the high-pressure cryocooling method

  5. Combined x-ray crystallography and computational modeling approach to investigate the Hsp90 C-terminal peptide binding to FKBP51.

    Science.gov (United States)

    Kumar, Rajnish; Moche, Martin; Winblad, Bengt; Pavlov, Pavel F

    2017-10-27

    FK506 binding protein of 51 kDa (FKBP51) is a heat shock protein 90 (Hsp90) co-chaperone involved in the regulation of steroid hormone receptors activity. It is known for its role in various regulatory pathways implicated in mood and stress-related disorders, cancer, obesity, Alzheimer's disease and corticosteroid resistant asthma. It consists of two FKBP12 like active peptidyl prolyl isomerase (PPIase) domains (an active FK1 and inactive FK2 domain) and one tetratricopeptide repeat (TPR) domain that mediates interaction with Hsp90 via its C-terminal MEEVD peptide. Here, we report a combined x-ray crystallography and molecular dynamics study to reveal the binding mechanism of Hsp90 MEEVD peptide to the TPR domain of FKBP51. The results demonstrated that the Hsp90 C-terminal peptide binds to the TPR domain of FKBP51 with the help of di-carboxylate clamp involving Lys272, Glu273, Lys352, Asn322, and Lys329 which are conserved throughout several di-carboxylate clamp TPR proteins. Interestingly, the results from molecular dynamics study are also in agreement to the complex structure where all the contacts between these two partners were consistent throughout the simulation period. In a nutshell, our findings provide new opportunity to engage this important protein-protein interaction target by small molecules designed by structure based drug design strategy.

  6. Interaction partners of PSD-93 studied by X-ray crystallography and fluorescence polarization spectroscopy

    DEFF Research Database (Denmark)

    Fiorentini, Monica; Bach, Anders; Strømgaard, Kristian

    2013-01-01

    PSD-93 (chapsyn-110, DLG2) is a member of the family of membrane-associated guanylate kinase (MAGUK) proteins. The MAGUK proteins are involved in receptor localization and signalling pathways. The best characterized MAGUK protein, PSD-95, is known to be involved in NMDA receptor signalling via it...

  7. C-shaped diastereomers containing cofacial thiophene-substituted quinoxaline rings: synthesis, photophysical properties, and X-ray crystallography.

    Science.gov (United States)

    DeBlase, Catherine R; Finke, Ryan T; Porras, Jonathan A; Tanski, Joseph M; Nadeau, Jocelyn M

    2014-05-16

    Synthesis and characterization of two diastereomeric C-shaped molecules containing cofacial thiophene-substituted quinoxaline rings are described. A previously known bis-α-diketone was condensed with an excess of 4-bromo-1,2-diaminobenzene in the presence of zinc acetate to give a mixture of two C-shaped diastereomers with cofacial bromine-substituted quinoxaline rings. After chromatographic separation, thiophene rings were installed by a microwave-assisted Suzuki coupling reaction, resulting in highly emissive diastereomeric compounds that were studied by UV-vis, fluorescence, and NMR spectroscopy, as well as X-ray crystallography. The unique symmetry of each diastereomer was confirmed by NMR spectroscopy. NMR data indicated that the syn isomer has restricted rotation about the bond connecting the thiophene and quinoxaline rings, which was also observed in the solid state. The spectroscopic properties of the C-shaped diastereomers were compared to a model compound containing only a single thiophene-substituted quinoxaline ring. Ground state intramolecular π-π interactions in solution were detected by NMR and UV-vis spectroscopy. Red-shifted emission bands, band broadening, and large Stokes shifts were observed, which collectively suggest excited state π-π interactions that produce excimer-like emissions, as well as a remarkable positive emission solvatochromism, indicating charge-transfer character in the excited state.

  8. A rapid alternative to X-ray crystallography for chiral determination: case studies of vibrational circular dichroism (VCD) to advance drug discovery projects.

    Science.gov (United States)

    Wesolowski, Steven S; Pivonka, Don E

    2013-07-15

    The absolute stereochemistry of chiral drugs is usually established via X-ray crystallography. However, vibrational circular dichroism (VCD) spectroscopy coupled with quantum mechanics simulations offers a rapid alternative to crystallography and is readily applied to both crystalline and non-crystalline samples. VCD is an effective complement to X-ray analysis of drug candidates, and it can be used as a high-throughput means of assessing absolute stereochemistry at all phases of the discovery process (hundreds of assignments per year). The practical implementation (or fee-for-service outsourcing) of VCD and selected case studies are illustrated with an emphasis on providing utility and impact to pharmaceutical discovery programs. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Exploring the atomic structure and conformational flexibility of a 320 Å long engineered viral fiber using X-ray crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Bhardwaj, Anshul [Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107 (United States); Casjens, Sherwood R. [University of Utah School of Medicine, Salt Lake City, UT 84112 (United States); Cingolani, Gino, E-mail: gino.cingolani@jefferson.edu [Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107 (United States)

    2014-02-01

    This study presents the crystal structure of a ∼320 Å long protein fiber generated by in-frame extension of its repeated helical coiled-coil core. Protein fibers are widespread in nature, but only a limited number of high-resolution structures have been determined experimentally. Unlike globular proteins, fibers are usually recalcitrant to form three-dimensional crystals, preventing single-crystal X-ray diffraction analysis. In the absence of three-dimensional crystals, X-ray fiber diffraction is a powerful tool to determine the internal symmetry of a fiber, but it rarely yields atomic resolution structural information on complex protein fibers. An 85-residue-long minimal coiled-coil repeat unit (MiCRU) was previously identified in the trimeric helical core of tail needle gp26, a fibrous protein emanating from the tail apparatus of the bacteriophage P22 virion. Here, evidence is provided that an MiCRU can be inserted in frame inside the gp26 helical core to generate a rationally extended fiber (gp26-2M) which, like gp26, retains a trimeric quaternary structure in solution. The 2.7 Å resolution crystal structure of this engineered fiber, which measures ∼320 Å in length and is only 20–35 Å wide, was determined. This structure, the longest for a trimeric protein fiber to be determined to such a high resolution, reveals the architecture of 22 consecutive trimerization heptads and provides a framework to decipher the structural determinants for protein fiber assembly, stability and flexibility.

  10. Exploring the atomic structure and conformational flexibility of a 320 Å long engineered viral fiber using X-ray crystallography

    International Nuclear Information System (INIS)

    Bhardwaj, Anshul; Casjens, Sherwood R.; Cingolani, Gino

    2014-01-01

    This study presents the crystal structure of a ∼320 Å long protein fiber generated by in-frame extension of its repeated helical coiled-coil core. Protein fibers are widespread in nature, but only a limited number of high-resolution structures have been determined experimentally. Unlike globular proteins, fibers are usually recalcitrant to form three-dimensional crystals, preventing single-crystal X-ray diffraction analysis. In the absence of three-dimensional crystals, X-ray fiber diffraction is a powerful tool to determine the internal symmetry of a fiber, but it rarely yields atomic resolution structural information on complex protein fibers. An 85-residue-long minimal coiled-coil repeat unit (MiCRU) was previously identified in the trimeric helical core of tail needle gp26, a fibrous protein emanating from the tail apparatus of the bacteriophage P22 virion. Here, evidence is provided that an MiCRU can be inserted in frame inside the gp26 helical core to generate a rationally extended fiber (gp26-2M) which, like gp26, retains a trimeric quaternary structure in solution. The 2.7 Å resolution crystal structure of this engineered fiber, which measures ∼320 Å in length and is only 20–35 Å wide, was determined. This structure, the longest for a trimeric protein fiber to be determined to such a high resolution, reveals the architecture of 22 consecutive trimerization heptads and provides a framework to decipher the structural determinants for protein fiber assembly, stability and flexibility

  11. Epitope-dependent mechanisms of CD27 neutralization revealed by X-ray crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Obmolova, Galina; Teplyakov, Alexey; Malia, Thomas J.; Wunderler, Nicole; Kwok, Deborah; Barone, Linda; Sweet, Raymond; Ort, Tatiana; Scully, Michael; Gilliland, Gary L. (Janssen)

    2017-03-01

    CD27 is a T and B cell co-stimulatory protein of the TNF receptor superfamily dependent on the availability of the TNF-like ligand CD70. Two anti-CD27 neutralizing monoclonal antibodies were obtained from mouse hybridoma and subsequently humanized and optimized for binding the target. The two antibodies are similar in terms of their CD27-binding affinity and ability to block NF-κB signaling, however their clearance rates in monkeys are very different. The pharmacokinetics profiles could be epitope dependent. To identify the epitopes, we determined the crystal structure of the ternary complex between CD27 and the Fab fragments of these non-competing antibodies. The structure reveals the binding modes of the antibodies suggesting that their mechanisms of action are distinctly different and provides a possible explanation of the in vivo data.

  12. Structural investigation of oxovanadium(IV) Schiff base complexes: X-ray crystallography, electrochemistry and kinetic of thermal decomposition.

    Science.gov (United States)

    Asadi, Mozaffar; Asadi, Zahra; Savaripoor, Nooshin; Dusek, Michal; Eigner, Vaclav; Shorkaei, Mohammad Ranjkesh; Sedaghat, Moslem

    2015-02-05

    A series of new VO(IV) complexes of tetradentate N2O2 Schiff base ligands (L(1)-L(4)), were synthesized and characterized by FT-IR, UV-vis and elemental analysis. The structure of the complex VOL(1)⋅DMF was also investigated by X-ray crystallography which revealed a vanadyl center with distorted octahedral coordination where the 2-aza and 2-oxo coordinating sites of the ligand were perpendicular to the "-yl" oxygen. The electrochemical properties of the vanadyl complexes were investigated by cyclic voltammetry. A good correlation was observed between the oxidation potentials and the electron withdrawing character of the substituents on the Schiff base ligands, showing the following trend: MeO5-H>5-Br>5-Cl. Furthermore, the kinetic parameters of thermal decomposition were calculated by using the Coats-Redfern equation. According to the Coats-Redfern plots the kinetics of thermal decomposition of studied complexes is of the first-order in all stages, the free energy of activation for each following stage is larger than the previous one and the complexes have good thermal stability. The preparation of VOL(1)⋅DMF yielded also another compound, one kind of vanadium oxide [VO]X, with different habitus of crystals, (platelet instead of prisma) and without L(1) ligand, consisting of a V10O28 cage, diaminium moiety and dimethylamonium as a counter ions. Because its crystal structure was also new, we reported it along with the targeted complex. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Low-Z polymer sample supports for fixed-target serial femtosecond X-ray crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Feld, Geoffrey K. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); National Institute of Environmental Health Science, Research Triangle Park, NC (United States); Heymann, Michael [Brandeis Univ., Waltham, MA (United States); Univ. of Hamburg and DESY, Hamburg (Germany); Benner, W. Henry [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Pardini, Tommaso [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Tsai, Ching -Ju [Paul Scherrer Inst. (PSI), Villigen (Switzerland); Boutet, Sebastien [SLAC National Accelerator Lab., Menlo Park, CA (United States); Coleman, Matthew A. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Hunter, Mark S. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); SLAC National Accelerator Lab., Menlo Park, CA (United States); Li, Xiaodan [Paul Scherrer Inst. (PSI), Villigen (Switzerland); Messerschmidt, Marc [SLAC National Accelerator Lab., Menlo Park, CA (United States); BioXFEL Science and Technology Center, Buffalo, NY (United States); Opathalage, Achini [Brandeis Univ., Waltham, MA (United States); Pedrini, Bill [Paul Scherrer Inst. (PSI), Villigen (Switzerland); Williams, Garth J. [SLAC National Accelerator Lab., Menlo Park, CA (United States); Krantz, Bryan A. [Univ. of California, Berkeley, CA (United States); Fraden, Seth [Brandeis Univ., Waltham, MA (United States); Hau-Riege, Stefan [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Evans, James E. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Segelke, Brent W. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Frank, Matthias [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2015-06-27

    X-ray free-electron lasers (XFELs) offer a new avenue to the structural probing of complex materials, including biomolecules. Delivery of precious sample to the XFEL beam is a key consideration, as the sample of interest must be serially replaced after each destructive pulse. The fixed-target approach to sample delivery involves depositing samples on a thin-film support and subsequent serial introduction via a translating stage. Some classes of biological materials, including two-dimensional protein crystals, must be introduced on fixed-target supports, as they require a flat surface to prevent sample wrinkling. A series of wafer and transmission electron microscopy (TEM)-style grid supports constructed of low-Z plastic have been custom-designed and produced. Aluminium TEM grid holders were engineered, capable of delivering up to 20 different conventional or plastic TEM grids using fixed-target stages available at the Linac Coherent Light Source (LCLS). As proof-of-principle, X-ray diffraction has been demonstrated from two-dimensional crystals of bacteriorhodopsin and three-dimensional crystals of anthrax toxin protective antigen mounted on these supports at the LCLS. In conclusion, the benefits and limitations of these low-Z fixed-target supports are discussed; it is the authors' belief that they represent a viable and efficient alternative to previously reported fixed-target supports for conducting diffraction studies with XFELs.

  14. The action of X-rays on plasmatic proteins (1962)

    International Nuclear Information System (INIS)

    Rinaldi, R.; Llory, J.; Suscillon, M.

    1962-01-01

    The agglomerate property of the human plasma on the erythrocytes is altered by X rays which is seen owing to an, increase of the sedimentation rate of red cells. When using this biological test: reversible agglomeration of the erythrocytes the authors tried to demonstrate the denaturation of the serum albumin under the action of the X rays. Further to this results we see that the doses of about 5. 10 5 roentgens denature completely the macromolecule of serum albumin. (authors) [fr

  15. High-resolution X-ray crystal structure of bovine H-protein using the high-pressure cryocooling method.

    Science.gov (United States)

    Higashiura, Akifumi; Ohta, Kazunori; Masaki, Mika; Sato, Masaru; Inaka, Koji; Tanaka, Hiroaki; Nakagawa, Atsushi

    2013-11-01

    Recently, many technical improvements in macromolecular X-ray crystallography have increased the number of structures deposited in the Protein Data Bank and improved the resolution limit of protein structures. Almost all high-resolution structures have been determined using a synchrotron radiation source in conjunction with cryocooling techniques, which are required in order to minimize radiation damage. However, optimization of cryoprotectant conditions is a time-consuming and difficult step. To overcome this problem, the high-pressure cryocooling method was developed (Kim et al., 2005) and successfully applied to many protein-structure analyses. In this report, using the high-pressure cryocooling method, the X-ray crystal structure of bovine H-protein was determined at 0.86 Å resolution. Structural comparisons between high- and ambient-pressure cryocooled crystals at ultra-high resolution illustrate the versatility of this technique. This is the first ultra-high-resolution X-ray structure obtained using the high-pressure cryocooling method.

  16. Conformational Flexibility of Proteins Involved in Ribosome Biogenesis: Investigations via Small Angle X-ray Scattering (SAXS

    Directory of Open Access Journals (Sweden)

    Dritan Siliqi

    2018-02-01

    Full Text Available The dynamism of proteins is central to their function, and several proteins have been described as flexible, as consisting of multiple domains joined by flexible linkers, and even as intrinsically disordered. Several techniques exist to study protein structures, but small angle X-ray scattering (SAXS has proven to be particularly powerful for the quantitative analysis of such flexible systems. In the present report, we have used SAXS in combination with X-ray crystallography to highlight their usefulness at characterizing flexible proteins, using as examples two proteins involved in different steps of ribosome biogenesis. The yeast BRCA2 and CDKN1A-interactig protein, Bcp1, is a chaperone for Rpl23 of unknown structure. We showed that it consists of a rigid, slightly elongated protein, with a secondary structure comprising a mixture of alpha helices and beta sheets. As an example of a flexible molecule, we studied the SBDS (Shwachman-Bodian-Diamond Syndrome protein that is involved in the cytoplasmic maturation of the 60S subunit and constitutes the mutated target in the Shwachman-Diamond Syndrome. In solution, this protein coexists in an ensemble of three main conformations, with the N- and C-terminal ends adopting different orientations with respect to the central domain. The structure observed in the protein crystal corresponds to an average of those predicted by the SAXS flexibility analysis.

  17. Direct methods in protein crystallography.

    Science.gov (United States)

    Karle, J

    1989-11-01

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

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

    Science.gov (United States)

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

    2012-10-08

    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.

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

    Directory of Open Access Journals (Sweden)

    Aaron J. Oakley

    2012-10-01

    Full Text Available 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.

  20. New carbocyclic nucleoside analogues with a bicyclo[2.2.1]heptane fragment as sugar moiety; synthesis, X-ray crystallography and anticancer activity.

    Science.gov (United States)

    Tănase, Constantin I; Drăghici, Constantin; Căproiu, Miron Teodor; Shova, Sergiu; Mathe, Christophe; Cocu, Florea G; Enache, Cristian; Maganu, Maria

    2014-01-01

    An amine group was synthesized starting from an optically active bicyclo[2.2.1]heptane compound, which was then used to build the 5 atoms ring of a key 6-chloropurine intermediate. This was then reacted with ammonia and selected amines obtaining new adenine- and 6-substituted adenine conformationally constrained carbocyclic nucleoside analogues with a bicyclo[2.2.1]heptane skeleton in the sugar moiety. X-ray crystallography confirmed an exo-coupling of base to the ring and a L configuration of the nucleoside analogues. The compounds were tested for anticancer activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Correlated single-crystal electronic absorption spectroscopy and X-ray crystallography at NSLS beamline X26-C

    International Nuclear Information System (INIS)

    Orville, A.M.; Buono, R.; Cowan, M.; Heroux, A.; Shea-McCarthy, G.; Schneider, D.K.; Skinner, J.M.; Skinner, M.J.; Stoner-Ma, D.; Sweet, R.M.

    2011-01-01

    The research philosophy and new capabilities installed at NSLS beamline X26-C to support electronic absorption and Raman spectroscopies coupled with X-ray diffraction are reviewed. This beamline is dedicated full time to multidisciplinary studies with goals that include revealing the relationship between the electronic and atomic structures in macromolecules. The beamline instrumentation has been fully integrated such that optical absorption spectra and X-ray diffraction images are interlaced. Therefore, optical changes induced by X-ray exposure can be correlated with X-ray diffraction data collection. The installation of Raman spectroscopy into the beamline is also briefly reviewed. Data are now routinely generated almost simultaneously from three complementary types of experiments from the same sample. The beamline is available now to the NSLS general user population.

  2. High-Resolution Detector For X-Ray Diffraction

    Science.gov (United States)

    Carter, Daniel C.; Withrow, William K.; Pusey, Marc L.; Yost, Vaughn H.

    1988-01-01

    Proposed x-ray-sensitive imaging detector offers superior spatial resolution, counting-rate capacity, and dynamic range. Instrument based on laser-stimulated luminescence and reusable x-ray-sensitive film. Detector scans x-ray film line by line. Extracts latent image in film and simultaneously erases film for reuse. Used primarily for protein crystallography. Principle adapted to imaging detectors for electron microscopy and fluorescence spectroscopy and general use in astronomy, engineering, and medicine.

  3. Characteristic Ligand-Induced Crystal Forms of HIV-1 Protease Complexes: A Novel Discovery of X-Ray Crystallography

    International Nuclear Information System (INIS)

    Olajuyigbe, Folasade M.; Geremia, Silvano

    2009-10-01

    Mixtures of saquinavir (SQV) and ritonavir (RTV) were cocrystallized with HIV-1 protease (PR) in an attempt to compare their relative potencies using a crystallographic approach and factors responsible for the respective crystal forms obtained were examined. The mixture ratio of the SQV/RTV was in the range of 1:1 to 1:50 with increasing concentration of dimethyl sulphoxide (DMSO) used. Two crystal forms of PR complexes were obtained. At concentrations of 0.8 and 1.2 % DMSO using 1:1 and 1:15 ratios of SQV/RTV, the crystal form was monoclinic while increasing the concentration of DMSO to 3.2 and 5.0% using 1:15 and 1:50 ratios of SQV/RTV, the orthorhombic crystal form was obtained. The high resolution X-ray crystal structures of the PR/ inhibitor complexes reveal that crystal forms with respective space groups are dependent on the occupancy of either SQV or RTV in the active site of the PR. The occupancy of either of the PR inhibitors in the active site of PR has interestingly demonstrated unique cooperativity effects in crystallization of protein-ligand complexes. The crystal forms obtained were also related to the concentration of DMSO and ammonium sulphate in crystallization, and storage conditions of purified PR. Surprisingly, the relative occupancies of these inhibitors in the active site suggested a competition between the two inhibitors which were not inhibition constants related. Analysis of the structures in both crystal forms show no difference in DMSO content but at higher concentration of DMSO (3.2 - 5.0%) in the orthorhombic crystal forms, there were protein-sulphate interactions which were absent in the monoclinic forms with lower concentration (0.8 - 1.2%) of DMSO. This work has clearly demonstrated that there is cooperativity in crystallization and the conditions of crystallization influence specific intermolecular contacts in crystal packing (crystal form). (author)

  4. A multiple CCD X-ray detector and its first operation with synchrotron radiation X-ray beam

    CERN Document Server

    Suzuki, M; Kumasaka, T; Sato, K; Toyokawa, H; Aries, I F; Jerram, P A; Ueki, T

    1999-01-01

    A 4x4 array structure of 16 identical CCD X-ray detector modules, called the multiple CCD X-ray detector system (MCCDX), was submitted to its first synchrotron radiation experiment at the protein crystallography station of the RIKEN beamline (BL45XU) at the SPring-8 facility. An X-ray diffraction pattern of cholesterol powder was specifically taken in order to investigate the overall system performance.

  5. Racemic & quasi-racemic protein crystallography enabled by chemical protein synthesis.

    Science.gov (United States)

    Kent, Stephen Bh

    2018-04-04

    A racemic protein mixture can be used to form centrosymmetric crystals for structure determination by X-ray diffraction. Both the unnatural d-protein and the corresponding natural l-protein are made by total chemical synthesis based on native chemical ligation-chemoselective condensation of unprotected synthetic peptide segments. Racemic protein crystallography is important for structure determination of the many natural protein molecules that are refractory to crystallization. Racemic mixtures facilitate the crystallization of recalcitrant proteins, and give diffraction-quality crystals. Quasi-racemic crystallization, using a single d-protein molecule, can facilitate the determination of the structures of a series of l-protein analog molecules. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. Clear as Crystal: The Story of the Braggs--How X-Ray Crystallography Has Contributed to Science

    Science.gov (United States)

    George, Robert; Patterson, John

    2014-01-01

    Here is a brief history of the work of two of Australia's most famous scientists, Sir William Bragg and his son Sir Lawrence Bragg. Jointly awarded the Nobel Prize in 1915 for their groundbreaking research into the use of X-rays to study the chemical structure and function of molecules, they have contributed to our heritage and to science at an…

  7. Small-angle X-ray scattering studies of thermally-induced globular protein gels

    International Nuclear Information System (INIS)

    Clark, A.H.; Tuffnell, C.D.

    1980-01-01

    Small-angle X-ray scattering has been applied to gels formed by heating globular proteins, in aqueous solution, above their unfolding temperatures. A number of BSA gels, previously characterised by electron microscopy, have been studied, and by setting up theoretical models for the scattering process, the X-ray data have been shown to be consistent with the microscope conclusions regarding network structure. It is concluded that the networks form by a linearly-directed aggregation of unfolded, disc-like, protein molecules, three-dimensional geometry being achieved by occasional branching, and/or cross-linking. Long-range inhomogeneities in network structure, easily observed by electron microscopy, and correlated with variations in pH or ionic strength, have an effect on X-ray scattering, and hence the X-ray method is sensitive not only to different network strand thicknesses, but to different degrees of uniformity as well. (author)

  8. What can we learn by computing 13Cα chemical shifts for X-ray protein models?

    International Nuclear Information System (INIS)

    Arnautova, Yelena A.; Vila, Jorge A.; Martin, Osvaldo A.; Scheraga, Harold A.

    2009-01-01

    The room-temperature X-ray structures of two proteins, solved at 1.8 and 1.9 Å resolution, are used to investigate whether a set of conformations, rather than a single X-ray structure, provides better agreement with both the X-ray data and the observed 13 C α chemical shifts in solution. The room-temperature X-ray structures of ubiquitin and of the RNA-binding domain of nonstructural protein 1 of influenza A virus solved at 1.8 and 1.9 Å resolution, respectively, were used to investigate whether a set of conformations rather than a single X-ray structure provides better agreement with both the X-ray data and the observed 13 C α chemical shifts in solution. For this purpose, a set of new conformations for each of these proteins was generated by fitting them to the experimental X-ray data deposited in the PDB. For each of the generated structures, which show R and R free factors similar to those of the deposited X-ray structure, the 13 C α chemical shifts of all residues in the sequence were computed at the DFT level of theory. The sets of conformations were then evaluated by their ability to reproduce the observed 13 C α chemical shifts by using the conformational average root-mean-square-deviation (ca-r.m.s.d.). For ubiquitin, the computed set of conformations is a better representation of the observed 13 C α chemical shifts in terms of the ca-r.m.s.d. than a single X-ray-derived structure. However, for the RNA-binding domain of nonstructural protein 1 of influenza A virus, consideration of an ensemble of conformations does not improve the agreement with the observed 13 C α chemical shifts. Whether an ensemble of conformations rather than any single structure is a more accurate representation of a protein structure in the crystal as well as of the observed 13 C α chemical shifts is determined by the dispersion of coordinates, in terms of the all-atom r.m.s.d. among the generated models; these generated models satisfy the experimental X-ray data with

  9. Characterization of Metalloproteins and Biomaterials by X-ray Absorption Spectroscopy and X-ray Diffraction

    DEFF Research Database (Denmark)

    Frankær, Christian Grundahl

    This thesis presents thework on combining complementary X-rays techniques for studying the structures of proteins and other biomaterials, and consists of three different projects: (i) Characterization of protein powders with X-ray powder diffraction (XRPD). (ii) The combination of X-ray...... crystallography and X-ray absorption spectroscopy (XAS) applied to studying different hexameric insulin conformations. (iii) The structures of polymorphs of strontium ranelate and the distribution of strontium in bone tissue. A procedure for fast identification and verification of protein powders using XRPD...... was correction for disordered bulk-solvent, but also correction for background and optimization of unit cell parameters have to be taken into account. A sample holder was designed for collecting powder diffraction data on a standard laboratory X-ray powder diffractometer. The background was reduced by use...

  10. Neutron protein crystallography in JAERI

    Indian Academy of Sciences (India)

    results have clearly suggested that hydrogen atoms and water molecules around ... resolution (from 1.5 Å to 2.0 Å) structural analyses of several proteins. .... system like PDB is not sufficient to study hydrogen-related studies such as hydrogen.

  11. Testing of gadolinium oxy-sulphide phosphors for use in CCD-based X-ray detectors for macromolecular crystallography

    CERN Document Server

    Pokric, M

    2002-01-01

    The resolution and detective quantum efficiency of CCD-based detectors used for X-ray diffraction is primarily affected by the layer of phosphor that converts incident X-ray photons into visible photons. The optimum thickness of this phosphor layer is strongly dependent on the fraction of absorbed incident X-ray photons and required spatial resolution. A range of terbium doped gadolinium oxy-sulphide (Gd sub 2 O sub 2 S : Tb) phosphor samples, provided by Applied Scintillation Technologies, have been evaluated for spatial resolution, light output and uniformity. The phosphor samples varied in coating weight (10-25 mg/cm sup 2), grain size (2.5, 4, 10 mu m), and applied coating (no coating, reflectors and absorbers). In addition, a non-uniform layer was introduced to some samples in order to provide an inherent diffusion layer. The experimental results showed that the introduction of a reflector increases the point spread function (PSF) and increases light yield up to 30%, while an absorber reduces the PSF tai...

  12. Protein folding and protein metallocluster studies using synchrotron small angler X-ray scattering

    International Nuclear Information System (INIS)

    Eliezer, D.

    1994-06-01

    Proteins, biological macromolecules composed of amino-acid building blocks, possess unique three dimensional shapes or conformations which are intimately related to their biological function. All of the information necessary to determine this conformation is stored in a protein's amino acid sequence. The problem of understanding the process by which nature maps protein amino-acid sequences to three-dimensional conformations is known as the protein folding problem, and is one of the central unsolved problems in biophysics today. The possible applications of a solution are broad, ranging from the elucidation of thousands of protein structures to the rational modification and design of protein-based drugs. The scattering of X-rays by matter has long been useful as a tool for the characterization of physical properties of materials, including biological samples. The high photon flux available at synchrotron X-ray sources allows for the measurement of scattering cross-sections of dilute and/or disordered samples. Such measurements do not yield the detailed geometrical information available from crystalline samples, but do allow for lower resolution studies of dynamical processes not observable in the crystalline state. The main focus of the work described here has been the study of the protein folding process using time-resolved small-angle x-ray scattering measurements. The original intention was to observe the decrease in overall size which must accompany the folding of a protein from an extended conformation to its compact native state. Although this process proved too fast for the current time-resolution of the technique, upper bounds were set on the probable compaction times of several small proteins. In addition, an interesting and unexpected process was detected, in which the folding protein passes through an intermediate state which shows a tendency to associate. This state is proposed to be a kinetic molten globule folding intermediate

  13. Structure of a 14-3-3 coordinated hexamer of the plant plasma membrane H+-ATPase by combining X-ray crystallography and electron cryomicroscopy

    NARCIS (Netherlands)

    Ottmann, C.; Marco, S.; Jaspert, N.; Marcon, C.; Schauer, N.; Weyand, M.; Vandermeeren, C.; Duby, G.; Boutry, M.; Wittinghofer, A.; Rigaud, J.-L.; Oecking, C.

    2007-01-01

    Regulatory 14-3-3 proteins activate the plant plasma membrane H+-ATPase by binding to its C-terminal autoinhibitory domain. This interaction requires phosphorylation of a C-terminal, mode III, recognition motif as well as an adjacent span of approximately 50 amino acids. Here we report the X-ray

  14. Preliminary small-angle X-ray scattering and X-ray diffraction studies of the BTB domain of lola protein from Drosophila melanogaster

    Science.gov (United States)

    Boyko, K. M.; Nikolaeva, A. Yu.; Kachalova, G. S.; Bonchuk, A. N.; Dorovatovskii, P. V.; Popov, V. O.

    2017-11-01

    The Drosophila genome has several dozens of transcription factors (TTK group) containing BTB domains assembled into octamers. The LOLA protein belongs to this family. The purification, crystallization, and preliminary X-ray diffraction and small-angle X-ray scattering (SAXS) studies of the BTB domain of this protein are reported. The crystallization conditions were found by the vapor-diffusion technique. A very low diffraction resolution (8.7 Å resolution) of the crystals was insufficient for the determination of the threedimensional structure of the BTB domain. The SAXS study demonstrated that the BTB domain of the LOLA protein exists as an octamer in solution.

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  16. Inhibition of d-xylose isomerase by polyols: atomic details by joint X-ray/neutron crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Kovalevsky, Andrey, E-mail: ayk@lanl.gov [Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States); Hanson, B. Leif [University of Toledo, 2801 West Bancroft Street, Toledo, OH 43606 (United States); Mason, Sax A. [Institut Laue–Langevin, 6 Rue Jules Horowitz, 38042 Grenoble (France); Forsyth, V. Trevor [Institut Laue–Langevin, 6 Rue Jules Horowitz, 38042 Grenoble (France); Keele University, Staffordshire (United Kingdom); Fisher, Zoe [Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States); Mustyakimov, Marat [Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States); Oak Ridge National Laboratory, PO Box 2008, MS 6475, Oak Ridge, TN 37831 (United States); Blakeley, Matthew P. [Institut Laue–Langevin, 6 Rue Jules Horowitz, 38042 Grenoble (France); Keen, David A. [Harwell Science and Innovation Campus, Didcot, Oxon OX11 0QX (United Kingdom); Langan, Paul [Oak Ridge National Laboratory, PO Box 2008, MS 6475, Oak Ridge, TN 37831 (United States); Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States)

    2012-09-01

    A joint X-ray/neutron structure of d-xylose isomerase in complex with the inhibitor sorbitol was determined at room temperature at an acidic pH of 5.9. Protonation of the O5 O atom of the sugar was directly observed in the nuclear density maps. Under acidic conditions sorbitol gains a water-mediated interaction with the enzyme active site, which may explain the increased potency of the inhibitor at low pH. d-Xylose isomerase (XI) converts the aldo-sugars xylose and glucose to their keto analogs xylulose and fructose, but is strongly inhibited by the polyols xylitol and sorbitol, especially at acidic pH. In order to understand the atomic details of polyol binding to the XI active site, a 2.0 Å resolution room-temperature joint X-ray/neutron structure of XI in complex with Ni{sup 2+} cofactors and sorbitol inhibitor at pH 5.9 and a room-temperature X-ray structure of XI containing Mg{sup 2+} ions and xylitol at the physiological pH of 7.7 were obtained. The protonation of oxygen O5 of the inhibitor, which was found to be deprotonated and negatively charged in previous structures of XI complexed with linear glucose and xylulose, was directly observed. The Ni{sup 2+} ions occupying the catalytic metal site (M2) were found at two locations, while Mg{sup 2+} in M2 is very mobile and has a high B factor. Under acidic conditions sorbitol gains a water-mediated interaction that connects its O1 hydroxyl to Asp257. This contact is not found in structures at basic pH. The new interaction that is formed may improve the binding of the inhibitor, providing an explanation for the increased affinity of the polyols for XI at low pH.

  17. Combining NMR and X-ray crystallography in fragment-based drug discovery: discovery of highly potent and selective BACE-1 inhibitors.

    Science.gov (United States)

    Wyss, Daniel F; Wang, Yu-Sen; Eaton, Hugh L; Strickland, Corey; Voigt, Johannes H; Zhu, Zhaoning; Stamford, Andrew W

    2012-01-01

    Fragment-based drug discovery (FBDD) has become increasingly popular over the last decade. We review here how we have used highly structure-driven fragment-based approaches to complement more traditional lead discovery to tackle high priority targets and those struggling for leads. Combining biomolecular nuclear magnetic resonance (NMR), X-ray crystallography, and molecular modeling with structure-assisted chemistry and innovative biology as an integrated approach for FBDD can solve very difficult problems, as illustrated in this chapter. Here, a successful FBDD campaign is described that has allowed the development of a clinical candidate for BACE-1, a challenging CNS drug target. Crucial to this achievement were the initial identification of a ligand-efficient isothiourea fragment through target-based NMR screening and the determination of its X-ray crystal structure in complex with BACE-1, which revealed an extensive H-bond network with the two active site aspartate residues. This detailed 3D structural information then enabled the design and validation of novel, chemically stable and accessible heterocyclic acylguanidines as aspartic acid protease inhibitor cores. Structure-assisted fragment hit-to-lead optimization yielded iminoheterocyclic BACE-1 inhibitors that possess desirable molecular properties as potential therapeutic agents to test the amyloid hypothesis of Alzheimer's disease in a clinical setting.

  18. X-ray repair cross complementing protein 1 in base excision repair

    DEFF Research Database (Denmark)

    Hanssen-Bauer, Audun; Solvang-Garten, Karin; Akbari, Mansour

    2012-01-01

    X-ray Repair Cross Complementing protein 1 (XRCC1) acts as a scaffolding protein in the converging base excision repair (BER) and single strand break repair (SSBR) pathways. XRCC1 also interacts with itself and rapidly accumulates at sites of DNA damage. XRCC1 can thus mediate the assembly of large...

  19. Development of an X-ray fluorescence holographic measurement system for protein crystals

    Energy Technology Data Exchange (ETDEWEB)

    Sato-Tomita, Ayana, E-mail: ayana.sato@jichi.ac.jp, E-mail: shibayam@jichi.ac.jp, E-mail: hayashi.koichi@nitech.ac.jp; Shibayama, Naoya, E-mail: ayana.sato@jichi.ac.jp, E-mail: shibayam@jichi.ac.jp, E-mail: hayashi.koichi@nitech.ac.jp; Okabe, Takahiro [Division of Biophysics, Department of Physiology, Jichi Medical University, Yakushiji, Shimotsuke 329-0498 (Japan); Happo, Naohisa [Department of Computer and Network Engineering, Graduate School of Information Sciences, Hiroshima City University, Asa-Minami-Ku, Hiroshima 731-3194 (Japan); Kimura, Koji [Department of Physical Science and Engineering, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555 (Japan); Matsushita, Tomohiro [Japan Synchrotron Radiation Research Institute (JASRI), SPring-8, Sayo, Hyogo 679-5198 (Japan); Park, Sam-Yong [Drug Design Laboratory, Department of Medical Life Science, Yokohama City University, Suehiro, Tsurumi, Yokohama 230-0045 (Japan); Sasaki, Yuji C. [Department of Advanced Material Science, Graduate School of Frontier Science, The University of Tokyo, Kashiwanoha, Kashiwa 277-8561 (Japan); Hayashi, Kouichi, E-mail: ayana.sato@jichi.ac.jp, E-mail: shibayam@jichi.ac.jp, E-mail: hayashi.koichi@nitech.ac.jp [Department of Physical Science and Engineering, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555 (Japan); Frontier Research Institute for Materials Science, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555 (Japan)

    2016-06-15

    Experimental procedure and setup for obtaining X-ray fluorescence hologram of crystalline metalloprotein samples are described. Human hemoglobin, an α{sub 2}β{sub 2} tetrameric metalloprotein containing the Fe(II) heme active-site in each chain, was chosen for this study because of its wealth of crystallographic data. A cold gas flow system was introduced to reduce X-ray radiation damage of protein crystals that are usually fragile and susceptible to damage. A χ-stage was installed to rotate the sample while avoiding intersection between the X-ray beam and the sample loop or holder, which is needed for supporting fragile protein crystals. Huge hemoglobin crystals (with a maximum size of 8 × 6 × 3 mm{sup 3}) were prepared and used to keep the footprint of the incident X-ray beam smaller than the sample size during the entire course of the measurement with the incident angle of 0°-70°. Under these experimental and data acquisition conditions, we achieved the first observation of the X-ray fluorescence hologram pattern from the protein crystals with minimal radiation damage, opening up a new and potential method for investigating the stereochemistry of the metal active-sites in biomacromolecules.

  20. Development of an X-ray fluorescence holographic measurement system for protein crystals

    International Nuclear Information System (INIS)

    Sato-Tomita, Ayana; Shibayama, Naoya; Okabe, Takahiro; Happo, Naohisa; Kimura, Koji; Matsushita, Tomohiro; Park, Sam-Yong; Sasaki, Yuji C.; Hayashi, Kouichi

    2016-01-01

    Experimental procedure and setup for obtaining X-ray fluorescence hologram of crystalline metalloprotein samples are described. Human hemoglobin, an α_2β_2 tetrameric metalloprotein containing the Fe(II) heme active-site in each chain, was chosen for this study because of its wealth of crystallographic data. A cold gas flow system was introduced to reduce X-ray radiation damage of protein crystals that are usually fragile and susceptible to damage. A χ-stage was installed to rotate the sample while avoiding intersection between the X-ray beam and the sample loop or holder, which is needed for supporting fragile protein crystals. Huge hemoglobin crystals (with a maximum size of 8 × 6 × 3 mm"3) were prepared and used to keep the footprint of the incident X-ray beam smaller than the sample size during the entire course of the measurement with the incident angle of 0°-70°. Under these experimental and data acquisition conditions, we achieved the first observation of the X-ray fluorescence hologram pattern from the protein crystals with minimal radiation damage, opening up a new and potential method for investigating the stereochemistry of the metal active-sites in biomacromolecules.

  1. A sample holder for in-house X-ray powder diffraction studies of protein powders

    DEFF Research Database (Denmark)

    Frankær, Christian Grundahl; Harris, Pernille; Ståhl, Kenny

    2011-01-01

    A sample holder for handling samples of protein for in-house X-ray powder diffraction (XRPD) analysis has been made and tested on lysozyme. The use of an integrated pinhole reduced the background, and good signal-to-noise ratios were obtained from only 7 l of sample, corresponding to approximatel...... 2-3 mg of dry protein. The sample holder is further adaptable to X-ray absorption spectroscopy (XAS) measurements. Both XRPD and XAS at the Zn K-edge were tested with hexameric Zn insulin....

  2. The charm of protein crystals--Structural biology at a glance in the International Year of Crystallography

    International Nuclear Information System (INIS)

    Su Xiaodong; Cao Qin

    2014-01-01

    Crystallography is a typical intellectual endeavor that has spanned human history for centuries. Through the persistent efforts of generations of scientists, crystallography has been transformed from a mathematical hypothesis to actual physical reality, mainly thanks to X-ray diffraction technology. 2014 is celebrated as the International Year of Crystallography (IYCr-2014), to commemorate that about 100 years ago, when Max von Laue in Germany and the father-and-son Braggs (William Henry Bragg and William Lawrence Bragg) in England pioneered the use of X-rays to determine the atomic structure of crystals; for this pioneering work they were awarded Nobel prizes for physics in the years of 1914 and 1915. This article is dedicated to the IYCr to describe the use of protein crystals, an application that has developed into protein crystallography and subsequently structural biology. In our overview of the history and future prospects of this field, we discuss in detail one example of caspase-6, to demonstrate how protein crystallography can help us understand the structure-function relationship of important proteins. (authors)

  3. Crystallization and preliminary X-ray analysis of the RAD protein from Antirrhinum majus

    International Nuclear Information System (INIS)

    Stevenson, Clare E. M.; Burton, Nicolas; Costa, Manuela; Nath, Utpal; Dixon, Ray A.; Coen, Enrico S.; Lawson, David M.

    2005-01-01

    An 8 kDa proteolytic fragment of the A. majus RADIALIS protein was crystallized and X-ray data were collected to 2 Å resolution. Crystals of the RADIALIS protein from Antirrhinum majus were grown by vapour diffusion after limited proteolysis. Mass spectrometry indicated that an 8 kDa fragment had been crystallized corresponding to the predicted MYB DNA-binding domain. X-ray data collected at room temperature were consistent with tetragonal symmetry, whereas data collected at 100 K using crystals cryoprotected by supplementing the mother liquor with ethylene glycol conformed to orthorhombic symmetry. It was subsequently shown that crystals soaked in cryoprotectants that were ‘osmolality-matched’ to the mother liquor retained tetragonal symmetry. Using these crystals, X-ray data were collected in-house to a maximum resolution of 2 Å

  4. Cyclic olefin homopolymer-based microfluidics for protein crystallization and in situ X-ray diffraction

    International Nuclear Information System (INIS)

    Emamzadah, Soheila; Petty, Tom J.; De Almeida, Victor; Nishimura, Taisuke; Joly, Jacques; Ferrer, Jean-Luc; Halazonetis, Thanos D.

    2009-01-01

    A cyclic olefin homopolymer-based microfluidics system has been established for protein crystallization and in situ X-ray diffraction. Microfluidics is a promising technology for the rapid identification of protein crystallization conditions. However, most of the existing systems utilize silicone elastomers as the chip material which, despite its many benefits, is highly permeable to water vapour. This limits the time available for protein crystallization to less than a week. Here, the use of a cyclic olefin homopolymer-based microfluidics system for protein crystallization and in situ X-ray diffraction is described. Liquid handling in this system is performed in 2 mm thin transparent cards which contain 500 chambers, each with a volume of 320 nl. Microbatch, vapour-diffusion and free-interface diffusion protocols for protein crystallization were implemented and crystals were obtained of a number of proteins, including chicken lysozyme, bovine trypsin, a human p53 protein containing both the DNA-binding and oligomerization domains bound to DNA and a functionally important domain of Arabidopsis Morpheus’ molecule 1 (MOM1). The latter two polypeptides have not been crystallized previously. For X-ray diffraction analysis, either the cards were opened to allow mounting of the crystals on loops or the crystals were exposed to X-rays in situ. For lysozyme, an entire X-ray diffraction data set at 1.5 Å resolution was collected without removing the crystal from the card. Thus, cyclic olefin homopolymer-based microfluidics systems have the potential to further automate protein crystallization and structural genomics efforts

  5. Femtosecond X-ray diffraction from two-dimensional protein crystals

    Directory of Open Access Journals (Sweden)

    Matthias Frank

    2014-03-01

    Full Text Available X-ray diffraction patterns from two-dimensional (2-D protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.

  6. Synthesis, spectroscopy, X-ray crystallography, DFT calculations, DNA binding and molecular docking of a propargyl arms containing Schiff base.

    Science.gov (United States)

    Balakrishnan, C; Subha, L; Neelakantan, M A; Mariappan, S S

    2015-11-05

    A propargyl arms containing Schiff base (L) was synthesized by the condensation of 1-[2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl]ethanone with trans-1,2-diaminocyclohexane. The structure of L was characterized by IR, (1)H NMR, (13)C NMR and UV-Vis spectroscopy and by single crystal X-ray diffraction analysis. The UV-Visible spectral behavior of L in different solvents exhibits positive solvatochromism. Density functional calculation of the L in gas phase was performed by using DFT (B3LYP) method with 6-31G basis set. The computed vibrational frequencies and NMR signals of L were compared with the experimental data. Tautomeric stability study inferred that the enolimine is more stable than the ketoamine form. The charge delocalization has been analyzed using natural bond orbital (NBO) analysis. Electronic absorption and emission spectral studies were used to study the binding of L with CT-DNA. The molecular docking was done to identify the interaction of L with A-DNA and B-DNA. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. X-CHIP: an integrated platform for high-throughput protein crystallization and on-the-chip X-ray diffraction data collection

    International Nuclear Information System (INIS)

    Kisselman, Gera; Qiu, Wei; Romanov, Vladimir; Thompson, Christine M.; Lam, Robert; Battaile, Kevin P.; Pai, Emil F.; Chirgadze, Nickolay Y.

    2011-01-01

    The X-CHIP (X-ray Crystallography High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process. The X-CHIP (X-ray Crystallization High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process. The system has been designed for crystallization condition screening, visual crystal inspection, initial X-ray screening and data collection in a high-throughput fashion. X-ray diffraction data acquisition can be performed directly on-the-chip at room temperature using an in situ approach. The capabilities of the chip eliminate the necessity for manual crystal handling and cryoprotection of crystal samples, while allowing data collection from multiple crystals in the same drop. This technology would be especially beneficial for projects with large volumes of data, such as protein-complex studies and fragment-based screening. The platform employs hydrophilic and hydrophobic concentric ring surfaces on a miniature plate transparent to visible light and X-rays to create a well defined and stable microbatch crystallization environment. The results of crystallization and data-collection experiments demonstrate that high-quality well diffracting crystals can be grown and high-resolution diffraction data sets can be collected using this technology. Furthermore, the quality of a single-wavelength anomalous dispersion data set collected with the X-CHIP at room temperature was sufficient to generate interpretable electron-density maps. This technology is highly resource-efficient owing to the use of nanolitre-scale drop volumes. It does not require any modification for most in-house and synchrotron beamline systems and offers

  8. X-CHIP: an integrated platform for high-throughput protein crystallization and on-the-chip X-ray diffraction data collection

    Energy Technology Data Exchange (ETDEWEB)

    Kisselman, Gera; Qiu, Wei; Romanov, Vladimir; Thompson, Christine M.; Lam, Robert [Ontario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, Ontario M5G 2C4 (Canada); Battaile, Kevin P. [Argonne National Laboratory, Argonne, Illinois 60439 (United States); Pai, Emil F.; Chirgadze, Nickolay Y., E-mail: nchirgad@uhnresearch.ca [Ontario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, Ontario M5G 2C4 (Canada); University of Toronto, Toronto, Ontario M5S 1A8 (Canada)

    2011-06-01

    The X-CHIP (X-ray Crystallography High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process. The X-CHIP (X-ray Crystallization High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process. The system has been designed for crystallization condition screening, visual crystal inspection, initial X-ray screening and data collection in a high-throughput fashion. X-ray diffraction data acquisition can be performed directly on-the-chip at room temperature using an in situ approach. The capabilities of the chip eliminate the necessity for manual crystal handling and cryoprotection of crystal samples, while allowing data collection from multiple crystals in the same drop. This technology would be especially beneficial for projects with large volumes of data, such as protein-complex studies and fragment-based screening. The platform employs hydrophilic and hydrophobic concentric ring surfaces on a miniature plate transparent to visible light and X-rays to create a well defined and stable microbatch crystallization environment. The results of crystallization and data-collection experiments demonstrate that high-quality well diffracting crystals can be grown and high-resolution diffraction data sets can be collected using this technology. Furthermore, the quality of a single-wavelength anomalous dispersion data set collected with the X-CHIP at room temperature was sufficient to generate interpretable electron-density maps. This technology is highly resource-efficient owing to the use of nanolitre-scale drop volumes. It does not require any modification for most in-house and synchrotron beamline systems and offers

  9. Development of the protein crystallography by synchrotron radiation

    International Nuclear Information System (INIS)

    Yamamoto, Masaki

    2014-01-01

    Since crystal structure determination of the first protein by Kendrew in 1959, protein crystallography developed into the leading role of the protein structure study by various technology developments. Especially the utilization of synchrotron radiation from the 1990s brought innovative progress of protein crystallography on the data quality and the phasing method and had expanded the samples targets including membrane proteins and suprarmolecular complexes. Here I give the outline of the history and the future prospects of the protein crystallography from the role of synchrotron radiation. (author)

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

    International Nuclear Information System (INIS)

    Westbrook, E. M.

    1999-01-01

    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

  11. X-ray crystallography and QM/MM investigation on the oligosaccharide synthesis mechanism of rice BGlu1 glycosynthases.

    Science.gov (United States)

    Wang, Jinhu; Pengthaisong, Salila; Cairns, James R Ketudat; Liu, Yongjun

    2013-02-01

    Nucleophile mutants of retaining β-glycosidase can act as glycosynthases to efficiently catalyze the synthesis of oligosaccharides. Previous studies proved that rice BGlu1 mutants E386G, E386S and E386A catalyze the oligosaccharide synthesis with different rates. The E386G mutant gave the fastest transglucosylation rate, which was approximately 3- and 19-fold faster than those of E386S and E386A. To account for the differences of their activities, in this paper, the X-ray crystal structures of BGlu1 mutants E386S and E386A were solved and compared with that of E386G mutant. However, they show quite similar active sites, which implies that their activities cannot be elucidated from the crystal structures alone. Therefore, a combined quantum mechanical/molecular mechanical (QM/MM) calculations were further performed. Our calculations reveal that the catalytic reaction follows a single-step mechanism, i.e., the extraction of proton by the acid/base, E176, and the formation of glycosidic bond are concerted. The energy barriers are calculated to be 19.9, 21.5 and 21.9kcal/mol for the mutants of E386G, E386S and E386A, respectively, which is consistent with the order of their experimental relative activities. But based on the calculated activation energies, 1.1kcal/mol energy difference may translate to nearly 100 fold rate difference. Although the rate limiting step in these mutants has not been established, considering the size of the product and the nature of the active site, it is likely that the product release, rather than chemistry, is rate limiting in these oligosaccharides synthesis catalyzed by BGlu1 mutants. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Structure-Based Design: Synthesis, X-ray Crystallography, and Biological Evaluation of N-Substituted-4-Hydroxy-2-Quinolone-3-Carboxamides as Potential Cytotoxic Agents.

    Science.gov (United States)

    Sabbah, Dima A; Hishmah, Bayan; Sweidan, Kamal; Bardaweel, Sanaa; AlDamen, Murad; Zhong, Haizhen A; Abu Khalaf, Reema; Hasan Ibrahim, Ameerah; Al-Qirim, Tariq; Abu Sheikha, Ghassan; Mubarak, Mohammad S

    2018-01-01

    Oncogenic potential of phosphatidylinositol 3-kinase (PI3Kα) has been highlighted as a therapeutic target for anticancer drug design. Target compounds were designed to address the effect of different substitution patterns at the N atom of the carboxamide moiety on the bioactivity of this series. Synthesis of the targeted compounds, crystallography, biological evaluation tests against human colon carcinoma (HCT-116), and Glide docking studies. A new series of N-substituted- 4-hydroxy-2-quinolone-3-carboxamides was prepared and characterized by means of FT-IR, 1H and 13C NMR, and elemental analysis. In addition, the identity of the core nucleus 5 was successfully characterized with the aid of X-ray crystallography. Biological activity of prepared compounds was investigated in vitro against human colon carcinoma (HCT-116) cell line. Results revealed that these compounds inhibit cell proliferation and induce apoptosis through an increase in caspase-3 activity and a decrease in DNA cellular content. Compounds 7, 14, and 17 which have H-bond acceptor moiety on p-position displayed promising PI3Kα inhibitory activity. On the other hand, derivatives tailored with bulky and hydrophobic motifs (16 and 18) on o- and m-positions exhibited moderate activity. Molecular docking studies against PI3Kα and caspase-3 showed an agreement between the predicted binding affinity (ΔGobsd) and IC50 values of the derivatives for the caspase-3 model. Furthermore, Glide docking studies against PI3Kα demonstrated that the newly synthesized compounds accommodate PI3Kα kinase catalytic domain and form H-bonding with key binding residues. The series exhibited a potential PI3Kα inhibitory activity in HCT-116 cell line. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  13. Localization of proteins and nucleic acids using soft x-ray microscopy

    International Nuclear Information System (INIS)

    Larabell, Carolyn A.; Yager, Deborah; Meyer-Ilse, Werner

    2000-01-01

    The high-resolution soft x-ray microscope (XM-1) at the Advanced Light Source was used to examine whole, hydrated mammalian cells, both chemically fixed and rapidly frozen and viewed in a cryostage. Using x-ray microscopy, high contrast information about the organization of the cytoplasm and nucleus of these cells was revealed at unsurpassed resolution. It is important to note that cryo-fixed cells have been examined in a state that most closely resembles their natural environment in that the cells were not exposed to chemical fixatives or chemical contrast enhancement reagents. We also used the power of soft x-ray microscopy to examine the localization of proteins and nucleic acids in whole, hydrated cells using silver-enhanced, immunogold labeling techniques. With this approach, we have obtained information about the distribution of such molecules with respect to cellular ultrastructure at five times better resolution than light microscopy. The power of soft x-ray microscopy to provide superb resolution information about the subcellular localization of proteins and nucleic acids places it in a commanding position to contribute to our understanding of the numerous molecules being identified through modern molecular biology techniques

  14. Imaging plate, a new type of x-ray area detector

    International Nuclear Information System (INIS)

    Kamiya, Nobuo; Amemiya, Yoshiyuki; Miyahara, Junji.

    1986-01-01

    In respective fields of X-ray crystallography, for the purpose of the efficient collection of reciprocal space information, two-dimensional X-ray detectors such as multiwire proportional chambers and X-ray television sets have been used together with conventional X-ray films. X-ray films are characterized by uniform sensitivity and high positional resolution over a wide area, but the sensitivity is low, and the range of action and the linearity of the sensitivity is problematic. They require the development process, accordingly lack promptitude. The MWPCs and X-ray television sets are superior in the sensitivity, its linearity, the range of action and promptitude, but interior in the uniformity and resolution to the films. Imaging plate is a new X-ray area detector developed by Fuji Photo Film Co., Ltd., for digital X-ray medical image diagnosis. This detector is superior in all the above mentioned performances, and it seems very useful also for X-ray crystallography. In this paper, the system composed of an imaging plate and its reader is described, and the basic performance as an X-ray area detector and the results of having recorded the diffraction images of protein crystals as the example of applying it to X-ray crystallography are reported. The imaging plate is that the crystalline fluorescent powder of BaFBr doped with Eu 2+ ions is applied on plastic films. (Kako, I.)

  15. Exploring the atomic structure and conformational flexibility of a 320 Å long engineered viral fiber using X-ray crystallography.

    Science.gov (United States)

    Bhardwaj, Anshul; Casjens, Sherwood R; Cingolani, Gino

    2014-02-01

    Protein fibers are widespread in nature, but only a limited number of high-resolution structures have been determined experimentally. Unlike globular proteins, fibers are usually recalcitrant to form three-dimensional crystals, preventing single-crystal X-ray diffraction analysis. In the absence of three-dimensional crystals, X-ray fiber diffraction is a powerful tool to determine the internal symmetry of a fiber, but it rarely yields atomic resolution structural information on complex protein fibers. An 85-residue-long minimal coiled-coil repeat unit (MiCRU) was previously identified in the trimeric helical core of tail needle gp26, a fibrous protein emanating from the tail apparatus of the bacteriophage P22 virion. Here, evidence is provided that an MiCRU can be inserted in frame inside the gp26 helical core to generate a rationally extended fiber (gp26-2M) which, like gp26, retains a trimeric quaternary structure in solution. The 2.7 Å resolution crystal structure of this engineered fiber, which measures ∼320 Å in length and is only 20-35 Å wide, was determined. This structure, the longest for a trimeric protein fiber to be determined to such a high resolution, reveals the architecture of 22 consecutive trimerization heptads and provides a framework to decipher the structural determinants for protein fiber assembly, stability and flexibility.

  16. Gold nanoparticles enhance the X-ray-induced degradation of human centrin 2 protein

    Energy Technology Data Exchange (ETDEWEB)

    Brun, Emilie [Laboratoire de Chimie Physique, CNRS UMR 8000, Universite Paris-Sud 11, Bat. 350, 91405 Orsay Cedex (France); Duchambon, Patricia; Blouquit, Yves [INSERM U759, Imagerie Integrative, Campus Universitaire d' Orsay, Bat. 112, Institut Curie, Centre de Recherche, Laboratoire R. Latarjet, Campus Universitaire d' Orsay, 91405 Orsay Cedex (France); Keller, Gerard [UMR CNRS 8612, Physico-Chimie-Pharmacotechnie-Biopharmacie, Universite Paris 11, Faculte de Pharmacie, 5 rue Jean-Baptiste Clement, 92296 Chatenay-Malabry (France); Sanche, Leon [Groupe en Sciences des Radiations, Departement de Medecine Nucleaire et Radiobiologie, Faculte de Medecine, Universite de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4 (Canada); Sicard-Roselli, Cecile [Laboratoire de Chimie Physique, CNRS UMR 8000, Universite Paris-Sud 11, Bat. 350, 91405 Orsay Cedex (France)], E-mail: cecile.sicard@u-psud.fr

    2009-03-15

    In the war against cancer, radiotherapy is a prominent tool but counterbalanced by the fact that it also induces damages in healthy tissues. Nanotechnologies could open a new possibility to decrease these side effects. In particular, gold nanoparticles (GNPs) could be used as radio-sensitizers. As the role of proteins in the processes leading to cell death cannot be neglected, their radio-sensitization by GNPs is of great interest. This is particularly true in the case of the human centrin 2 protein, which has been proposed to be involved in DNA repair processes. To investigate this effect, we quantified for the first time the degradation of this protein in a gold colloidal solution when submitted to X-rays. We showed that the X-ray-induced degradation of the human centrin 2 protein is enhanced 1.5-fold in the presence of GNPs, even though no covalent bond exists between protein and GNPs. Among the conditions tested, the maximum enhancement was found with the higher GNP:protein ratio of 2x10{sup -4} and with the higher X-ray energy of 49 keV.

  17. Gold nanoparticles enhance the X-ray-induced degradation of human centrin 2 protein

    International Nuclear Information System (INIS)

    Brun, Emilie; Duchambon, Patricia; Blouquit, Yves; Keller, Gerard; Sanche, Leon; Sicard-Roselli, Cecile

    2009-01-01

    In the war against cancer, radiotherapy is a prominent tool but counterbalanced by the fact that it also induces damages in healthy tissues. Nanotechnologies could open a new possibility to decrease these side effects. In particular, gold nanoparticles (GNPs) could be used as radio-sensitizers. As the role of proteins in the processes leading to cell death cannot be neglected, their radio-sensitization by GNPs is of great interest. This is particularly true in the case of the human centrin 2 protein, which has been proposed to be involved in DNA repair processes. To investigate this effect, we quantified for the first time the degradation of this protein in a gold colloidal solution when submitted to X-rays. We showed that the X-ray-induced degradation of the human centrin 2 protein is enhanced 1.5-fold in the presence of GNPs, even though no covalent bond exists between protein and GNPs. Among the conditions tested, the maximum enhancement was found with the higher GNP:protein ratio of 2x10 -4 and with the higher X-ray energy of 49 keV

  18. Catalytic Mechanism of Nitrile Hydratase Proposed by Time-resolved X-ray Crystallography Using a Novel Substrate, tert-Butylisonitrile*S⃞

    Science.gov (United States)

    Hashimoto, Koichi; Suzuki, Hiroyuki; Taniguchi, Kayoko; Noguchi, Takumi; Yohda, Masafumi; Odaka, Masafumi

    2008-01-01

    Nitrile hydratases (NHases) have an unusual iron or cobalt catalytic center with two oxidized cysteine ligands, cysteine-sulfinic acid and cysteine-sulfenic acid, catalyzing the hydration of nitriles to amides. Recently, we found that the NHase of Rhodococcus erythropolis N771 exhibited an additional catalytic activity, converting tert-butylisonitrile (tBuNC) to tert-butylamine. Taking advantage of the slow reactivity of tBuNC and the photoreactivity of nitrosylated NHase, we present the first structural evidence for the catalytic mechanism of NHase with time-resolved x-ray crystallography. By monitoring the reaction with attenuated total reflectance-Fourier transform infrared spectroscopy, the product from the isonitrile carbon was identified as a CO molecule. Crystals of nitrosylated inactive NHase were soaked with tBuNC. The catalytic reaction was initiated by photo-induced denitrosylation and stopped by flash cooling. tBuNC was first trapped at the hydrophobic pocket above the iron center and then coordinated to the iron ion at 120 min. At 440 min, the electron density of tBuNC was significantly altered, and a new electron density was observed near the isonitrile carbon as well as the sulfenate oxygen of αCys114. These results demonstrate that the substrate was coordinated to the iron and then attacked by a solvent molecule activated by αCys114-SOH. PMID:18948265

  19. Synthesis, characterization, X-ray crystallography, acetyl cholinesterase inhibition and antioxidant activities of some novel ketone derivatives of gallic hydrazide-derived Schiff bases.

    Science.gov (United States)

    Gwaram, Nura Suleiman; Ali, Hapipah Mohd; Abdulla, Mahmood Ameen; Buckle, Michael J C; Sukumaran, Sri Devi; Chung, Lip Yong; Othman, Rozana; Alhadi, Abeer A; Yehye, Wageeh A; Hadi, A Hamid A; Hassandarvish, Pouya; Khaledi, Hamid; Abdelwahab, Siddig Ibrahim

    2012-02-28

    Alzheimer's disease (AD) is the most common form of dementia among older people and the pathogenesis of this disease is associated with oxidative stress. Acetylcholinesterase inhibitors with antioxidant activities are considered potential treatments for AD. Some novel ketone derivatives of gallic hydrazide-derived Schiff bases were synthesized and examined for their antioxidant activities and in vitro and in silico acetyl cholinesterase inhibition. The compounds were characterized using spectroscopy and X-ray crystallography. The ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays revealed that all the compounds have strong antioxidant activities. N-(1-(5-bromo-2-hydroxyphenyl)-ethylidene)-3,4,5-trihydroxybenzohydrazide (2) was the most potent inhibitor of human acetyl cholinesterase, giving an inhibition rate of 77% at 100 μM. Molecular docking simulation of the ligand-enzyme complex suggested that the ligand may be positioned in the enzyme's active-site gorge, interacting with residues in the peripheral anionic subsite (PAS) and acyl binding pocket (ABP). The current work warrants further preclinical studies to assess the potential for these novel compounds for the treatment of AD.

  20. Implementing an X-ray validation pipeline for the Protein Data Bank

    International Nuclear Information System (INIS)

    Gore, Swanand; Velankar, Sameer; Kleywegt, Gerard J.

    2012-01-01

    The implementation of a validation pipeline, based on community recommendations, for future depositions of X-ray crystal structures in the Protein Data Bank is described. There is an increasing realisation that the quality of the biomacromolecular structures deposited in the Protein Data Bank (PDB) archive needs to be assessed critically using established and powerful validation methods. The Worldwide Protein Data Bank (wwPDB) organization has convened several Validation Task Forces (VTFs) to advise on the methods and standards that should be used to validate all of the entries already in the PDB as well as all structures that will be deposited in the future. The recommendations of the X-ray VTF are currently being implemented in a software pipeline. Here, ongoing work on this pipeline is briefly described as well as ways in which validation-related information could be presented to users of structural data

  1. Implementing an X-ray validation pipeline for the Protein Data Bank

    Energy Technology Data Exchange (ETDEWEB)

    Gore, Swanand; Velankar, Sameer; Kleywegt, Gerard J., E-mail: gerard@ebi.ac.uk [EMBL–EBI, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD (United Kingdom)

    2012-04-01

    The implementation of a validation pipeline, based on community recommendations, for future depositions of X-ray crystal structures in the Protein Data Bank is described. There is an increasing realisation that the quality of the biomacromolecular structures deposited in the Protein Data Bank (PDB) archive needs to be assessed critically using established and powerful validation methods. The Worldwide Protein Data Bank (wwPDB) organization has convened several Validation Task Forces (VTFs) to advise on the methods and standards that should be used to validate all of the entries already in the PDB as well as all structures that will be deposited in the future. The recommendations of the X-ray VTF are currently being implemented in a software pipeline. Here, ongoing work on this pipeline is briefly described as well as ways in which validation-related information could be presented to users of structural data.

  2. The structural chemistry of metallocorroles: combined X-ray crystallography and quantum chemistry studies afford unique insights.

    Science.gov (United States)

    Thomas, Kolle E; Alemayehu, Abraham B; Conradie, Jeanet; Beavers, Christine M; Ghosh, Abhik

    2012-08-21

    Although they share some superficial structural similarities with porphyrins, corroles, trianionic ligands with contracted cores, give rise to fundamentally different transition metal complexes in comparison with the dianionic porphyrins. Many metallocorroles are formally high-valent, although a good fraction of them are also noninnocent, with significant corrole radical character. These electronic-structural characteristics result in a variety of fascinating spectroscopic behavior, including highly characteristic, paramagnetically shifted NMR spectra and textbook cases of charge-transfer spectra. Although our early research on corroles focused on spectroscopy, we soon learned that the geometric structures of metallocorroles provide a fascinating window into their electronic-structural characteristics. Thus, we used X-ray structure determinations and quantum chemical studies, chiefly using DFT, to obtain a comprehensive understanding of metallocorrole geometric and electronic structures. This Account describes our studies of the structural chemistry of metallocorroles. At first blush, the planar or mildly domed structure of metallocorroles might appear somewhat uninteresting particularly when compared to metalloporphyrins. Metalloporphyrins, especially sterically hindered ones, are routinely ruffled or saddled, but the missing meso carbon apparently makes the corrole skeleton much more resistant to nonplanar distortions. Ruffling, where the pyrrole rings are alternately twisted about the M-N bonds, is energetically impossible for metallocorroles. Saddling is also uncommon; thus, a number of sterically hindered, fully substituted metallocorroles exhibit almost perfectly planar macrocycle cores. Against this backdrop, copper corroles stand out as an important exception. As a result of an energetically favorable Cu(d(x2-y2))-corrole(π) orbital interaction, copper corroles, even sterically unhindered ones, are inherently saddled. Sterically hindered substituents

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

    International Nuclear Information System (INIS)

    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.

    2004-01-01

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

    Science.gov (United States)

    MacDowell, Alastair A; Celestre, Rich 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, Tom; Berger, James M; Agard, David A; Padmore, Howard A

    2004-11-01

    At the Advanced Light Source, three protein crystallography beamlines have been built that use as a source one of the three 6 T 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, owing 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. Structure of liposome encapsulating proteins characterized by X-ray scattering and shell-modeling

    International Nuclear Information System (INIS)

    Hirai, Mitsuhiro; Kimura, Ryota; Takeuchi, Kazuki; Hagiwara, Yoshihiko; Kawai-Hirai, Rika; Ohta, Noboru; Igarashi, Noriyuki; Shimuzu, Nobutaka

    2013-01-01

    Wide-angle X-ray scattering data using a third-generation synchrotron radiation source are presented. Lipid liposomes are promising drug delivery systems because they have superior curative effects owing to their high adaptability to a living body. Lipid liposomes encapsulating proteins were constructed and the structures examined using synchrotron radiation small- and wide-angle X-ray scattering (SR-SWAXS). The liposomes were prepared by a sequential combination of natural swelling, ultrasonic dispersion, freeze-throw, extrusion and spin-filtration. The liposomes were composed of acidic glycosphingolipid (ganglioside), cholesterol and phospholipids. By using shell-modeling methods, the asymmetric bilayer structure of the liposome and the encapsulation efficiency of proteins were determined. As well as other analytical techniques, SR-SWAXS and shell-modeling methods are shown to be a powerful tool for characterizing in situ structures of lipid liposomes as an important candidate of drug delivery systems

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

    International Nuclear Information System (INIS)

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

    2004-01-01

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

  8. 1,4,8,11-Tetra[2-aryl-1-diazenyl]-1,4,8,11-tetraazacyclotetradecanes - synthesis, characterization, and x-ray crystallography of the first tetrakistriazenes to be reported

    Energy Technology Data Exchange (ETDEWEB)

    Clarke, J.D.; Vaughan, K. [Dept. of Chemistry, Saint Mary' s Univ., Halifax, Nova Scotia (Canada)], E-mail: keith.vaughan@smu.ca; Bertolasi, V. [Dipartimento di Chimica and Centro di Strutturistica Diffrattometrica, Universita' di Ferrara, Ferrara (Italy)

    2006-10-15

    The reactions of a series of arene diazonium salts with 1,4,8,11-tetraazacyclotetradecane (cyclam) afford the novel compounds, the 1,4,8,11-tetra[2-aryl-1-diazenyl]-1,4,8,11-tetraazacyclotetradecanes (1a-1f), which are the first examples of tetrakistriazenes to be reported. The tetrakistriazenes were characterized by IR spectroscopy, proton and carbon NMR, elemental analysis, high resolution electrospray mass spectrometry, and X-ray crystallography. The analogous reaction of a diazonium salt with 1,4,7-triazacyclononane or 1,5,9-triazacyclododecane yields the tristriazenes 2, 3a, and 3b. The structures of compounds 1c and 1e were solved by X-ray crystallography at low temperature (150 K). Both molecules display a conformation where the four phenyltriazenyl groups point alternately upwards and downwards with respect to the mean macrocyclic plane. (author)

  9. 1,4,8,11-Tetra[2-aryl-1-diazenyl]-1,4,8,11-tetraazacyclotetradecanes - synthesis, characterization, and x-ray crystallography of the first tetrakistriazenes to be reported

    International Nuclear Information System (INIS)

    Clarke, J.D.; Vaughan, K.; Bertolasi, V.

    2006-01-01

    The reactions of a series of arene diazonium salts with 1,4,8,11-tetraazacyclotetradecane (cyclam) afford the novel compounds, the 1,4,8,11-tetra[2-aryl-1-diazenyl]-1,4,8,11-tetraazacyclotetradecanes (1a-1f), which are the first examples of tetrakistriazenes to be reported. The tetrakistriazenes were characterized by IR spectroscopy, proton and carbon NMR, elemental analysis, high resolution electrospray mass spectrometry, and X-ray crystallography. The analogous reaction of a diazonium salt with 1,4,7-triazacyclononane or 1,5,9-triazacyclododecane yields the tristriazenes 2, 3a, and 3b. The structures of compounds 1c and 1e were solved by X-ray crystallography at low temperature (150 K). Both molecules display a conformation where the four phenyltriazenyl groups point alternately upwards and downwards with respect to the mean macrocyclic plane. (author)

  10. The use of Co2+ for crystallization and structure determination, using a conventional monochromatic X-ray source, of flax rust avirulence protein

    International Nuclear Information System (INIS)

    Gunčar, Gregor; Wang, Ching-I A.; Forwood, Jade K.; Teh, Trazel; Catanzariti, Ann-Maree; Ellis, Jeffrey G.; Dodds, Peter N.; Kobe, Boštjan

    2007-01-01

    It is demonstrated that anomalous diffraction based on the signal from a cobalt ion measured on a conventional monochromatic X-ray source can be used to determine the structure of a protein with a novel fold (M. lini avirulence protein AvrL567-A). The approach could be applicable to many metal-binding proteins, particularly when synchrotron radiation is not readily available. Metal-binding sites are ubiquitous in proteins and can be readily utilized for phasing. It is shown that a protein crystal structure can be solved using single-wavelength anomalous diffraction based on the anomalous signal of a cobalt ion measured on a conventional monochromatic X-ray source. The unique absorption edge of cobalt (1.61 Å) is compatible with the Cu Kα wavelength (1.54 Å) commonly available in macromolecular crystallography laboratories. This approach was applied to the determination of the structure of Melampsora lini avirulence protein AvrL567-A, a protein with a novel fold from the fungal pathogen flax rust that induces plant disease resistance in flax plants. This approach using cobalt ions may be applicable to all cobalt-binding proteins and may be advantageous when synchrotron radiation is not readily available

  11. Visualizing a protein quake with time-resolved X-ray scattering at a free-electron laser

    DEFF Research Database (Denmark)

    Arnlund, David; Johansson, Linda C.; Wickstrand, Cecilia

    2014-01-01

    We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast glob...

  12. Cloning, purification, crystallization and preliminary X-ray studies of flagellar hook scaffolding protein FlgD from Pseudomonas aeruginosa PAO1

    International Nuclear Information System (INIS)

    Luo, Miao; Niu, Siqiang; Yin, Yibing; Huang, Ailong; Wang, Deqiang

    2009-01-01

    In order to better elucidate the functions of FlgD in flagellar hook biosynthesis, the three-dimensional structure of FlgD is being determined by X-ray crystallography. Here, the expression, purification, crystallization and preliminary crystallographic analysis of FlgD from P. aeruginosa are reported. FlgD regulates the assembly of the hook cap structure to prevent leakage of hook monomers into the medium and hook monomer polymerization and also plays a role in determination of the correct hook length, with the help of the FliK protein. In order to better elucidate the functions of FlgD in flagellar hook biosynthesis, the three-dimensional structure of FlgD is being determined by X-ray crystallography. Here, the expression, purification, crystallization and preliminary crystallographic analysis of FlgD from P. aeruginosa are reported. The crystal belonged to space group I222 and diffracted to a resolution of 2.5 Å, with unit-cell parameters a = 116.47, b = 118.71, c = 118.85 Å. The crystals are most likely to contain three molecules in the asymmetric unit, with a V M value of 2.73 Å 3 Da −1

  13. CCD-based X-ray detectors for X-ray diffraction studies

    International Nuclear Information System (INIS)

    Ito, K.; Amemiya, Y.

    1999-01-01

    CCD-based X-ray detectors are getting to be used for X-ray diffraction studies especially in the studies where real time (automated) measurements and time-resolved measurements are required. Principles and designs of two typical types of CCD-based detectors are described; one is ths system in which x-ray image intensifiers are coupled to maximize the detective quantum efficiency for time-resolved measurements, and the other is the system in which tapered optical fibers are coupled for the reduction of the image into the CCD, which is optimized for automated measurements for protein crystallography. These CCD-based X-ray detectors have an image distortion and non-uniformity of response to be corrected by software. Correction schemes which we have developed are also described. (author)

  14. Resolution of a protein sequence ambiguity by X-ray crystallographic and mass spectrometric methods

    International Nuclear Information System (INIS)

    Keefe, L.J.; Lattman, E.E.; Wolkow, C.; Woods, A.; Chevrier, M.; Cotter, R.J.

    1992-01-01

    Ambiguities in amino acid sequences are a potential problem in X-ray crystallographic studies of proteins. Amino acid side chains often cannot be reliably identified from the electron density. Many protein crystal structures that are now being solved are simple variants of a known wild-type structure. Thus, cloning artifacts or other untoward events can readily lead to cases in which the proposed sequence is not correct. An example is presented showing that mass spectrometry provides an excellent tool for analyzing suspected errors. The X-ray crystal structure of an insertion mutant of Staphylococcal nuclease has been solved to 1.67 A resolution and refined to a crystallographic R value of 0.170. A single residue has been inserted in the C-terminal α helix. The inserted amino acid was believed to be an alanine residue, but the final electron density maps strongly indicated that a glycine had been inserted instead. To confirm the observations from the X-ray data, matrix-assisted laser desorption mass spectrometry was employed to verify the glycine insertion. This mass spectrometric technique has sufficient mass accuracy to detect the methyl group that distinguishes glycine from alanine and can be extended to the more common situation in which crystallographic measurements suggest a problem with the sequence, but cannot pinpoint its location or nature. (orig.)

  15. Resolution of a protein sequence ambiguity by X-ray crystallographic and mass spectrometric methods

    Energy Technology Data Exchange (ETDEWEB)

    Keefe, L.J.; Lattman, E.E. (Dept. of Biophysics and Biophysical Chemistry, Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States)); Wolkow, C.; Woods, A.; Chevrier, M.; Cotter, R.J. (Middle Atlantic Mass Spectrometry Lab., Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States))

    1992-04-01

    Ambiguities in amino acid sequences are a potential problem in X-ray crystallographic studies of proteins. Amino acid side chains often cannot be reliably identified from the electron density. Many protein crystal structures that are now being solved are simple variants of a known wild-type structure. Thus, cloning artifacts or other untoward events can readily lead to cases in which the proposed sequence is not correct. An example is presented showing that mass spectrometry provides an excellent tool for analyzing suspected errors. The X-ray crystal structure of an insertion mutant of Staphylococcal nuclease has been solved to 1.67 A resolution and refined to a crystallographic R value of 0.170. A single residue has been inserted in the C-terminal {alpha} helix. The inserted amino acid was believed to be an alanine residue, but the final electron density maps strongly indicated that a glycine had been inserted instead. To confirm the observations from the X-ray data, matrix-assisted laser desorption mass spectrometry was employed to verify the glycine insertion. This mass spectrometric technique has sufficient mass accuracy to detect the methyl group that distinguishes glycine from alanine and can be extended to the more common situation in which crystallographic measurements suggest a problem with the sequence, but cannot pinpoint its location or nature. (orig.).

  16. Macromolecular crystallography using synchrotron radiation

    International Nuclear Information System (INIS)

    Bartunik, H.D.; Phillips, J.C.; Fourme, R.

    1982-01-01

    The use of synchrotron X-ray sources in macromolecular crystallography is described. The properties of synchrotron radiation relevant to macromolecular crystallography are examined. The applications discussed include anomalous dispersion techniques, the acquisition of normal and high resolution data, and kinetic studies of structural changes in macromolecules; protein data are presented illustrating these applications. The apparatus used is described including information on the electronic detectors, the monitoring of the incident beam and crystal cooling. (U.K.)

  17. Measurements of accurate x-ray scattering data of protein solutions using small stationary sample cells

    Science.gov (United States)

    Hong, Xinguo; Hao, Quan

    2009-01-01

    In this paper, we report a method of precise in situ x-ray scattering measurements on protein solutions using small stationary sample cells. Although reduction in the radiation damage induced by intense synchrotron radiation sources is indispensable for the correct interpretation of scattering data, there is still a lack of effective methods to overcome radiation-induced aggregation and extract scattering profiles free from chemical or structural damage. It is found that radiation-induced aggregation mainly begins on the surface of the sample cell and grows along the beam path; the diameter of the damaged region is comparable to the x-ray beam size. Radiation-induced aggregation can be effectively avoided by using a two-dimensional scan (2D mode), with an interval as small as 1.5 times the beam size, at low temperature (e.g., 4 °C). A radiation sensitive protein, bovine hemoglobin, was used to test the method. A standard deviation of less than 5% in the small angle region was observed from a series of nine spectra recorded in 2D mode, in contrast to the intensity variation seen using the conventional stationary technique, which can exceed 100%. Wide-angle x-ray scattering data were collected at a standard macromolecular diffraction station using the same data collection protocol and showed a good signal/noise ratio (better than the reported data on the same protein using a flow cell). The results indicate that this method is an effective approach for obtaining precise measurements of protein solution scattering.

  18. Measurements of accurate x-ray scattering data of protein solutions using small stationary sample cells

    International Nuclear Information System (INIS)

    Hong Xinguo; Hao Quan

    2009-01-01

    In this paper, we report a method of precise in situ x-ray scattering measurements on protein solutions using small stationary sample cells. Although reduction in the radiation damage induced by intense synchrotron radiation sources is indispensable for the correct interpretation of scattering data, there is still a lack of effective methods to overcome radiation-induced aggregation and extract scattering profiles free from chemical or structural damage. It is found that radiation-induced aggregation mainly begins on the surface of the sample cell and grows along the beam path; the diameter of the damaged region is comparable to the x-ray beam size. Radiation-induced aggregation can be effectively avoided by using a two-dimensional scan (2D mode), with an interval as small as 1.5 times the beam size, at low temperature (e.g., 4 deg. C). A radiation sensitive protein, bovine hemoglobin, was used to test the method. A standard deviation of less than 5% in the small angle region was observed from a series of nine spectra recorded in 2D mode, in contrast to the intensity variation seen using the conventional stationary technique, which can exceed 100%. Wide-angle x-ray scattering data were collected at a standard macromolecular diffraction station using the same data collection protocol and showed a good signal/noise ratio (better than the reported data on the same protein using a flow cell). The results indicate that this method is an effective approach for obtaining precise measurements of protein solution scattering.

  19. Studies of protein structure in solution and protein folding using synchrotron small-angle x-ray scattering

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Lingling [Stanford Univ., CA (United States)

    1996-04-01

    Synchrotron small angle x-ray scattering (SAXS) has been applied to the structural study of several biological systems, including the nitrogenase complex, the heat shock cognate protein (hsc70), and lysozyme folding. The structural information revealed from the SAXS experiments is complementary to information obtained by other physical and biochemical methods, and adds to our knowledge and understanding of these systems.

  20. Imaging metals in proteins by combining electrophoresis with rapid x-ray fluorescence mapping

    International Nuclear Information System (INIS)

    Finney, L.; Chishti, Y.; Khare, T.; Giometti, C.; Levina, A.; Lay, P.A.; Vogt, S.

    2010-01-01

    Growing evidence points toward a very dynamic role for metals in biology. This suggests that physiological circumstance may mandate metal ion redistribution among ligands. This work addresses a critical need for technology that detects, identifies, and measures the metal-containing components of complex biological matrixes. We describe a direct, user-friendly approach for identifying and quantifying metal?protein adducts in complex samples using native- or SDS-PAGE, blotting, and rapid synchrotron X-ray fluorescence mapping with micro-XANES (X-ray absorption near-edge structure) of entire blots. The identification and quantification of each metal bound to a protein spot has been demonstrated, and the technique has been applied in two exemplary cases. In the first, the speciation of the in vitro binding of exogenous chromium to blood serum proteins was influenced markedly by both the oxidation state of chromium exposed to the serum proteins and the treatment conditions, which is of relevance to the biochemistry of Cr dietary supplements. In the second case, in vivo changes in endogenous metal speciation were examined to probe the influence of oxygen depletion on iron speciation in Shewanella oneidensis.

  1. In vacuo X-ray data collection from graphene-wrapped protein crystals

    International Nuclear Information System (INIS)

    Warren, Anna J.; Crawshaw, Adam D.; Trincao, Jose; Aller, Pierre; Alcock, Simon; Nistea, Ioana; Salgado, Paula S.; Evans, Gwyndaf

    2015-01-01

    A method is reported for collecting room-temperature data from protein crystals under vacuum by protecting them with a thin graphene layer. The measurement of diffraction data from macromolecular crystal samples held in vacuo holds the promise of a very low X-ray background and zero absorption of incident and scattered beams, leading to better data and the potential for accessing very long X-ray wavelengths (>3 Å) for native sulfur phasing. Maintaining the hydration of protein crystals under vacuum is achieved by the use of liquid jets, as with serial data collection at free-electron lasers, or is side-stepped by cryocooling the samples, as implemented at new synchrotron beamlines. Graphene has been shown to protect crystals from dehydration by creating an extremely thin layer that is impermeable to any exchanges with the environment. Furthermore, owing to its hydrophobicity, most of the aqueous solution surrounding the crystal is excluded during sample preparation, thus eliminating most of the background caused by liquid. Here, it is shown that high-quality data can be recorded at room temperature from graphene-wrapped protein crystals in a rough vacuum. Furthermore, it was observed that graphene protects crystals exposed to different relative humidities and a chemically harsh environment

  2. In vacuo X-ray data collection from graphene-wrapped protein crystals

    Energy Technology Data Exchange (ETDEWEB)

    Warren, Anna J. [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Crawshaw, Adam D. [Newcastle University, Newcastle upon Tyne NE2 4HH (United Kingdom); Trincao, Jose; Aller, Pierre; Alcock, Simon; Nistea, Ioana [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Salgado, Paula S. [Newcastle University, Newcastle upon Tyne NE2 4HH (United Kingdom); Evans, Gwyndaf, E-mail: gwyndaf.evans@diamond.ac.uk [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom)

    2015-09-26

    A method is reported for collecting room-temperature data from protein crystals under vacuum by protecting them with a thin graphene layer. The measurement of diffraction data from macromolecular crystal samples held in vacuo holds the promise of a very low X-ray background and zero absorption of incident and scattered beams, leading to better data and the potential for accessing very long X-ray wavelengths (>3 Å) for native sulfur phasing. Maintaining the hydration of protein crystals under vacuum is achieved by the use of liquid jets, as with serial data collection at free-electron lasers, or is side-stepped by cryocooling the samples, as implemented at new synchrotron beamlines. Graphene has been shown to protect crystals from dehydration by creating an extremely thin layer that is impermeable to any exchanges with the environment. Furthermore, owing to its hydrophobicity, most of the aqueous solution surrounding the crystal is excluded during sample preparation, thus eliminating most of the background caused by liquid. Here, it is shown that high-quality data can be recorded at room temperature from graphene-wrapped protein crystals in a rough vacuum. Furthermore, it was observed that graphene protects crystals exposed to different relative humidities and a chemically harsh environment.

  3. Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array

    Energy Technology Data Exchange (ETDEWEB)

    Lyubimov, Artem Y. [Stanford University, Stanford, CA 94305 (United States); Stanford University, Stanford, CA 94305 (United States); Stanford University, Stanford, CA 94305 (United States); Stanford University, Stanford, CA 94305 (United States); Stanford University, Stanford, CA 94305 (United States); Murray, Thomas D. [University of California, Berkeley, CA 94720 (United States); Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Koehl, Antoine [Stanford University, Stanford, CA 94305 (United States); Araci, Ismail Emre [Stanford University, Stanford, CA 94305 (United States); Stanford University, Stanford, CA 94305 (United States); Uervirojnangkoorn, Monarin; Zeldin, Oliver B. [Stanford University, Stanford, CA 94305 (United States); Stanford University, Stanford, CA 94305 (United States); Stanford University, Stanford, CA 94305 (United States); Stanford University, Stanford, CA 94305 (United States); Stanford University, Stanford, CA 94305 (United States); Cohen, Aina E.; Soltis, S. Michael; Baxter, Elizabeth L. [SLAC National Accelerator Laboratory, Stanford, CA 94305 (United States); Brewster, Aaron S.; Sauter, Nicholas K. [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Brunger, Axel T., E-mail: brunger@stanford.edu [Stanford University, Stanford, CA 94305 (United States); Stanford University, Stanford, CA 94305 (United States); Stanford University, Stanford, CA 94305 (United States); Stanford University, Stanford, CA 94305 (United States); Stanford University, Stanford, CA 94305 (United States); Berger, James M., E-mail: brunger@stanford.edu [Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Stanford University, Stanford, CA 94305 (United States)

    2015-04-01

    A microfluidic platform has been developed for the capture and X-ray analysis of protein microcrystals, affording a means to improve the efficiency of XFEL and synchrotron experiments. X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat for conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.

  4. X-ray Excited Optical Fluorescence and Diffraction Imaging of Reactivity and Crystallinity in a Zeolite Crystal : Crystallography and Molecular Spectroscopy in One

    NARCIS (Netherlands)

    Ristanovic, Zoran; Hofmann, Jan P; Richard, Marie-Ingrid; Jiang, Tao; Chahine, Gilbert A; Schülli, Tobias U; Meirer, Florian; Weckhuysen, Bert M

    2016-01-01

    Structure-activity relationships in heterogeneous catalysis are challenging to be measured on a single-particle level. For the first time, one X-ray beam is used to determine the crystallographic structure and reactivity of a single zeolite crystal. The method generates μm-resolved X-ray diffraction

  5. Electronic damage in S atoms in a native protein crystal induced by an intense X-ray free-electron laser pulse

    Directory of Open Access Journals (Sweden)

    L. Galli

    2015-07-01

    Full Text Available Current hard X-ray free-electron laser (XFEL sources can deliver doses to biological macromolecules well exceeding 1 GGy, in timescales of a few tens of femtoseconds. During the pulse, photoionization can reach the point of saturation in which certain atomic species in the sample lose most of their electrons. This electronic radiation damage causes the atomic scattering factors to change, affecting, in particular, the heavy atoms, due to their higher photoabsorption cross sections. Here, it is shown that experimental serial femtosecond crystallography data collected with an extremely bright XFEL source exhibit a reduction of the effective scattering power of the sulfur atoms in a native protein. Quantitative methods are developed to retrieve information on the effective ionization of the damaged atomic species from experimental data, and the implications of utilizing new phasing methods which can take advantage of this localized radiation damage are discussed.

  6. The small angle x-ray scattering of globular proteins in solution during heat denaturation

    Science.gov (United States)

    Banuelos, Jose; Urquidi, Jacob

    2008-10-01

    The ability of proteins to change their conformation in response to changes in their environment has consequences in biological processes like metabolism, chemical regulation in cells, and is believed to play a role in the onset of several neurodegenerative diseases. Factors such as a change in temperature, pressure, and the introduction of ions into the aqueous environment of a protein can give rise to the folding/unfolding of a protein. As a protein unfolds, the ratio of nonpolar to polar groups exposed to water changes, affecting a protein's thermodynamic properties. Using small angle x-ray scattering (SAXS), we are currently studying the intermediate protein conformations that arise during the folding/unfolding process as a function of temperature for five globular proteins. Trends in the observed intermediate structures of these globular proteins, along with correlations with data on protein thermodynamics may help elucidate shared characteristics between all proteins in the folding/unfolding process. Experimental design considerations will be discussed and preliminary results for some of these systems will be presented.

  7. Implementing an X-ray validation pipeline for the Protein Data Bank.

    Science.gov (United States)

    Gore, Swanand; Velankar, Sameer; Kleywegt, Gerard J

    2012-04-01

    There is an increasing realisation that the quality of the biomacromolecular structures deposited in the Protein Data Bank (PDB) archive needs to be assessed critically using established and powerful validation methods. The Worldwide Protein Data Bank (wwPDB) organization has convened several Validation Task Forces (VTFs) to advise on the methods and standards that should be used to validate all of the entries already in the PDB as well as all structures that will be deposited in the future. The recommendations of the X-ray VTF are currently being implemented in a software pipeline. Here, ongoing work on this pipeline is briefly described as well as ways in which validation-related information could be presented to users of structural data.

  8. Crystallization and initial X-ray analysis of polyhydroxyalkanoate granule-associated protein from Aeromonas hydrophila

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Minglian; Li, Zhenguo; Zheng, Wei; Lou, Zhiyong [MOE Key Laboratory of Protein Science, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084 (China); Chen, Guo-Qiang, E-mail: chengq@stu.edu.cn [MOE Key Laboratory of Protein Science, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084 (China); Multidisciplinary Research Center, Shantou University, Shantou 515063, Guangdong (China)

    2006-08-01

    The phasin PhaP{sub Ah} from A. hydrophila strain 4AK4 was crystallized using the hanging-drop vapour-diffusion method. Polyhydroxyalkanoate (PHA) granule-associated proteins (phasins) were discovered in PHA-accumulating bacteria. They play a crucial role as a structural protein during initial PHA-granule formation and granule growth and also serve as interfaces for granule stabilization in vivo. The phasin PhaP{sub Ah} from Aeromonas hydrophila strain 4AK4 was crystallized using the hanging-drop vapour-diffusion method. Single crystals were cryocooled for X-ray diffraction analysis. The phasin crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 80.8, b = 108.9, c = 134.4 Å.

  9. Induced variation in protein mutants after multiple EMS and X-ray treatments

    International Nuclear Information System (INIS)

    Walther, H.; Seibold, K.H.

    1978-01-01

    Results from two experiments with spring barley in the M 3 and M 8 generations gave information on the efficiency of selected mutants, improved in protein yield by different mutagenic treatments. A selection rate of 1-2% was found to be realistic according to a 5% significance level. However, differences between mutagenic treatments with EMS and X-rays and between varieties were found to be not only incidental. To evaluate the results within a protein mutation breeding programme a bivariate selection model was applied and was found to allow clear decisions on mutagenic improvements in protein production, measured in g protein/m 2 . When substituting protein yield in g/seed for protein yield in g/m 2 in the early generations, all relations to protein and grain yield in g/m 2 were found to be low and negative. We conclude that this substituted selection character can be of only limited aid. But very high positive correlations exist between protein yield/m 2 , lysine yield/m 2 and grain yield/m 2 , which means that these selection characters would render a more reliable basis for selection in early generations. (author)

  10. Deficiency in plasma protein synthesis caused by x-ray-induced lethal albino alleles in mouse

    International Nuclear Information System (INIS)

    Garland, R.C.; Satrustegui, J.; Gluecksohn-Waelsch, S.; Cori, C.F.

    1976-01-01

    Plasma protein synthesis was studied in mice bearing x-ray induced lethal mutations at the albino locus. Newborn albino mutants showed a decrease in each of the three principal plasma proteins, albumin, α-fetoprotein, and transferrin, when compared with colored littermate controls. Incorporation of [ 14 C] leucine into plasma proteins of the newborn albinos 30 min after injection was only 1 / 5 that of the controls, but incorporation into total liver protein was only slightly diminished. Incorporation of [ 14 C] leucine into an albumin fraction obtained by immunoprecipitation from livers incubated in vitro in an amino acid mixture was also strongly diminished. Thus, the liver of 18-day-old albino fetuses incorporated into this fraction 1 / 3 and that of newborn albinos 1 / 8 as much as the controls, but in both cases the incorporation into total liver protein was only 25 percent less than in the respective controls. These results indicate that the rather severe structural abnormalities observed in the mutants in the endoplasmic reticulum and the Golgi apparatus are not associated with a general deficiency of hepatic protein synthesis. Instead the data from this and previous work show that the progressive deficiency from fetal life to birth involves certain specific proteins represented by several perinatally developing enzymes and by plasma proteins. It is suggested that the mutational effects observed in these mice are due to deletions involving regulatory rather than structural genes at or near the albino locus

  11. Effect of caffeine on the expression of a major X-ray induced protein in human tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Hughes, E.N.; Boothman, D.A. (Univ. of Pennsylvania School of Medicine, Philadelphia (USA))

    1991-03-01

    We have examined the effect of caffeine on the concomitant processes of the repair of potentially lethal damage (PLD) and the synthesis of X-ray-induced proteins in the human malignant melanoma cell line, Ul-Mel. Caffeine administered at a dose of 5mM after X radiation not only inhibited PLD repair but also markedly reduced the level of XIP269, a major X-ray-induced protein whose expression has been shown to correlate with the capacity to repair PLD. The expression of the vast majority of other cellular proteins, including seven other X-ray-induced proteins, remained unchanged following caffeine treatment. A possible role for XIP269 in cell cycle delay following DNA damage by X irradiation is discussed.

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

    Science.gov (United States)

    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. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Preliminary X-ray crystallographic studies of yeast mitochondrial protein Tom70p

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Yunkun [Department of Cell Biology, Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States); McCombs, Debbie; Nagy, Lisa; DeLucas, Lawrence [Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States); Sha, Bingdong, E-mail: bdsha@uab.edu [Department of Cell Biology, Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States)

    2006-03-01

    Tom70p is an important translocase of the outer membrane complex member and a major surface receptor of the protein-translocation machinery in the outer mitochondrial membrane. To investigate the mechanism by which Tom70p functions to deliver the mitochondrial protein precursors, the cytosolic fragment of yeast Tom70p (cTom70p) has been crystallized. Protein translocations across mitochondrial membranes play critical roles in mitochondrion biogenesis. Protein transport from the cell cytosol to the mitochondrial matrix is carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tom70p is an important TOM-complex member and a major surface receptor of the protein-translocation machinery in the outer mitochondrial membrane. To investigate the mechanism by which Tom70p functions to deliver the mitochondrial protein precursors, the cytosolic fragment of yeast Tom70p (cTom70p) was crystallized. The crystals diffract to 3.2 Å using a synchrotron X-ray source and belong to space group P2{sub 1}, with unit-cell parameters a = 44.89, b = 168.78, c = 83.41 Å, α = 90.00, β = 102.74, γ = 90.00°. There are two Tom70p molecules in one asymmetric unit, which corresponds to a solvent content of approximately 51%. Structure determination by MAD methods is under way.

  14. X-ray data processing

    OpenAIRE

    Powell, Harold R.

    2017-01-01

    The method of molecular structure determination by X-ray crystallography is a little over a century old. The history is described briefly, along with developments in X-ray sources and detectors. The fundamental processes involved in measuring diffraction patterns on area detectors, i.e. autoindexing, refining crystal and detector parameters, integrating the reflections themselves and putting the resultant measurements on to a common scale are discussed, with particular reference to the most c...

  15. Cloning, Expression, Purification, Crystallization and Preliminary X-ray Analysis of Mycoplasma Genitalium Protein MG289

    Energy Technology Data Exchange (ETDEWEB)

    Sippel, K.; Boehlein, S; Sakai, Y; Quirit, J; Agbandje-McKenna, M; Rosser, C; McKenna, R

    2009-01-01

    Mycoplasma genitalium is a human pathogen that is associated with nongonococcal urethritis in men and cervicitis in women. The cloning, expression, purification and crystallization of the protein MG289 from M. genitalium strain G37 are reported here. Crystals of MG289 diffracted X-rays to 2.8 {angstrom} resolution. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 49.7, b = 90.9, c = 176.1 {angstrom}. The diffraction data after processing had an overall R{sub merge} of 8.7%. The crystal structure of Cypl, the ortholog of MG289 from M. hyorhinis, has recently been determined, providing a reasonable phasing model; molecular replacement is currently under way.

  16. Role of X-ray-inducible genes and proteins in adaptive survival responses

    International Nuclear Information System (INIS)

    Meyers, M.; Schea, R.A.; Petrowski, A.E.; Seabury, H.; McLaughlin, P.W.; Lee, I.; Lee, S.W.; Boothman, D.A.

    1992-01-01

    Certain X-ray-inducible genes and their corresponding protein products, appearing following low priming doses of ionizing radiation may subsequently give rise to an adaptive survival response, ultimately leading to increased radioresistance. Further, this adaptive radioresistance may be due to increased DNA repair (or misrepair) processes. Ultimately, the function of low-dose-induced cDNA clones within the cell is hoped to elucidate to follow the effects of specific gene turn-off on adaptive responses. Future research must determine the various functions of adaptive response gene products so that the beneficial or deleterious consequences of adaptive responses, which increases resistance to ionizing radiation, can be determined. (author). 19 refs., 1 fig

  17. Preparation, crystallization and preliminary X-ray analysis of YjcG protein from Bacillus subtilis

    International Nuclear Information System (INIS)

    Li, Dan; Chan, Chiomui; Liang, Yu-He; Zheng, Xiaofeng; Li, Lanfen; Su, Xiao-Dong

    2005-01-01

    B. subtilis YjcG protein was expressed, purified and crystallized. A complete diffraction data set was collected at BSRF beamline 3W1A and processed to 2.3 Å resolution. Bacillus subtilis YjcG is a functionally uncharacterized protein with 171 residues that has no structural homologue in the Protein Data Bank. However, it shows sequence homology to bacterial and archaeal 2′–5′ RNA ligases. In order to identify its exact function via structural studies, the yjcG gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET21-DEST. The protein was expressed in a soluble form in Escherichia coli and was purified to homogeneity. Crystals suitable for X-ray analysis were obtained that diffracted to 2.3 Å and belonged to space group C2, with unit-cell parameters a = 99.66, b = 73.93, c = 61.77 Å, β = 113.56°

  18. Calculation of accurate small angle X-ray scattering curves from coarse-grained protein models

    Directory of Open Access Journals (Sweden)

    Stovgaard Kasper

    2010-08-01

    Full Text Available Abstract Background Genome sequencing projects have expanded the gap between the amount of known protein sequences and structures. The limitations of current high resolution structure determination methods make it unlikely that this gap will disappear in the near future. Small angle X-ray scattering (SAXS is an established low resolution method for routinely determining the structure of proteins in solution. The purpose of this study is to develop a method for the efficient calculation of accurate SAXS curves from coarse-grained protein models. Such a method can for example be used to construct a likelihood function, which is paramount for structure determination based on statistical inference. Results We present a method for the efficient calculation of accurate SAXS curves based on the Debye formula and a set of scattering form factors for dummy atom representations of amino acids. Such a method avoids the computationally costly iteration over all atoms. We estimated the form factors using generated data from a set of high quality protein structures. No ad hoc scaling or correction factors are applied in the calculation of the curves. Two coarse-grained representations of protein structure were investigated; two scattering bodies per amino acid led to significantly better results than a single scattering body. Conclusion We show that the obtained point estimates allow the calculation of accurate SAXS curves from coarse-grained protein models. The resulting curves are on par with the current state-of-the-art program CRYSOL, which requires full atomic detail. Our method was also comparable to CRYSOL in recognizing native structures among native-like decoys. As a proof-of-concept, we combined the coarse-grained Debye calculation with a previously described probabilistic model of protein structure, TorusDBN. This resulted in a significant improvement in the decoy recognition performance. In conclusion, the presented method shows great promise for

  19. A new small-angle X-ray scattering set-up on the crystallography beamline I711 at MAX-lab

    DEFF Research Database (Denmark)

    Knaapila, M.; Svensson, C.; Barauskas, J.

    2009-01-01

    A small-angle X-ray scattering (SAXS) set-up has recently been developed at beamline I711 at the MAX II storage ring in Lund (Sweden). An overview of the required modifications is presented here together with a number of application examples. The accessible q range in a SAXS experiment is 0.009-0...

  20. A Versatile System for High-Throughput In Situ X-ray Screening and Data Collection of Soluble and Membrane-Protein Crystals

    Energy Technology Data Exchange (ETDEWEB)

    Broecker, Jana; Klingel, Viviane; Ou, Wei-Lin; Balo, Aidin R.; Kissick, David J.; Ogata, Craig M.; Kuo, Anling; Ernst, Oliver P.

    2016-10-12

    In recent years, in situ data collection has been a major focus of progress in protein crystallography. Here, we introduce the Mylar in situ method using Mylar-based sandwich plates that are inexpensive, easy to make and handle, and show significantly less background scattering than other setups. A variety of cognate holders for patches of Mylar in situ sandwich films corresponding to one or more wells makes the method robust and versatile, allows for storage and shipping of entire wells, and enables automated crystal imaging, screening, and goniometerbased X-ray diffraction data-collection at room temperature and under cryogenic conditions for soluble and membrane-protein crystals grown in or transferred to these plates. We validated the Mylar in situ method using crystals of the water-soluble proteins hen egg-white lysozyme and sperm whale myoglobin as well as the 7-transmembrane protein bacteriorhodopsin from Haloquadratum walsbyi. In conjunction with current developments at synchrotrons, this approach promises high-resolution structural studies of membrane proteins to become faster and more routine.

  1. Protein preparation, crystallization and preliminary X-ray analysis of Trypanosoma cruzi nucleoside diphosphate kinase 1

    International Nuclear Information System (INIS)

    Gómez Barroso, J. A.; Pereira, H.; Miranda, M.; Pereira, C.; Garratt, R. C.; Aguilar, C. F.

    2010-01-01

    T. cruzi TcNDPK1 was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl 2 , 20% PEG 3350. Data were collected to 3.5 Å resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 Å. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease

  2. X-ray diffraction analysis and in vitro characterization of the UAM2 protein from Oryza sativa

    DEFF Research Database (Denmark)

    Welner, Ditte Hededam; Tsai, Alex Yi-Lin; DeGiovanni, Andy M.

    2017-01-01

    protein from Oryza sativa (OsUAM2) were undertaken. Here, X-ray diffraction data are reported, as well as analysis of the oligomeric state in the crystal and in solution. OsUAM2 crystallizes readily but forms highly radiation-sensitive crystals with limited diffraction power, requiring careful low......, a requirement for a structural metal ion....

  3. Unique structure and dynamics of the EphA5 ligand binding domain mediate its binding specificity as revealed by X-ray crystallography, NMR and MD simulations.

    Directory of Open Access Journals (Sweden)

    Xuelu Huan

    Full Text Available The 16 EphA and EphB receptors represent the largest family of receptor tyrosine kinases, and their interactions with 9 ephrin-A and ephrin-B ligands initiate bidirectional signals controlling many physiological and pathological processes. Most interactions occur between receptor and ephrins of the same class, and only EphA4 can bind all A and B ephrins. To understand the structural and dynamic principles that enable Eph receptors to utilize the same jellyroll β-sandwich fold to bind ephrins, the VAPB-MSP domain, peptides and small molecules, we have used crystallography, NMR and molecular dynamics (MD simulations to determine the first structure and dynamics of the EphA5 ligand-binding domain (LBD, which only binds ephrin-A ligands. Unexpectedly, despite being unbound, the high affinity ephrin-binding pocket of EphA5 resembles that of other Eph receptors bound to ephrins, with a helical conformation over the J-K loop and an open pocket. The openness of the pocket is further supported by NMR hydrogen/deuterium exchange data and MD simulations. Additionally, the EphA5 LBD undergoes significant picosecond-nanosecond conformational exchanges over the loops, as revealed by NMR and MD simulations, but lacks global conformational exchanges on the microsecond-millisecond time scale. This is markedly different from the EphA4 LBD, which shares 74% sequence identity and 87% homology. Consequently, the unbound EphA5 LBD appears to comprise an ensemble of open conformations that have only small variations over the loops and appear ready to bind ephrin-A ligands. These findings show how two proteins with high sequence homology and structural similarity are still able to achieve distinctive binding specificities through different dynamics, which may represent a general mechanism whereby the same protein fold can serve for different functions. Our findings also suggest that a promising strategy to design agonists/antagonists with high affinity and selectivity

  4. Protein structure determination by single-wavelength anomalous diffraction phasing of X-ray free-electron laser data

    Directory of Open Access Journals (Sweden)

    Karol Nass

    2016-05-01

    Full Text Available Serial femtosecond crystallography (SFX at X-ray free-electron lasers (XFELs offers unprecedented possibilities for macromolecular structure determination of systems that are prone to radiation damage. However, phasing XFEL data de novo is complicated by the inherent inaccuracy of SFX data, and only a few successful examples, mostly based on exceedingly strong anomalous or isomorphous difference signals, have been reported. Here, it is shown that SFX data from thaumatin microcrystals can be successfully phased using only the weak anomalous scattering from the endogenous S atoms. Moreover, a step-by-step investigation is presented of the particular problems of SAD phasing of SFX data, analysing data from a derivative with a strong anomalous signal as well as the weak signal from endogenous S atoms.

  5. Protein NMR Structures Refined with Rosetta Have Higher Accuracy Relative to Corresponding X-ray Crystal Structures

    Science.gov (United States)

    2014-01-01

    We have found that refinement of protein NMR structures using Rosetta with experimental NMR restraints yields more accurate protein NMR structures than those that have been deposited in the PDB using standard refinement protocols. Using 40 pairs of NMR and X-ray crystal structures determined by the Northeast Structural Genomics Consortium, for proteins ranging in size from 5–22 kDa, restrained Rosetta refined structures fit better to the raw experimental data, are in better agreement with their X-ray counterparts, and have better phasing power compared to conventionally determined NMR structures. For 37 proteins for which NMR ensembles were available and which had similar structures in solution and in the crystal, all of the restrained Rosetta refined NMR structures were sufficiently accurate to be used for solving the corresponding X-ray crystal structures by molecular replacement. The protocol for restrained refinement of protein NMR structures was also compared with restrained CS-Rosetta calculations. For proteins smaller than 10 kDa, restrained CS-Rosetta, starting from extended conformations, provides slightly more accurate structures, while for proteins in the size range of 10–25 kDa the less CPU intensive restrained Rosetta refinement protocols provided equally or more accurate structures. The restrained Rosetta protocols described here can improve the accuracy of protein NMR structures and should find broad and general for studies of protein structure and function. PMID:24392845

  6. Development of high-performance X-ray transparent crystallization plates for in situ protein crystal screening and analysis

    Energy Technology Data Exchange (ETDEWEB)

    Soliman, Ahmed S. M.; Warkentin, Matthew [Cornell University, Ithaca, New York (United States); Apker, Benjamin [MiTeGen LLC, Ithaca, New York (United States); Thorne, Robert E., E-mail: ret6@cornell.edu [Cornell University, Ithaca, New York (United States); MiTeGen LLC, Ithaca, New York (United States)

    2011-07-01

    An optically, UV and X-ray transparent crystallization plate suitable for in situ analysis has been developed. The plate uses contact line pinning rather than wells to confine the liquids. X-ray transparent crystallization plates based upon a novel drop-pinning technology provide a flexible, simple and inexpensive approach to protein crystallization and screening. The plates consist of open cells sealed top and bottom by thin optically, UV and X-ray transparent films. The plates do not need wells or depressions to contain liquids. Instead, protein drops and reservoir solution are held in place by rings with micrometre dimensions that are patterned onto the bottom film. These rings strongly pin the liquid contact lines, thereby improving drop shape and position uniformity, and thus crystallization reproducibility, and simplifying automated image analysis of drop contents. The same rings effectively pin solutions containing salts, proteins, cryoprotectants, oils, alcohols and detergents. Strong pinning by rings allows the plates to be rotated without liquid mixing to 90° for X-ray data collection or to be inverted for hanging-drop crystallization. The plates have the standard SBS format and are compatible with standard liquid-handling robots.

  7. Study of the earth's deep interior and crystallography. X-ray and neutron diffraction experiments under high pressures

    International Nuclear Information System (INIS)

    Yagi, Takehiko

    2014-01-01

    History of the study of the Earth's deep interior was reviewed. In order to understand Earth's deep interior from the view point of materials science, X-ray diffraction under high pressure and high temperature played very important role. Use of synchrotron radiation dramatically advanced this experimental technique and it is now possible to make precise X-ray study under the P-T conditions corresponding even to the center of the Earth. In order to clarify the behavior of light elements such as hydrogen, however, studies using neutron diffraction are also required. A new neutron beam line dedicated for high-pressure science is constructed at J-PARC and is now ready for use. (author)

  8. Catalysis of GTP hydrolysis by small GTPases at atomic detail by integration of X-ray crystallography, experimental, and theoretical IR spectroscopy.

    Science.gov (United States)

    Rudack, Till; Jenrich, Sarah; Brucker, Sven; Vetter, Ingrid R; Gerwert, Klaus; Kötting, Carsten

    2015-10-02

    Small GTPases regulate key processes in cells. Malfunction of their GTPase reaction by mutations is involved in severe diseases. Here, we compare the GTPase reaction of the slower hydrolyzing GTPase Ran with Ras. By combination of time-resolved FTIR difference spectroscopy and QM/MM simulations we elucidate that the Mg(2+) coordination by the phosphate groups, which varies largely among the x-ray structures, is the same for Ran and Ras. A new x-ray structure of a Ran·RanBD1 complex with improved resolution confirmed this finding and revealed a general problem with the refinement of Mg(2+) in GTPases. The Mg(2+) coordination is not responsible for the much slower GTPase reaction of Ran. Instead, the location of the Tyr-39 side chain of Ran between the γ-phosphate and Gln-69 prevents the optimal positioning of the attacking water molecule by the Gln-69 relative to the γ-phosphate. This is confirmed in the RanY39A·RanBD1 crystal structure. The QM/MM simulations provide IR spectra of the catalytic center, which agree very nicely with the experimental ones. The combination of both methods can correlate spectra with structure at atomic detail. For example the FTIR difference spectra of RasA18T and RanT25A mutants show that spectral differences are mainly due to the hydrogen bond of Thr-25 to the α-phosphate in Ran. By integration of x-ray structure analysis, experimental, and theoretical IR spectroscopy the catalytic center of the x-ray structural models are further refined to sub-Å resolution, allowing an improved understanding of catalysis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Catalysis of GTP Hydrolysis by Small GTPases at Atomic Detail by Integration of X-ray Crystallography, Experimental, and Theoretical IR Spectroscopy*

    Science.gov (United States)

    Rudack, Till; Jenrich, Sarah; Brucker, Sven; Vetter, Ingrid R.; Gerwert, Klaus; Kötting, Carsten

    2015-01-01

    Small GTPases regulate key processes in cells. Malfunction of their GTPase reaction by mutations is involved in severe diseases. Here, we compare the GTPase reaction of the slower hydrolyzing GTPase Ran with Ras. By combination of time-resolved FTIR difference spectroscopy and QM/MM simulations we elucidate that the Mg2+ coordination by the phosphate groups, which varies largely among the x-ray structures, is the same for Ran and Ras. A new x-ray structure of a Ran·RanBD1 complex with improved resolution confirmed this finding and revealed a general problem with the refinement of Mg2+ in GTPases. The Mg2+ coordination is not responsible for the much slower GTPase reaction of Ran. Instead, the location of the Tyr-39 side chain of Ran between the γ-phosphate and Gln-69 prevents the optimal positioning of the attacking water molecule by the Gln-69 relative to the γ-phosphate. This is confirmed in the RanY39A·RanBD1 crystal structure. The QM/MM simulations provide IR spectra of the catalytic center, which agree very nicely with the experimental ones. The combination of both methods can correlate spectra with structure at atomic detail. For example the FTIR difference spectra of RasA18T and RanT25A mutants show that spectral differences are mainly due to the hydrogen bond of Thr-25 to the α-phosphate in Ran. By integration of x-ray structure analysis, experimental, and theoretical IR spectroscopy the catalytic center of the x-ray structural models are further refined to sub-Å resolution, allowing an improved understanding of catalysis. PMID:26272610

  10. Nanoflow electrospinning serial femtosecond crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Sierra, Raymond G.; Laksmono, Hartawan [SLAC National Accelerator Laboratory, Menlo Park, CA 94025 (United States); Kern, Jan [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); SLAC National Accelerator Laboratory, Menlo Park, CA 94025 (United States); Tran, Rosalie; Hattne, Johan [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Alonso-Mori, Roberto [SLAC National Accelerator Laboratory, Menlo Park, CA 94025 (United States); Lassalle-Kaiser, Benedikt [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Glöckner, Carina; Hellmich, Julia [Technische Universität Berlin, Strasse des 17 Juni 135, 10623 Berlin (Germany); Schafer, Donald W. [SLAC National Accelerator Laboratory, Menlo Park, CA 94025 (United States); Echols, Nathaniel; Gildea, Richard J.; Grosse-Kunstleve, Ralf W. [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Sellberg, Jonas [SLAC National Accelerator Laboratory, Menlo Park, CA 94025 (United States); Stockholm University, S-106 91 Stockholm (Sweden); McQueen, Trevor A. [Stanford University, Stanford, CA 94025 (United States); Fry, Alan R.; Messerschmidt, Marc M.; Miahnahri, Alan; Seibert, M. Marvin; Hampton, Christina Y.; Starodub, Dmitri; Loh, N. Duane; Sokaras, Dimosthenis; Weng, Tsu-Chien [SLAC National Accelerator Laboratory, Menlo Park, CA 94025 (United States); Zwart, Petrus H. [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Glatzel, Pieter [European Synchrotron Radiation Facility, Grenoble (France); Milathianaki, Despina; White, William E. [SLAC National Accelerator Laboratory, Menlo Park, CA 94025 (United States); Adams, Paul D. [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Williams, Garth J.; Boutet, Sébastien [SLAC National Accelerator Laboratory, Menlo Park, CA 94025 (United States); Zouni, Athina [Technische Universität Berlin, Strasse des 17 Juni 135, 10623 Berlin (Germany); Messinger, Johannes [Umeå Universitet, Umeå (Sweden); Sauter, Nicholas K. [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Bergmann, Uwe [SLAC National Accelerator Laboratory, Menlo Park, CA 94025 (United States); Yano, Junko; Yachandra, Vittal K. [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Bogan, Michael J., E-mail: mbogan@slac.stanford.edu [SLAC National Accelerator Laboratory, Menlo Park, CA 94025 (United States); SLAC National Accelerator Laboratory, Menlo Park, CA 94025 (United States)

    2012-11-01

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

  11. Nanoflow electrospinning serial femtosecond crystallography

    International Nuclear Information System (INIS)

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

    2012-01-01

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

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

    Science.gov (United States)

    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

  13. New carbocyclic N(6)-substituted adenine and pyrimidine nucleoside analogues with a bicyclo[2.2.1]heptane fragment as sugar moiety; synthesis, antiviral, anticancer activity and X-ray crystallography.

    Science.gov (United States)

    Tănase, Constantin I; Drăghici, Constantin; Cojocaru, Ana; Galochkina, Anastasia V; Orshanskaya, Jana R; Zarubaev, Vladimir V; Shova, Sergiu; Enache, Cristian; Maganu, Maria

    2015-10-01

    New nucleoside analogues with an optically active bicyclo[2.2.1]heptane skeleton as sugar moiety and 6-substituted adenine were synthesized by alkylation of 6-chloropurine intermediate. Thymine and uracil analogs were synthesized by building the pyrimidine ring on amine 1. X-ray crystallography confirmed an exo-coupling of the thymine to the ring and an L configuration of the nucleoside analogue. The library of compounds was tested for their inhibitory activity against influenza virus A∖California/07/09 (H1N1)pdm09 and coxsackievirus B4 in cell culture. Compounds 13a and 13d are the most promising for their antiviral activity against influenza, and compound 3c against coxsackievirus B4. Compounds 3b and 3g were tested for anticancer activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. 1,3-Oxazole-based selective picomolar inhibitors of cytosolic human carbonic anhydrase II alleviate ocular hypertension in rabbits: Potency is supported by X-ray crystallography of two leads.

    Science.gov (United States)

    Ferraroni, Marta; Lucarini, Laura; Masini, Emanuela; Korsakov, Mikhail; Scozzafava, Andrea; Supuran, Claudiu T; Krasavin, Mikhail

    2017-09-01

    Two lead 1,3-oxazole-based carbonic anhydrase inhibitors (CAIs) earlier identified as selective, picomolar inhibitors of hCA II (a cytosolic target for treatment of glaucoma) have been investigated further. Firstly, they were found to be conveniently synthesized on multigram scale, which enables further development. These compounds were found to be comparable in efficacy to dorzolamide eye drops when applied in the eye drop form as well. Finally, the reasons for unusually high potency of these compounds became understood from their high-resolution X-ray crystallography structures. These data significantly expand our understanding of heterocycle-based primary sulfonamides, many of which have recently emerged from our labs - particularly, from the corneal permeability standpoint. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Azo coupling of 4-nitrophenyldiazonium chloride with aliphatic nucleophiles: an integrated organic synthesis and X-ray crystallography experiment; Acoplamento de cloreto de 4-nitrofenildiazonio com nucleofilos alifaticos: experimento integrado de sintese organica e cristalografia de raios X

    Energy Technology Data Exchange (ETDEWEB)

    Cunha, Silvio; Marques, Monique F.; Rocha, Valeria, E-mail: silviodc@ufba.br [Universidade Federal da Bahia (UFBA), Salvador, BA (Brazil). Instituto de Quimica; Lariucci, Carlito; Vencato, Ivo [Universidade Federal de Goiania (UFG), GO (Brazil). Instituto de Fisica

    2013-11-01

    This article describes an undergraduate experiment for the synthesis of p-nitrophenyldiazonium chloride and its coupling with acetylacetone and two enaminones, 4-phenylamino-pent-3-en-2-one and 4-amino-pent-3-en-2-one, in an adaptation of a previously reported synthetic protocol. The azo dyes 4-(E)-phenylamino-3-[(E)-2-(4-nitrophenylazo)]-3-penten-2-one and 4-(E)-amino-3-[(E)-2-(4-nitrophenylazo)]-3-penten-2-one were obtained, and the solid state structure of this latter azo compound was characterized by single crystal X-ray diffraction studies. This two-week integrated laboratory approach involves simple synthetic experiments and microwave chemistry in the organic laboratory plus crystallography analysis, suitable for novice students on undergraduate experimental chemistry courses. (author)

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

    Energy Technology Data Exchange (ETDEWEB)

    Kurpiewska, Katarzyna, E-mail: kurpiews@chemia.uj.edu.pl [Jagiellonian University, Faculty of Chemistry, Department of Crystal Chemistry and Crystal Physics, Protein Crystallography Group, Ingardena 3, 30-060 Kraków (Poland); Dziubek, Kamil; Katrusiak, Andrzej [Adam Mickiewicz University, Faculty of Chemistry, Department of Materials Chemistry, Umultowska 89b, 61-61 Poznań (Poland); Font, Josep [School of Medical Science, University of Sydney, NSW 2006 (Australia); Ribò, Marc; Vilanova, Maria [Universitat de Girona, Laboratorid’Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Campus de Montilivi, 17071 Girona (Spain); Lewiński, Krzysztof [Jagiellonian University, Faculty of Chemistry, Department of Crystal Chemistry and Crystal Physics, Protein Crystallography Group, Ingardena 3, 30-060 Kraków (Poland)

    2016-04-01

    Highlights: • A unique crystallographic studies of wild-type and mutated form of the same protein under high pressure. • Compressibility of RNase A molecule is significantly affected by a single amino acid substitution. • High pressure protein crystallography helps understanding protein flexibility and identify conformational substrates. - Abstract: 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.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    Highlights: • A unique crystallographic studies of wild-type and mutated form of the same protein under high pressure. • Compressibility of RNase A molecule is significantly affected by a single amino acid substitution. • High pressure protein crystallography helps understanding protein flexibility and identify conformational substrates. - Abstract: 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.

  18. X-ray Free Electron Laser Determination of Crystal Structures of Dark and Light States of a Reversibly Photoswitching Fluorescent Protein at Room Temperature

    Directory of Open Access Journals (Sweden)

    Christopher D. M. Hutchison

    2017-09-01

    Full Text Available The photochromic fluorescent protein Skylan-NS (Nonlinear Structured illumination variant mEos3.1H62L is a reversibly photoswitchable fluorescent protein which has an unilluminated/ground state with an anionic and cis chromophore conformation and high fluorescence quantum yield. Photo-conversion with illumination at 515 nm generates a meta-stable intermediate with neutral trans-chromophore structure that has a 4 h lifetime. We present X-ray crystal structures of the cis (on state at 1.9 Angstrom resolution and the trans (off state at a limiting resolution of 1.55 Angstrom from serial femtosecond crystallography experiments conducted at SPring-8 Angstrom Compact Free Electron Laser (SACLA at 7.0 keV and 10.5 keV, and at Linac Coherent Light Source (LCLS at 9.5 keV. We present a comparison of the data reduction and structure determination statistics for the two facilities which differ in flux, beam characteristics and detector technologies. Furthermore, a comparison of droplet on demand, grease injection and Gas Dynamic Virtual Nozzle (GDVN injection shows no significant differences in limiting resolution. The photoconversion of the on- to the off-state includes both internal and surface exposed protein structural changes, occurring in regions that lack crystal contacts in the orthorhombic crystal form.

  19. Probing the reaction mechanism of IspH protein by x-ray structure analysis

    KAUST Repository

    Gräwert, Tobias

    2009-12-28

    Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) represent the two central intermediates in the biosynthesis of isoprenoids. The recently discovereddeoxyxylulose 5-phosphate pathway generates a mixture of IPP and DMAPP in its final step by reductive dehydroxylation of 1-hydroxy-2-methyl- 2-butenyl 4-diphosphate. This conversion is catalyzed by IspH protein comprising a central iron-sulfur cluster as electron transfer cofactor in the active site. The five crystal structures of IspH in complex with substrate, converted substrate, products and PPi reported in this article provide unique insights into the mechanism of this enzyme. While IspH protein crystallizes with substrate bound to a [4Fe-4S] cluster, crystals of IspH in complex with IPP, DMAPP or inorganic pyrophosphate feature [3Fe-4S] clusters. The IspH:substrate complex reveals a hairpin conformation of the ligand with the C(1) hydroxyl group coordinated to the unique site in a [4Fe-4S] cluster of aconitase type. The resulting alkoxide complex is coupled to a hydrogen-bonding network, which serves as proton reservoir via a Thr167 proton relay. Prolonged x-ray irradiation leads to cleavage of the C(1)-O bond (initiated by reducing photo electrons). The data suggest a reaction mechanism involving a combination of Lewis-acid activation and proton coupled electron transfer. The resulting allyl radical intermediate can acquire a second electron via the iron-sulfur cluster. The reaction may be terminated by the transfer of a proton from the β-phosphate of the substrate to C(1) (affording DMAPP) or C(3) (affording IPP).

  20. Probing the reaction mechanism of IspH protein by x-ray structure analysis

    KAUST Repository

    Grä wert, Tobias; Span, Ingrid; Eisenreich, Wolfgang; Rohdich, Felix; Eppinger, Jö rg; Bacher, Adelbert; Groll, Michael

    2009-01-01

    Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) represent the two central intermediates in the biosynthesis of isoprenoids. The recently discovereddeoxyxylulose 5-phosphate pathway generates a mixture of IPP and DMAPP in its final step by reductive dehydroxylation of 1-hydroxy-2-methyl- 2-butenyl 4-diphosphate. This conversion is catalyzed by IspH protein comprising a central iron-sulfur cluster as electron transfer cofactor in the active site. The five crystal structures of IspH in complex with substrate, converted substrate, products and PPi reported in this article provide unique insights into the mechanism of this enzyme. While IspH protein crystallizes with substrate bound to a [4Fe-4S] cluster, crystals of IspH in complex with IPP, DMAPP or inorganic pyrophosphate feature [3Fe-4S] clusters. The IspH:substrate complex reveals a hairpin conformation of the ligand with the C(1) hydroxyl group coordinated to the unique site in a [4Fe-4S] cluster of aconitase type. The resulting alkoxide complex is coupled to a hydrogen-bonding network, which serves as proton reservoir via a Thr167 proton relay. Prolonged x-ray irradiation leads to cleavage of the C(1)-O bond (initiated by reducing photo electrons). The data suggest a reaction mechanism involving a combination of Lewis-acid activation and proton coupled electron transfer. The resulting allyl radical intermediate can acquire a second electron via the iron-sulfur cluster. The reaction may be terminated by the transfer of a proton from the β-phosphate of the substrate to C(1) (affording DMAPP) or C(3) (affording IPP).

  1. Small angle X-ray scattering and transmission electron microscopy study of the Lactobacillus brevis S-layer protein

    Energy Technology Data Exchange (ETDEWEB)

    Jaeaeskelaeinen, Pentti [Department of Biomedical Engineering and Computational Science, PO Box 2200, FI-02015 Aalto University School of Science and Technology (Finland); Engelhardt, Peter [Haartman Institute, Department of Pathology, PO Box 21, FIN-00014 University of Helsinki (Finland); Hynoenen, Ulla; Palva, Airi [Department of Basic Veterinary Sciences, Division of Microbiology, FIN-00014 University of Helsinki (Finland); Torkkeli, Mika; Serimaa, Ritva, E-mail: ritva.serimaa@helsinki.f [Department of Physics, POB 64, 00014 University of Helsinki (Finland)

    2010-10-01

    The structure of self-assembly domain containing recombinant truncation mutants of Lactobacillus brevis surface layer protein SlpA in aqueous solution was studied using small-angle X-ray scattering and transmission electron microscopy. The proteins were found out to interact with each other forming stable globular oligomers of about 10 monomers. The maximum diameter of the oligomers varied between 75 A and 435 A.

  2. Electron crystallography of three dimensional protein crystals

    NARCIS (Netherlands)

    Georgieva, Dilyana

    2008-01-01

    This thesis describes an investigation of the potential of electron diffraction for studying three dimensional sub-micro-crystals of proteins and pharmaceuticals. A prerequisite for using electron diffraction for structural studies is the predictable availability of tiny crystals. A method for

  3. Ten Good Reasons for the Use of the Tellurium-Centered Anderson-Evans Polyoxotungstate in Protein Crystallography.

    Science.gov (United States)

    Bijelic, Aleksandar; Rompel, Annette

    2017-06-20

    Protein crystallography represents at present the most productive and most widely used method to obtain structural information on target proteins and protein-ligand complexes within the atomic resolution range. The knowledge obtained in this way is essential for understanding the biology, chemistry, and biochemistry of proteins and their functions but also for the development of compounds of high pharmacological and medicinal interest. Here, we address the very central problem in protein crystallography: the unpredictability of the crystallization process. Obtaining protein crystals that diffract to high resolutions represents the essential step to perform any structural study by X-ray crystallography; however, this method still depends basically on trial and error making it a very time- and resource-consuming process. The use of additives is an established process to enable or improve the crystallization of proteins in order to obtain high quality crystals. Therefore, a more universal additive addressing a wider range of proteins is desirable as it would represent a huge advance in protein crystallography and at the same time drastically impact multiple research fields. This in turn could add an overall benefit for the entire society as it profits from the faster development of novel or improved drugs and from a deeper understanding of biological, biochemical, and pharmacological phenomena. With this aim in view, we have tested several compounds belonging to the emerging class of polyoxometalates (POMs) for their suitability as crystallization additives and revealed that the tellurium-centered Anderson-Evans polyoxotungstate [TeW 6 O 24 ] 6- (TEW) was the most suitable POM-archetype. After its first successful application as a crystallization additive, we repeatedly reported on TEW's positive effects on the crystallization behavior of proteins with a particular focus on the protein-TEW interactions. As electrostatic interactions are the main force for TEW binding

  4. Magnetic x-ray microdiffraction

    Energy Technology Data Exchange (ETDEWEB)

    Evans, Paul G [Computer-Aided Engineering Center, University of Wisconsin, Madison, WI 53706 (United States); Isaacs, Eric D [Center for Nanoscale Materials, Argonne National Laboratory, Argonne, IL 60439 (United States)

    2006-08-07

    Magnetic x-ray microdiffraction uses the structural specificity of x-ray diffraction to probe complex magnetic structures at the length scales relevant to physical phenomena including domain dynamics and phase transitions. Conventional magnetic crystallography techniques such as neutron or x-ray diffraction lack this spatial resolution. The combination of both reciprocal space and real space resolution with a rich magnetic cross section allows new microscopy techniques to be developed and applied to magnetism at the scale of single domains. Potential applications include a wide range of magnetic problems in nanomagnetism, the interaction of strain, polarization and magnetization in complex oxides and spatially resolved studies of magnetic phase transitions. We present the physical basis for x-ray microdiffraction and magnetic scattering processes, review microdiffraction domain imaging techniques in antiferromagnetic and ferromagnetic materials and discuss potential directions for studies. (topical review)

  5. Purification, crystallization and X-ray diffraction analysis of human dynamin-related protein 1 GTPase-GED fusion protein

    International Nuclear Information System (INIS)

    Klinglmayr, Eva; Wenger, Julia; Mayr, Sandra; Bossy-Wetzel, Ella; Puehringer, Sandra

    2012-01-01

    The crystallization and initial diffraction analysis of human Drp1 GTPase-GED fusion protein are reported. The mechano-enzyme dynamin-related protein 1 plays an important role in mitochondrial fission and is implicated in cell physiology. Dysregulation of Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage. Drp1 shares structural and functional similarities with dynamin 1 with respect to domain organization, ability to self-assemble into spiral-like oligomers and GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1 have greatly improved the understanding of this prototypical member of the dynamin superfamily. However, high-resolution structural information for full-length human Drp1 covering the GTPase domain, the middle domain and the GTPase effector domain (GED) is still lacking. In order to obtain mechanistic insights into the catalytic activity, a nucleotide-free GTPase-GED fusion protein of human Drp1 was expressed, purified and crystallized. Initial X-ray diffraction experiments yielded data to 2.67 Å resolution. The hexagonal-shaped crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 53.59, b = 151.65, c = 43.53 Å, one molecule per asymmetric unit and a solvent content of 42%. Expression of selenomethionine-labelled protein is currently in progress. Here, the expression, purification, crystallization and X-ray diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which form a basis for more detailed structural and biophysical analysis

  6. Solid state structural investigations of the bis(chalcone) compound with single crystal X-ray crystallography, DFT, gamma-ray spectroscopy and chemical spectroscopy methods

    Science.gov (United States)

    Yakalı, Gül; Biçer, Abdullah; Eke, Canel; Cin, Günseli Turgut

    2018-04-01

    A bis(chalcone), (2E,6E)-2,6-bis((E)-3phenylallidene)cyclohexanone, was characterized by 1H NMR, 13C NMR, FTIR, UV-Vis spectroscopy, gamma-ray spectroscopy and single crystal X- ray structural analysis. The optimized molecular structure of the compound is calculated using DFT/B3LYP with 6-31G (d,p) level. The calculated geometrical parameters are in good agreement with the experimental data obtained from our reported X-ray structure. The powder and single crystal compounds were gama-irradiated using clinical electron linear accelerator and 60Co gamma-ray source, respectively. Spectral studies (1H NMR, 13C NMR, FTIR and UV-Vis) of powder chalcone compound were also investigated before and after irradiation. Depending on the irradiation notable changes were observed in spectral features powder sample. Single crystal X-ray diffraction investigation shows that both unirradiated and irradiated single crystal samples crystallizes in a orthorhombic crystal system in the centrosymmetric space group Pbcn and exhibits an C-H..O intramolecular and intermolecular hydrogen bonds. The crystal packing is stabilised by strong intermolecular bifurcate C-H..O hydrogen bonds and π…π stacking interactions. The asymmetric unit of the title compound contains one-half of a molecule. The other half of the molecule is generated with (1-x,y,-3/2-z) symmetry operator. The molecule is almost planar due to having π conjugated system of chalcones. However, irradiated single crystal compound showed significant changes lattice parameters, crystal volume and density. According to results of gamma-ray spectroscopy, radioactive elements of powder compound which are 123Sb(n,g),124Sb,57Fe(g,p),56Mn, 55Mn(g,n), and 54Mn were determined using photoactivation analysis. However, the most intensive gamma-ray energy signals are 124Sb.

  7. Lanthanide complexes of macrocyclic polyoxovanadates by VO4 units: synthesis, characterization, and structure elucidation by X-ray crystallography and EXAFS spectroscopy.

    Science.gov (United States)

    Nishio, Masaki; Inami, Shinnosuke; Katayama, Misaki; Ozutsumi, Kazuhiko; Hayashi, Yoshihito

    2012-01-16

    Reactions of a tetravanadate anion, [V(4)O(12)](4-), with a series of lanthanide(III) salts yield three types of lanthanide complexes of macrocyclic polyoxovanadates: (Et(4)N)(6)[Ln(III)V(9)O(27)] [Ln = Nd (1), Sm (2), Eu (3), Gd (4), Tb (5), Dy (6)], (Et(4)N)(5)[(H(2)O)Ho(III)(V(4)O(12))(2)] (7), and (Et(4)N)(7)[Ln(III)V(10)O(30)] [Ln = Er (8), Tm (9), Yb (10), Lu (11)]. Lanthanide complexes 1-11 are isolated and characterized by IR, elemental analysis, single-crystal X-ray diffraction, and extended X-ray absorption fine structure spectroscopy (EXAFS). Lanthanide complexes 1-6 are composed of a square-antiprism eight-coordinated Ln(III) center with a macrocyclic polyoxovanadate that is constructed from nine VO(4) tetrahedra through vertex sharing. The structure of 7 is composed of a seven-coordinated Ho(III) center, which exhibits a capped trigonal-prism coordination environment by the sandwiching of two cyclic tetravanadates with a capping H(2)O ligand. Lanthanide complexes 8-11 have a six-coordinated Ln(III) center with a 10-membered vanadate ligand. The structural trend to adopt a larger coordination number for a larger lanthanide ion among the three types of structures is accompanied by a change in the vanadate ring sizes. These lanthanide complexes are examined by EXAFS spectroscopies on lanthanide L(III) absorption edges, and the EXAFS oscillations of each of the samples in the solid state and in acetonitrile are identical. The Ln-O and Ln···V bond lengths obtained from fits of the EXAFS data are consistent with the data from the single-crystal X-ray studies, reflecting retention of the structures in acetonitrile.

  8. Compact structure of ribosomal protein S4 in solution as revealed by small-angle X-ray scattering

    International Nuclear Information System (INIS)

    Serdyuk, I.N.; Sarkisyan, M.A.; Gogia, Z.V.

    1981-01-01

    The authors report the results of a small-angle X-ray scattering study of ribosomal protein preparations obtained by neutron scattering method. The theoretical resolution of the diffractometer (Kratky camera, the entrance slit 80 μm, the receiving slit 190 μm, the sample-detector distance 20.4 cm) was the same as the resolution of X-ray diffractometers, on which high rsub(g) values for ribosomal proteins were obtained. They used protein S4 adjusted to 20 mg/ml without any essential loss of solubility. The scattering indicatrix obtained in a wide range of angles has demonstrated that the X-ray rsub(g) obtained here coincides with the earlier obtained neutron rsub(g) and the outer part of the scattering curve is similar to that of slightly elongated compact bodies. They conclude that all discrepancies between their data on the study of ribosomal protein structure in solution and other data are not connected with the characteristics of the instruments used but only with the quality of the protein preparations. (Auth.)

  9. Protein Crystallography: A 'Must' Technology for Drug Design

    International Nuclear Information System (INIS)

    Matsuzaki, Takao

    2004-01-01

    The history of drug-related protein crystallography and drug design is reviewed to show that 'Lead Generation' is high-lighted in the pharmaceutical industry nowadays. A new drug design method has been developed. The method gave very high success rate; 10-60 % gave < 100 μM, 90 % gave < 10 mM. The crystal structures of drug-protein complexes have become even more important to give solid experimental bases for e.g. 1,000 designed structures and to find the new mechanisms of drug action

  10. Generation of high brightness x-ray source and its medical applications

    International Nuclear Information System (INIS)

    Fujii, Sadao; Muro, Mikio; Oku, Yasunari; Daido, Hiroyuki; Takahashi, Kenjiro

    2001-01-01

    Laser produced plasmas are one of the most feasible sources to be used for industrial applications, especially medical applications: Angiography, Protein crystallography, X-ray microscopy and XAFS. In the present paper, laser requirements are clarified for the medical and life science fields and then we estimate both the photon energy spectra and the number of photons based on Monte-Carlo simulation. (author)

  11. Structural Basis of X-ray-Induced Transient Photo-bleaching in a Photoactivatable Green Fluorescent Protein

    Energy Technology Data Exchange (ETDEWEB)

    Adam, V. [European Synchrotron Radiation Facility, 6 rue Jules Horowitz, BP 220, 38043 Grenoble Cedex (France); Carpentier, Ph.; Lelimousin, M.; Darnault, C.; Bourgeois, D. [IBS, Institut de Biologie Structurale Jean-Pierre Ebel, CEA, CNRS, UniVersite Joseph Fourier, 41 rue Jules Horowitz, 38027 Grenoble (France); Violot, S. [Laboratoire de Physiologie Cellulaire Vegetale, Institut de Recherches en Technologie et Sciences pour le ViVant, CEA, CNRS, INRA, UniVersite Joseph Fourier, 17 rue des Martyrs, F-38054 Grenoble (France); Nienhaus, U. [Institute of Applied Physics and Center for Functional nano-structures (CFN), Karlsruhe Institute of Technology, 76128 Karlsruhe (Germany); Nienhaus, U. [Department of Physics, UniVersity of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (US)

    2009-07-01

    We have observed the photoactivatable fluorescent protein IrisFP in a transient dark state with near-atomic resolution. This dark state is assigned to a radical species that either relaxes to the ground state or evolves into a permanently bleached chromophore. We took advantage of X-rays to populate the radical, which presumably forms under illumination with visible light by an electron-transfer reaction in the triplet state. The combined X-ray diffraction and in crystallo UV-vis absorption, fluorescence, and Raman data reveal that radical formation in IrisFP involves pronounced but reversible distortion of the chromophore, suggesting a transient loss of {pi} conjugation. These results reveal that the methylene bridge of the chromophore is the Achilles' heel of fluorescent proteins and help unravel the mechanisms of blinking and photo-bleaching in FPs, which are of importance in the rational design of photo-stable variants. and is also partly reversible. (authors)

  12. Investigation of molecular mechanisms of action of chelating drugs on protein-lipid model membranes by X-ray fluorescence

    International Nuclear Information System (INIS)

    Novikova, N. N.; Zheludeva, S. I.; Koval'chuk, M. V.; Stepina, N. D.; Erko, A. I.; Yur'eva, E. A.

    2009-01-01

    Protein-lipid films based on the enzyme alkaline phosphatase were subjected to the action of chelating drugs, which are used for accelerating the removal of heavy metals from the human body, and the elemental composition of the resulting films was investigated. Total-reflection X-ray fluorescence measurements were performed at the Berlin Electron Storage Ring Company for Synchrotron Radiation (BESSY) in Germany. A comparative estimation of the protective effect of four drugs (EDTA, succimer, xydiphone, and mediphon) on membrane-bound enzymes damaged by lead ions was made. The changes in the elemental composition of the protein-lipid films caused by high doses of chelating drugs were investigated. It was shown that state-of-the-art X-ray techniques can, in principle, be used to develop new methods for the in vitro evaluation of the efficiency of drugs, providing differential data on their actions.

  13. Exploring structural variability in X-ray crystallographic models using protein local optimization by torsion-angle sampling

    International Nuclear Information System (INIS)

    Knight, Jennifer L.; Zhou, Zhiyong; Gallicchio, Emilio; Himmel, Daniel M.; Friesner, Richard A.; Arnold, Eddy; Levy, Ronald M.

    2008-01-01

    Torsion-angle sampling, as implemented in the Protein Local Optimization Program (PLOP), is used to generate multiple structurally variable single-conformer models which are in good agreement with X-ray data. An ensemble-refinement approach to differentiate between positional uncertainty and conformational heterogeneity is proposed. Modeling structural variability is critical for understanding protein function and for modeling reliable targets for in silico docking experiments. Because of the time-intensive nature of manual X-ray crystallographic refinement, automated refinement methods that thoroughly explore conformational space are essential for the systematic construction of structurally variable models. Using five proteins spanning resolutions of 1.0–2.8 Å, it is demonstrated how torsion-angle sampling of backbone and side-chain libraries with filtering against both the chemical energy, using a modern effective potential, and the electron density, coupled with minimization of a reciprocal-space X-ray target function, can generate multiple structurally variable models which fit the X-ray data well. Torsion-angle sampling as implemented in the Protein Local Optimization Program (PLOP) has been used in this work. Models with the lowest R free values are obtained when electrostatic and implicit solvation terms are included in the effective potential. HIV-1 protease, calmodulin and SUMO-conjugating enzyme illustrate how variability in the ensemble of structures captures structural variability that is observed across multiple crystal structures and is linked to functional flexibility at hinge regions and binding interfaces. An ensemble-refinement procedure is proposed to differentiate between variability that is a consequence of physical conformational heterogeneity and that which reflects uncertainty in the atomic coordinates

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

    Science.gov (United States)

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

    2017-07-01

    Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5-20 µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A 2A adenosine receptor (A 2A AR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8 000 000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suites Cheetah and CrystFEL enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A 2A AR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A 2A AR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5-20 µm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS

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

    Directory of Open Access Journals (Sweden)

    Jose M. Martin-Garcia

    2017-07-01

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

  16. Intergrown new zeolite beta polymorphs with interconnected 12-ring channels solved by combining electron crystallography and single-crystal X-ray diffraction

    KAUST Repository

    Yu, Zhengbao

    2012-10-09

    Two new polymorphs of zeolite beta, denoted as SU-78A and SU-78B, were synthesized by employing dicyclohexylammonium hydroxides as organic structure-directing agents. The structure was solved by combining transmission electron microscopy and single-crystal X-ray diffraction. SU-78 is an intergrowth of SU-78A and SU-78B and contains interconnected 12-ring channels in three directions. The two polymorphs are built from the same building layer, similar to that for the zeolite beta family. The layer stacking in SU-78, however, is different from those in zeolite beta polymorph A, B, and C, showing new zeolite framework topologies. SU-78 is thermally stable up to 600 °C. © 2012 American Chemical Society.

  17. Detection of Reaction Intermediates in Mg2+-Dependent DNA Synthesis and RNA Degradation by Time-Resolved X-Ray Crystallography.

    Science.gov (United States)

    Samara, Nadine L; Gao, Yang; Wu, Jinjun; Yang, Wei

    2017-01-01

    Structures of enzyme-substrate/product complexes have been studied for over four decades but have been limited to either before or after a chemical reaction. Recently using in crystallo catalysis combined with X-ray diffraction, we have discovered that many enzymatic reactions in nucleic acid metabolism require additional metal ion cofactors that are not present in the substrate or product state. By controlling metal ions essential for catalysis, the in crystallo approach has revealed unprecedented details of reaction intermediates. Here we present protocols used for successful studies of Mg 2+ -dependent DNA polymerases and ribonucleases that are applicable to analyses of a variety of metal ion-dependent reactions. © 2017 Elsevier Inc. All rights reserved.

  18. Hydrogen bonds of DsrD protein revealed by neutron crystallography

    International Nuclear Information System (INIS)

    Chatake, Toshiyuki; Higuchi, Yoshiki; Mizuno, Nobuhiro; Tanaka, Ichiro; Niimura, Nobuo; Morimoto, Yukio

    2008-01-01

    Hydrogen bonds of DNA-binding protein DsrD have been determined by neutron diffraction. In terms of proton donors and acceptors, DsrD protein shows striking differences from other proteins. The features of hydrogen bonds in DsrD protein from sulfate-reducing bacteria have been investigated by neutron protein crystallography. The function of DsrD has not yet been elucidated clearly, but its X-ray crystal structure revealed that it comprises a winged-helix motif and shows the highest structural homology to the DNA-binding proteins. Since any neutron structure of a DNA recognition protein has not yet been obtained, here detailed information on the hydrogen bonds in the winged-helix-motif protein is given and the following features found. (i) The number of hydrogen bonds per amino acid of DsrD is relatively fewer than for other proteins for which neutron structures were determined previously. (ii) Hydrogen bonds are localized between main-chain and main-chain atoms; there are few hydrogen bonds between main-chain and side-chain atoms and between side-chain and side-chain atoms. (iii) Hydrogen bonds inducted by protonation of specific amino acid residues (Glu50) seem to play an essential role in the dimerization of DsrD. The former two points are related to the function of the DNA-binding protein; the three-dimensional structure was mainly constructed by hydrogen bonds in main chains, while the side chains appeared to be used for another role. The latter point would be expected to contribute to the crystal growth of DsrD

  19. Multichannel X-ray detectors

    Energy Technology Data Exchange (ETDEWEB)

    Khabakhpashev, A

    1980-10-01

    A typical design is discussed of multiwire proportional counters and their characteristic feature is explained, ie., the possibility of showing one or two coordinates of the X-ray quantum absorption site. The advantages of such instruments are listed, such as increased sensitivity of determination, the possibility of recording radiations of a different intensity, the possibility of on-line data processing and of the digital display of results. The fields of application include X-ray structural analysis in solid state physics, crystallography, molecular biology, astronomy, materials testing, and medicine.

  20. X-ray data processing.

    Science.gov (United States)

    Powell, Harold R

    2017-10-31

    The method of molecular structure determination by X-ray crystallography is a little over a century old. The history is described briefly, along with developments in X-ray sources and detectors. The fundamental processes involved in measuring diffraction patterns on area detectors, i.e. autoindexing, refining crystal and detector parameters, integrating the reflections themselves and putting the resultant measurements on to a common scale are discussed, with particular reference to the most commonly used software in the field. © 2017 The Author(s).

  1. Batch crystallization of rhodopsin for structural dynamics using an X-ray free-electron laser

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Wenting; Nogly, Przemyslaw; Rheinberger, Jan; Kick, Leonhard M.; Gati, Cornelius; Nelson, Garrett; Deupi, Xavier; Standfuss, Jörg; Schertler, Gebhard; Panneels, Valérie, E-mail: valerie.panneels@psi.ch [Paul Scherrer Institute, OFLC/103, 5232 Villigen-PSI (Switzerland)

    2015-06-27

    A new batch preparation method is presented for high-density micrometre-sized crystals of the G protein-coupled receptor rhodopsin for use in time-resolved serial femtosecond crystallography at an X-ray free-electron laser using a liquid jet. Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols: (i) screening the low-salt crystallization conditions preferred for serial crystallography by vapour diffusion, (ii) optimization of batch crystallization, (iii) testing the crystal size and quality using second-harmonic generation (SHG) imaging and X-ray powder diffraction and (iv) production of millilitres of rhodopsin crystal suspension in batches for serial crystallography tests; these crystals diffracted at an XFEL at the Linac Coherent Light Source using a liquid-jet setup.

  2. Quantum effets in nonresonant X-ray scattering

    Energy Technology Data Exchange (ETDEWEB)

    Slowik, Jan Malte

    2015-11-15

    Due to their versatile properties, X rays are a unique tool to investigate the structure and dynamics of matter. X-ray scattering is the fundamental principle of many imaging techniques. Examples are X-ray crystallography, which recently celebrated one hundred years and is currently the leading method in structure determination of proteins, as well as X-ray phase contrast imaging (PCI), which is an imaging technique with countless applications in biology, medicine, etc. The technological development of X-ray free electron lasers (XFEL) has brought X-ray imaging at the edge of a new scientific revolution. XFELs offer ultrashort X-ray pulses with unprecedented high X-ray fluence and excellent spatial coherence properties. These properties make them an outstanding radiation source for X-ray scattering experiments, providing ultrafast temporal resolution as well as atomic spatial resolution. However, the radiation-matter interaction in XFEL experiments also advances into a novel regime. This demands a sound theoretical fundament to describe and explore the new experimental possibilities. This dissertation is dedicated to the theoretical study of nonresonant X-ray scattering. As the first topic, I consider the near-field imaging by propagation based X-ray phase contrast imaging (PCI). I devise a novel theory of PCI, in which radiation and matter are quantized. Remarkably, the crucial interference term automatically excludes contributions from inelastic scattering. This explains the success of the classical description thus far. The second topic of the thesis is the X-ray imaging of coherent electronic motion, where quantum effects become particularly apparent. The electron density of coherent electronic wave packets - important in charge transfer and bond breaking - varies in time, typically on femto- or attosecond time scales. In the near future, XFELs are envisaged to provide attosecond X-ray pulses, opening the possibility for time-resolved ultrafast X-ray scattering

  3. Quantum effets in nonresonant X-ray scattering

    International Nuclear Information System (INIS)

    Slowik, Jan Malte

    2015-11-01

    Due to their versatile properties, X rays are a unique tool to investigate the structure and dynamics of matter. X-ray scattering is the fundamental principle of many imaging techniques. Examples are X-ray crystallography, which recently celebrated one hundred years and is currently the leading method in structure determination of proteins, as well as X-ray phase contrast imaging (PCI), which is an imaging technique with countless applications in biology, medicine, etc. The technological development of X-ray free electron lasers (XFEL) has brought X-ray imaging at the edge of a new scientific revolution. XFELs offer ultrashort X-ray pulses with unprecedented high X-ray fluence and excellent spatial coherence properties. These properties make them an outstanding radiation source for X-ray scattering experiments, providing ultrafast temporal resolution as well as atomic spatial resolution. However, the radiation-matter interaction in XFEL experiments also advances into a novel regime. This demands a sound theoretical fundament to describe and explore the new experimental possibilities. This dissertation is dedicated to the theoretical study of nonresonant X-ray scattering. As the first topic, I consider the near-field imaging by propagation based X-ray phase contrast imaging (PCI). I devise a novel theory of PCI, in which radiation and matter are quantized. Remarkably, the crucial interference term automatically excludes contributions from inelastic scattering. This explains the success of the classical description thus far. The second topic of the thesis is the X-ray imaging of coherent electronic motion, where quantum effects become particularly apparent. The electron density of coherent electronic wave packets - important in charge transfer and bond breaking - varies in time, typically on femto- or attosecond time scales. In the near future, XFELs are envisaged to provide attosecond X-ray pulses, opening the possibility for time-resolved ultrafast X-ray scattering

  4. Skull x-ray

    Science.gov (United States)

    X-ray - head; X-ray - skull; Skull radiography; Head x-ray ... There is low radiation exposure. X-rays are monitored and regulated to provide the minimum amount of radiation exposure needed to produce the image. Most ...

  5. Neck x-ray

    Science.gov (United States)

    X-ray - neck; Cervical spine x-ray; Lateral neck x-ray ... There is low radiation exposure. X-rays are monitored so that the lowest amount of radiation is used to produce the image. Pregnant women and ...

  6. Femtosecond X-ray diffraction from an aerosolized beam of protein nanocrystals.

    Science.gov (United States)

    Awel, Salah; Kirian, Richard A; Wiedorn, Max O; Beyerlein, Kenneth R; Roth, Nils; Horke, Daniel A; Oberthür, Dominik; Knoska, Juraj; Mariani, Valerio; Morgan, Andrew; Adriano, Luigi; Tolstikova, Alexandra; Xavier, P Lourdu; Yefanov, Oleksandr; Aquila, Andrew; Barty, Anton; Roy-Chowdhury, Shatabdi; Hunter, Mark S; James, Daniel; Robinson, Joseph S; Weierstall, Uwe; Rode, Andrei V; Bajt, Saša; Küpper, Jochen; Chapman, Henry N

    2018-02-01

    High-resolution Bragg diffraction from aerosolized single granulovirus nanocrystals using an X-ray free-electron laser is demonstrated. The outer dimensions of the in-vacuum aerosol injector components are identical to conventional liquid-microjet nozzles used in serial diffraction experiments, which allows the injector to be utilized with standard mountings. As compared with liquid-jet injection, the X-ray scattering background is reduced by several orders of magnitude by the use of helium carrier gas rather than liquid. Such reduction is required for diffraction measurements of small macromolecular nanocrystals and single particles. High particle speeds are achieved, making the approach suitable for use at upcoming high-repetition-rate facilities.

  7. Phosphor Scanner For Imaging X-Ray Diffraction

    Science.gov (United States)

    Carter, Daniel C.; Hecht, Diana L.; Witherow, William K.

    1992-01-01

    Improved optoelectronic scanning apparatus generates digitized image of x-ray image recorded in phosphor. Scanning fiber-optic probe supplies laser light stimulating luminescence in areas of phosphor exposed to x rays. Luminescence passes through probe and fiber to integrating sphere and photomultiplier. Sensitivity and resolution exceed previously available scanners. Intended for use in x-ray crystallography, medical radiography, and molecular biology.

  8. Structure of a complex of uridine phosphorylase from Yersinia pseudotuberculosis with the modified bacteriostatic antibacterial drug determined by X-ray crystallography and computer analysis

    Energy Technology Data Exchange (ETDEWEB)

    Balaev, V. V.; Lashkov, A. A., E-mail: alashkov83@gmail.com; Gabdoulkhakov, A. G.; Seregina, T. A.; Dontsova, M. V.; Mikhailov, A. M. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2015-03-15

    Pseudotuberculosis and bubonic plague are acute infectious diseases caused by the bacteria Yersinia pseudotuberculosis and Yersinia pestis. These diseases are treated, in particular, with trimethoprim and its modified analogues. However, uridine phosphorylases (pyrimidine nucleoside phosphorylases) that are present in bacterial cells neutralize the action of trimethoprim and its modified analogues on the cells. In order to reveal the character of the interaction of the drug with bacterial uridine phosphorylase, the atomic structure of the unligated molecule of uridine-specific pyrimidine nucleoside phosphorylase from Yersinia pseudotuberculosis (YptUPh) was determined by X-ray diffraction at 1.7 Å resolution with high reliability (R{sub work} = 16.2, R{sub free} = 19.4%; r.m.s.d. of bond lengths and bond angles are 0.006 Å and 1.005°, respectively; DPI = 0.107 Å). The atoms of the amino acid residues of the functionally important secondary-structure elements—the loop L9 and the helix H8—of the enzyme YptUPh were located. The three-dimensional structure of the complex of YptUPh with modified trimethoprim—referred to as 53I—was determined by the computer simulation. It was shown that 53I is a pseudosubstrate of uridine phosphorylases, and its pyrimidine-2,4-diamine group is located in the phosphate-binding site of the enzyme YptUPh.

  9. Structure of a complex of uridine phosphorylase from Yersinia pseudotuberculosis with the modified bacteriostatic antibacterial drug determined by X-ray crystallography and computer analysis

    International Nuclear Information System (INIS)

    Balaev, V. V.; Lashkov, A. A.; Gabdoulkhakov, A. G.; Seregina, T. A.; Dontsova, M. V.; Mikhailov, A. M.

    2015-01-01

    Pseudotuberculosis and bubonic plague are acute infectious diseases caused by the bacteria Yersinia pseudotuberculosis and Yersinia pestis. These diseases are treated, in particular, with trimethoprim and its modified analogues. However, uridine phosphorylases (pyrimidine nucleoside phosphorylases) that are present in bacterial cells neutralize the action of trimethoprim and its modified analogues on the cells. In order to reveal the character of the interaction of the drug with bacterial uridine phosphorylase, the atomic structure of the unligated molecule of uridine-specific pyrimidine nucleoside phosphorylase from Yersinia pseudotuberculosis (YptUPh) was determined by X-ray diffraction at 1.7 Å resolution with high reliability (R work = 16.2, R free = 19.4%; r.m.s.d. of bond lengths and bond angles are 0.006 Å and 1.005°, respectively; DPI = 0.107 Å). The atoms of the amino acid residues of the functionally important secondary-structure elements—the loop L9 and the helix H8—of the enzyme YptUPh were located. The three-dimensional structure of the complex of YptUPh with modified trimethoprim—referred to as 53I—was determined by the computer simulation. It was shown that 53I is a pseudosubstrate of uridine phosphorylases, and its pyrimidine-2,4-diamine group is located in the phosphate-binding site of the enzyme YptUPh

  10. Structure of a complex of uridine phosphorylase from Yersinia pseudotuberculosis with the modified bacteriostatic antibacterial drug determined by X-ray crystallography and computer analysis

    Science.gov (United States)

    Balaev, V. V.; Lashkov, A. A.; Gabdoulkhakov, A. G.; Seregina, T. A.; Dontsova, M. V.; Mikhailov, A. M.

    2015-03-01

    Pseudotuberculosis and bubonic plague are acute infectious diseases caused by the bacteria Yersinia pseudotuberculosis and Yersinia pestis. These diseases are treated, in particular, with trimethoprim and its modified analogues. However, uridine phosphorylases (pyrimidine nucleoside phosphorylases) that are present in bacterial cells neutralize the action of trimethoprim and its modified analogues on the cells. In order to reveal the character of the interaction of the drug with bacterial uridine phosphorylase, the atomic structure of the unligated molecule of uridine-specific pyrimidine nucleoside phosphorylase from Yersinia pseudotuberculosis ( YptUPh) was determined by X-ray diffraction at 1.7 Å resolution with high reliability ( R work = 16.2, R free = 19.4%; r.m.s.d. of bond lengths and bond angles are 0.006 Å and 1.005°, respectively; DPI = 0.107 Å). The atoms of the amino acid residues of the functionally important secondary-structure elements—the loop L9 and the helix H8—of the enzyme YptUPh were located. The three-dimensional structure of the complex of YptUPh with modified trimethoprim—referred to as 53I—was determined by the computer simulation. It was shown that 53I is a pseudosubstrate of uridine phosphorylases, and its pyrimidine-2,4-diamine group is located in the phosphate-binding site of the enzyme YptUPh.

  11. DNA binding, antioxidant, cytotoxicity (MTT, lactate dehydrogenase, NO), and cellular uptake studies of structurally different nickel(II) thiosemicarbazone complexes: synthesis, spectroscopy, electrochemistry, and X-ray crystallography.

    Science.gov (United States)

    Prabhakaran, R; Kalaivani, P; Huang, R; Poornima, P; Vijaya Padma, V; Dallemer, F; Natarajan, K

    2013-02-01

    Three new nickel(II) thiosemicarbazone complexes have been synthesized and characterized by analytical, spectral, and single-crystal X-ray diffraction studies. In complex 1, the ligand 2-hydroxy-1-naphthaldehydethiosemicarbazone coordinated as a monobasic tridentate donor, whereas in complexes 2 and 3, the ligands salicylaldehyde-4(N)-ethylthiosemicarbazone and 2-hydroxy-1-naphthaldehyde-4(N)-ethylthiosemicarbazone coordinated as a dibasic tridentate donor. The DNA binding ability of the complexes in calf thymus DNA was explored by absorption and emission titration experiments. The antioxidant property of the new complexes was evaluated to test their free-radical scavenging ability. In vitro cytotoxicity assays were performed for the new complexes in A549 and HepG2 cell lines. The new compounds overcome cisplatin resistance in the A549 cell line and they were also active in the HepG2 cell line. The cellular uptake study showed the accumulation of the complexes in tumor cells depended on the nature of the ligand attached to the nickel ion.

  12. Synthesis, spectra and X-ray crystallography of dipyridin-2-ylmethanone oxime and its CuX2(oxime)2 complexes: Thermal, Hirshfeld surface and DFT analysis

    Science.gov (United States)

    Warad, Ismail; Abdoh, Muneer; Al Ali, Anas; Shivalingegowda, Naveen; Kumara, Karthik; Zarrouk, Abdelkader; Lokanath, Neartur Krishnappagowda

    2018-02-01

    Dipyridin-2-ylmethanone oxime (C11H9N3O), was prepared using di-2-pyridyl ketone. The oxime ligand and its neutral CuX2 (oxime)2 (X = Cl or Br) complexes have been identified with the aid of several spectroscopic techniques such as: IR, EI-MS, EA, UV-visible, TG, 1H-NMR and finally the structure of the free oxime ligand was confirmed by X-ray diffraction studies. The oxime crystallizes in the monoclinic space group P21/c, with cell parameters a = 8.8811 (8) Å, b = 10.6362 (8) Å, c = 11.2050 (8) Å, β = 109.085 (4) º, V = 1000.26 (14) Å3 and Z = 4. The molecular conformation is stabilized by a strong intramolecular Osbnd H⋯N hydrogen bonding between the hydroxyl group of the oxime moiety and the nitrogen of the pyridine ring. Since the oxime structure was solved by XRD, the ligand structure parameters like bond length and angles were compared to the DFT computed one, the UV-visible to TD-SCF and Hirshfeld surface to MEP analysis.

  13. Novel Tacrine-Benzofuran Hybrids as Potent Multitarget-Directed Ligands for the Treatment of Alzheimer's Disease: Design, Synthesis, Biological Evaluation, and X-ray Crystallography.

    Science.gov (United States)

    Zha, Xiaoming; Lamba, Doriano; Zhang, Lili; Lou, Yinghan; Xu, Changxu; Kang, Di; Chen, Li; Xu, Yungen; Zhang, Luyong; De Simone, Angela; Samez, Sarah; Pesaresi, Alessandro; Stojan, Jure; Lopez, Manuela G; Egea, Javier; Andrisano, Vincenza; Bartolini, Manuela

    2016-01-14

    Twenty-six new tacrine-benzofuran hybrids were designed, synthesized, and evaluated in vitro on key molecular targets for Alzheimer's disease. Most hybrids exhibited good inhibitory activities on cholinesterases and β-amyloid self-aggregation. Selected compounds displayed significant inhibition of human β-secretase-1 (hBACE-1). Among the 26 hybrids, 2e showed the most interesting profile as a subnanomolar selective inhibitor of human acetylcholinesterase (hAChE) (IC50 = 0.86 nM) and a good inhibitor of both β-amyloid aggregation (hAChE- and self-induced, 61.3% and 58.4%, respectively) and hBACE-1 activity (IC50 = 1.35 μM). Kinetic studies showed that 2e acted as a slow, tight-binding, mixed-type inhibitor, while X-ray crystallographic studies highlighted the ability of 2e to induce large-scale structural changes in the active-site gorge of Torpedo californica AChE (TcAChE), with significant implications for structure-based drug design. In vivo studies confirmed that 2e significantly ameliorates performances of scopolamine-treated ICR mice. Finally, 2e administration did not exhibit significant hepatotoxicity.

  14. Synthesis, X-ray crystallography characterization, vibrational spectroscopic, molecular electrostatic potential maps, thermodynamic properties studies of N,N'-di(p-thiazole)formamidine.

    Science.gov (United States)

    Rofouei, M K; Fereyduni, E; Sohrabi, N; Shamsipur, M; Attar Gharamaleki, J; Sundaraganesan, N

    2011-01-01

    In this work, we will report a combined experimental and theoretical study on molecular and vibrational structure of N,N'-di(p-thiazole)formamidine (DpTF). DpTF has been synthesized and characterized by elemental analysis, FT-IR, FT-Raman, 1H NMR, 13C NMR spectroscopy and X-ray single crystal diffraction. The FT-IR and FT-Raman spectra of DpTF were recorded in the solid phase. The optimized geometry was calculated by HF and B3LYP methods using 6-31G(d) basis set. The FT-IR and FT-Raman spectra of DpTF was calculated at the HF/B3LYP/6-31G(d) level and were interpreted in terms of potential energy distribution (PED) analysis. The scaled theoretical wavenumber showed very good agreement with the experimental values. A detailed interpretation of the infrared and Raman spectra of DpTF was reported. On the basis of vibrational analyses, the thermodynamic properties of the title compound at different temperatures have been calculated, revealing the correlations between Cp,m°, Sm°, Hm° and temperatures. Furthermore, molecular electrostatic potential maps (MESP) and total dipole moment properties of the compound have been calculated. Copyright © 2010 Elsevier B.V. All rights reserved.

  15. Smarter Drugs: How Protein Crystallography Revolutionizes Drug Design

    International Nuclear Information System (INIS)

    Smith, Clyde

    2005-01-01

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

  16. Protein preparation, crystallization and preliminary X-ray crystallographic analysis of SMU.961 protein from the caries pathogen Streptococcus mutans

    International Nuclear Information System (INIS)

    Gao, Xiong-Zhuo; Li, Lan-Fen; Su, Xiao-Dong; Zhao, XiaoJun; Liang, Yu-He

    2007-01-01

    The SMU.961 protein from S. mutans was crystallized and preliminary characterization of the crystals, which diffracted to 2.9 Å resolution, shows them to belong to space group C2. The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni 2+ -chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 Å resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 Å, β = 98.82°

  17. Protein preparation, crystallization and preliminary X-ray crystallographic analysis of SMU.961 protein from the caries pathogen Streptococcus mutans

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Xiong-Zhuo [Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China); National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Li, Lan-Fen; Su, Xiao-Dong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Zhao, XiaoJun, E-mail: zhaoxj@scu.edu.cn [Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China); Liang, Yu-He, E-mail: zhaoxj@scu.edu.cn [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China)

    2007-10-01

    The SMU.961 protein from S. mutans was crystallized and preliminary characterization of the crystals, which diffracted to 2.9 Å resolution, shows them to belong to space group C2. The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni{sup 2+}-chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 Å resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 Å, β = 98.82°.

  18. Assessment of blood serum immunoglobulin and C-reactive protein concentrations in workers of X-ray diagnostics units

    International Nuclear Information System (INIS)

    Klucinski, P.; Martirosian, G.; Mazur, B.; Kaufman, J.; Hrycek, A.; Cieslak, P.

    2005-01-01

    Workers of X-rays departments are occupationally exposed to long-term low levels of ionizing radiation. The aim of the study was to investigate the influence of occupational exposure of low-level X-ray radiation on immunoglobulin and C-reactive protein (CRP) concentrations in radiology workers. In the study group of 41 X-ray department workers and the control group composed of 32 persons, immunoglobulins (IgM, IgG,IgA) and CPR concentrations were analyzed. The study group was subdivided by gender and smoking habit. A significant decrease in IgG level was found in the workers and the female subgroup. The same observation was made when smokers and nonsmokers of both groups were compared. Smoking workers showed lower concentrations of IgA than non-smokers. Occupational exposure to low levels of ionizing radiation is associated with suppressive influence on the immunoglobulin production, especially IgG. In addition, smoking decreases the production of IgA in radiology workers. (author)

  19. Crystallization and preliminary X-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2

    International Nuclear Information System (INIS)

    Rodríguez Guilbe, María M.; Alfaro Malavé, Elisa C.; Akerboom, Jasper; Marvin, Jonathan S.; Looger, Loren L.; Schreiter, Eric R.

    2008-01-01

    The genetically encoded fluorescent calcium-indicator protein GCaMP2 was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution and the structure was solved by molecular replacement. Fluorescent proteins and their engineered variants have played an important role in the study of biology. The genetically encoded calcium-indicator protein GCaMP2 comprises a circularly permuted fluorescent protein coupled to the calcium-binding protein calmodulin and a calmodulin target peptide, M13, derived from the intracellular calmodulin target myosin light-chain kinase and has been used to image calcium transients in vivo. To aid rational efforts to engineer improved variants of GCaMP2, this protein was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 126.1, b = 47.1, c = 68.8 Å, β = 100.5° and one GCaMP2 molecule in the asymmetric unit. The structure was phased by molecular replacement and refinement is currently under way

  20. The protein micro-crystallography beamlines for targeted protein research program

    International Nuclear Information System (INIS)

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

    2010-01-01

    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. (author)

  1. Osmium(III) analogues of KP1019: Electrochemical and chemical synthesis, spectroscopic characterization, x-ray crystallography, hydrolytic stability, and antiproliferative activity

    KAUST Repository

    Kuhn, Paul-Steffen

    2014-10-20

    A one-electron reduction of osmium(IV) complexes trans-[OsIVCl4(Hazole)2], where Hazole = 1H-pyrazole ([1]0), 2H-indazole ([2]0), 1H-imidazole ([3]0), and 1H-benzimidazole ([4]0), afforded a series of eight new complexes as osmium analogues of KP1019, a lead anticancer drug in clinical trials, with the general formula (cation)[trans-OsIIICl4(Hazole)2], where cation = H2pz+ (H2pz[1]), H2ind+ (H2ind[2]), H2im+ (H2im[3]), Ph4P+ (Ph4P[3]), nBu4N+ (nBu4N[3]), H2bzim+ (H2bzim[4]), Ph4P+ (Ph4P[4]), and nBu4N+ (nBu4N[4]). All complexes were characterized by elemental analysis, 1H NMR spectroscopy, electrospray ionization mass spectrometry, UV-vis spectroscopy, cyclic voltammetry, while H2pz[1], H2ind[2], and nBu4[3], in addition, by X-ray diffraction. The reduced species [1]- and [4]- are stable in aqueous media in the absence of air oxygen and do not react with small biomolecules such as amino acids and the nucleotide 5′-dGMP. Cell culture experiments in five different human cancer cell lines (HeLa, A549, FemX, MDA-MB-453, and LS-174) and one noncancerous cell line (MRC-5) were performed, and the results were discussed and compared to those for KP1019 and cisplatin. Benzannulation in complexes with similar structure enhances antitumor activity by several orders of magnitude, implicating different mechanisms of action of the tested compounds. In particular, complexes H2ind[2] and H2bzim[4] exhibited significant antiproliferative activity in vitro when compared to H2pz[1] and H2im[3]. (Chemical Equation Presented).

  2. Osmium(III) analogues of KP1019: electrochemical and chemical synthesis, spectroscopic characterization, X-ray crystallography, hydrolytic stability, and antiproliferative activity.

    Science.gov (United States)

    Kuhn, Paul-Steffen; Büchel, Gabriel E; Jovanović, Katarina K; Filipović, Lana; Radulović, Siniša; Rapta, Peter; Arion, Vladimir B

    2014-10-20

    A one-electron reduction of osmium(IV) complexes trans-[Os(IV)Cl4(Hazole)2], where Hazole = 1H-pyrazole ([1](0)), 2H-indazole ([2](0)), 1H-imidazole ([3](0)), and 1H-benzimidazole ([4](0)), afforded a series of eight new complexes as osmium analogues of KP1019, a lead anticancer drug in clinical trials, with the general formula (cation)[trans-Os(III)Cl4(Hazole)2], where cation = H2pz(+) (H2pz[1]), H2ind(+) (H2ind[2]), H2im(+) (H2im[3]), Ph4P(+) (Ph4P[3]), nBu4N(+) (nBu4N[3]), H2bzim(+) (H2bzim[4]), Ph4P(+) (Ph4P[4]), and nBu4N(+) (nBu4N[4]). All complexes were characterized by elemental analysis, (1)H NMR spectroscopy, electrospray ionization mass spectrometry, UV-vis spectroscopy, cyclic voltammetry, while H2pz[1], H2ind[2], and nBu4[3], in addition, by X-ray diffraction. The reduced species [1](-) and [4](-) are stable in aqueous media in the absence of air oxygen and do not react with small biomolecules such as amino acids and the nucleotide 5'-dGMP. Cell culture experiments in five different human cancer cell lines (HeLa, A549, FemX, MDA-MB-453, and LS-174) and one noncancerous cell line (MRC-5) were performed, and the results were discussed and compared to those for KP1019 and cisplatin. Benzannulation in complexes with similar structure enhances antitumor activity by several orders of magnitude, implicating different mechanisms of action of the tested compounds. In particular, complexes H2ind[2] and H2bzim[4] exhibited significant antiproliferative activity in vitro when compared to H2pz[1] and H2im[3].

  3. Osmium(III) analogues of KP1019: Electrochemical and chemical synthesis, spectroscopic characterization, x-ray crystallography, hydrolytic stability, and antiproliferative activity

    KAUST Repository

    Kuhn, Paul-Steffen; Bü chel, Gabriel E.; Jovanović, Katarina K.; Filipović, Lana; Radulović, Siniša S.; Rapta, Peter; Arion, Vladimir B.

    2014-01-01

    A one-electron reduction of osmium(IV) complexes trans-[OsIVCl4(Hazole)2], where Hazole = 1H-pyrazole ([1]0), 2H-indazole ([2]0), 1H-imidazole ([3]0), and 1H-benzimidazole ([4]0), afforded a series of eight new complexes as osmium analogues of KP1019, a lead anticancer drug in clinical trials, with the general formula (cation)[trans-OsIIICl4(Hazole)2], where cation = H2pz+ (H2pz[1]), H2ind+ (H2ind[2]), H2im+ (H2im[3]), Ph4P+ (Ph4P[3]), nBu4N+ (nBu4N[3]), H2bzim+ (H2bzim[4]), Ph4P+ (Ph4P[4]), and nBu4N+ (nBu4N[4]). All complexes were characterized by elemental analysis, 1H NMR spectroscopy, electrospray ionization mass spectrometry, UV-vis spectroscopy, cyclic voltammetry, while H2pz[1], H2ind[2], and nBu4[3], in addition, by X-ray diffraction. The reduced species [1]- and [4]- are stable in aqueous media in the absence of air oxygen and do not react with small biomolecules such as amino acids and the nucleotide 5′-dGMP. Cell culture experiments in five different human cancer cell lines (HeLa, A549, FemX, MDA-MB-453, and LS-174) and one noncancerous cell line (MRC-5) were performed, and the results were discussed and compared to those for KP1019 and cisplatin. Benzannulation in complexes with similar structure enhances antitumor activity by several orders of magnitude, implicating different mechanisms of action of the tested compounds. In particular, complexes H2ind[2] and H2bzim[4] exhibited significant antiproliferative activity in vitro when compared to H2pz[1] and H2im[3]. (Chemical Equation Presented).

  4. Unusual saccharin-N,O (carbonyl) coordination in mixed-ligand copper(II) complexes: Synthesis, X-ray crystallography and biological activity

    Science.gov (United States)

    Mokhtaruddin, Nur Shuhada Mohd; Yusof, Enis Nadia Md; Ravoof, Thahira B. S. A.; Tiekink, Edward R. T.; Veerakumarasivam, Abhi; Tahir, Mohamed Ibrahim Mohamed

    2017-07-01

    Three tridentate Schiff bases containing N and S donor atoms were synthesized via the condensation reaction between S-2-methylbenzyldithiocarbazate with 2-acetyl-4-methylpyridine (S2APH); 4-methyl-3-thiosemicarbazide with 2-acetylpyridine (MT2APH) and 4-ethyl-3-thiosemicarbazide with 2-acetylpyridine (ET2APH). Three new, binuclear and mixed-ligand copper(II) complexes with the general formula, [Cu(sac)(L)]2 (sac = saccharinate anion; L = anion of the Schiff base) were then synthesized, and subsequently characterized by IR and UV/Vis spectroscopy as well as by molar conductivity and magnetic susceptibility measurements. The Schiff bases were also spectroscopically characterized using NMR and MS to further confirm their structures. The spectroscopic data indicated that the Schiff bases behaved as a tridentate NNS donor ligands coordinating via the pyridyl-nitrogen, azomethine-nitrogen and thiolate-sulphur atoms. Magnetic data indicated a square pyramidal environment for the complexes and the conductivity values showed that the complexes were essentially non-electrolytes in DMSO. The X-ray crystallographic analysis of one complex, [Cu(sac)(S2AP)]2 showed that the Cu(II) atom was coordinated to the thiolate-S, azomethine-N and pyridyl-N donors of the S2AP Schiff base and to the saccharinate-N from one anion, as well as to the carbonyl-O atom from a symmetry related saccharinate anion yielding a centrosymmetric binuclear complex with a penta-coordinate, square pyramidal geometry. All the copper(II) saccharinate complexes were found to display strong cytotoxic activity against the MCF-7 and MDA-MB-231 human breast cancer cell lines.

  5. Total external reflection X-ray fluorescence analysis of protein-metal ion interactions in biological systems

    Science.gov (United States)

    Novikova, N. N.; Kovalchuk, M. V.; Yur'eva, E. A.; Konovalov, O. V.; Rogachev, A. V.; Stepina, N. D.; Sukhorukov, V. S.; Tsaregorodtsev, A. D.; Chukhrai, E. S.; Yakunin, S. N.

    2012-09-01

    This paper presents the results of an investigation into hemoglobin-based protein films that were formed on a liquid surface. X-ray standing wave measurements were performed at the ID 10 beamline of the European Synchrotron Radiation Facility (ESRF) and at the Langmuir station of the Kurchatov Synchrotron Radiation Source. It was found that the ability of the protein to bind metal ions is substantially increased due to the conformational rearrangements of protein macromolecules caused by various damaging effects. The elemental composition of protein preparations, which were isolated from children and adults with chronic metabolic diseases accompanied by endogenous intoxication, was analyzed. The results of the investigations offer evidence that an increase in the ligand-binding properties of the protein molecules, which was observed in model experiments using protein films, is a common trait and corresponds to in vivo processes accompanying metabolic disturbances in the body.

  6. Status of the digital pixel array detector for protein crystallography

    CERN Document Server

    Datte, P; Beuville, E; Endres, N; Druillole, F; Luo, L; Millaud, J E; Xuong, N H

    1999-01-01

    A two-dimensional photon counting digital pixel array detector is being designed for static and time resolved protein crystallography. The room temperature detector will significantly enhance monochromatic and polychromatic protein crystallographic through-put data rates by more than three orders of magnitude. The detector has an almost infinite photon counting dynamic range and exhibits superior spatial resolution when compared to present crystallographic phosphor imaging plates or phosphor coupled CCD detectors. The detector is a high resistivity N-type Si with a pixel pitch of 150x150 mu m, and a thickness of 300 mu m, and is bump bonded to an application specific integrated circuit. The event driven readout of the detector is based on the column architecture and allows an independent pixel hit rate above 1 million photons/s/pixel. The device provides energy discrimination and sparse data readout which yields minimal dead-time. This type of architecture allows a continuous (frameless) data acquisition, a f...

  7. Structure of HI-6*sarin-acetylcholinesterase determined by X-ray crystallography and molecular dynamics simulation: reactivator mechanism and design.

    Directory of Open Access Journals (Sweden)

    Fredrik Ekström

    2009-06-01

    Full Text Available Organophosphonates such as isopropyl metylphosphonofluoridate (sarin are extremely toxic as they phosphonylate the catalytic serine residue of acetylcholinesterase (AChE, an enzyme essential to humans and other species. Design of effective AChE reactivators as antidotes to various organophosphonates requires information on how the reactivators interact with the phosphonylated AChEs. However, such information has not been available hitherto because of three main challenges. First, reactivators are generally flexible in order to change from the ground state to the transition state for reactivation; this flexibility discourages determination of crystal structures of AChE in complex with effective reactivators that are intrinsically disordered. Second, reactivation occurs upon binding of a reactivator to the phosphonylated AChE. Third, the phosphorous conjugate can develop resistance to reactivation. We have identified crystallographic conditions that led to the determination of a crystal structure of the sarin(nonaged-conjugated mouse AChE in complex with [(E-[1-[(4-carbamoylpyridin-1-ium-1-ylmethoxymethyl]pyridin-2-ylidene]methyl]-oxoazanium dichloride (HI-6 at a resolution of 2.2 A. In this structure, the carboxyamino-pyridinium ring of HI-6 is sandwiched by Tyr124 and Trp286, however, the oxime-pyridinium ring is disordered. By combining crystallography with microsecond molecular dynamics simulation, we determined the oxime-pyridinium ring structure, which shows that the oxime group of HI-6 can form a hydrogen-bond network to the sarin isopropyl ether oxygen, and a water molecule is able to form a hydrogen bond to the catalytic histidine residue and subsequently deprotonates the oxime for reactivation. These results offer insights into the reactivation mechanism of HI-6 and design of better reactivators.

  8. Synthesis and X-ray Crystallography of [Mg(H2O)6][AnO2(C2H5COO)3]2 (An = U, Np, or Pu).

    Science.gov (United States)

    Serezhkin, Viktor N; Grigoriev, Mikhail S; Abdulmyanov, Aleksey R; Fedoseev, Aleksandr M; Savchenkov, Anton V; Serezhkina, Larisa B

    2016-08-01

    Synthesis and X-ray crystallography of single crystals of [Mg(H2O)6][AnO2(C2H5COO)3]2, where An = U (I), Np (II), or Pu (III), are reported. Compounds I-III are isostructural and crystallize in the trigonal crystal system. The structures of I-III are built of hydrated magnesium cations [Mg(H2O)6](2+) and mononuclear [AnO2(C2H5COO)3](-) complexes, which belong to the AB(01)3 crystallochemical group of uranyl complexes (A = AnO2(2+), B(01) = C2H5COO(-)). Peculiarities of intermolecular interactions in the structures of [Mg(H2O)6][UO2(L)3]2 complexes depending on the carboxylate ion L (acetate, propionate, or n-butyrate) are investigated using the method of molecular Voronoi-Dirichlet polyhedra. Actinide contraction in the series of U(VI)-Np(VI)-Pu(VI) in compounds I-III is reflected in a decrease in the mean An═O bond lengths and in the volume and sphericity degree of Voronoi-Dirichlet polyhedra of An atoms.

  9. Direct imaging electron microscopy (EM) methods in modern structural biology: overview and comparison with X-ray crystallography and single-particle cryo-EM reconstruction in the studies of large macromolecules.

    Science.gov (United States)

    Miyaguchi, Katsuyuki

    2014-10-01

    Determining the structure of macromolecules is important for understanding their function. The fine structure of large macromolecules is currently studied primarily by X-ray crystallography and single-particle cryo-electron microscopy (EM) reconstruction. Before the development of these techniques, macromolecular structure was often examined by negative-staining, rotary-shadowing and freeze-etching EM, which are categorised here as 'direct imaging EM methods'. In this review, the results are summarised by each of the above techniques and compared with respect to four macromolecules: the ryanodine receptor, cadherin, rhodopsin and the ribosome-translocon complex (RTC). The results of structural analysis of the ryanodine receptor and cadherin are consistent between each technique. The results obtained for rhodopsin vary to some extent within each technique and between the different techniques. Finally, the results for RTC are inconsistent between direct imaging EM and other analytical techniques, especially with respect to the space within RTC, the reasons for which are discussed. Then, the role of direct imaging EM methods in modern structural biology is discussed. Direct imaging methods should support and verify the results obtained by other analytical methods capable of solving three-dimensional molecular architecture, and they should still be used as a primary tool for studying macromolecule structure in vivo. © 2014 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

  10. Humidity control and hydrophilic glue coating applied to mounted protein crystals improves X-ray diffraction experiments

    Science.gov (United States)

    Baba, Seiki; Hoshino, Takeshi; Ito, Len; Kumasaka, Takashi

    2013-01-01

    Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection. PMID:23999307

  11. Cloning, recombinant production, crystallization and preliminary X-ray diffraction analysis of SDF2-like protein from Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Radzimanowski, Jens; Ravaud, Stephanie; Schott, Andrea; Strahl, Sabine; Sinning, Irmgard

    2009-01-01

    Overexpression, purification, crystallization and preliminary X-ray diffraction of the stromal-cell-derived factor 2-like protein of Arabidopsis thaliana are reported. The crystals belonged to the space group P6 1 and diffracted to 1.95 Å resolution. The stromal-cell-derived factor 2-like protein of Arabidopsis thaliana (AtSDL) has been shown to be highly up-regulated in response to unfolded protein response (UPR) inducing reagents, suggesting that it plays a crucial role in the plant UPR pathway. AtSDL has been cloned, overexpressed, purified and crystallized using the vapour-diffusion method. Two crystal forms have been obtained under very similar conditions. The needle-shaped crystals did not diffract X-rays, while the other form diffracted to 1.95 Å resolution using a synchrotron-radiation source and belonged to the hexagonal space group P6 1 , with unit-cell parameters a = b = 96.1, c = 69.3 Å

  12. Automated microfluidic sample-preparation platform for high-throughput structural investigation of proteins by small-angle X-ray scattering

    DEFF Research Database (Denmark)

    Lafleur, Josiane P.; Snakenborg, Detlef; Nielsen, Søren Skou

    2011-01-01

    A new microfluidic sample-preparation system is presented for the structural investigation of proteins using small-angle X-ray scattering (SAXS) at synchrotrons. The system includes hardware and software features for precise fluidic control, sample mixing by diffusion, automated X-ray exposure...... control, UV absorbance measurements and automated data analysis. As little as 15 l of sample is required to perform a complete analysis cycle, including sample mixing, SAXS measurement, continuous UV absorbance measurements, and cleaning of the channels and X-ray cell with buffer. The complete analysis...

  13. Proteins on surfaces investigated by microbeam grazing incidence small angle X-ray scattering

    Energy Technology Data Exchange (ETDEWEB)

    Gebhardt, Ronald; Riekel, Christian; Burghammer, Manfred [European Synchrotron Radiation Facility, B.P. 220, F-38043 Grenoble Cedex (France); Vendrely, Charlotte [Universite de Cergy-Pontoise, ERRMECE, F-95000, Cergy-Pontoise (France); Mueller-Buschbaum, Peter [TU Muenchen, Physik Department E13, Muenchen (Germany)

    2009-07-01

    Grazing incidence small angle scattering with a 1 micron x-ray beam ({mu}GISAXS) is applied to study structural ordering of casein micelles and fibroin in solution cast films. {mu}GISAXS scans provide the possibility to locate highly ordered areas and to investigate variation in the molecular packing. In the case of the casein micelles, ordered film structures have been generated by decreasing their natural size dispersion. While dynamic light scattering was used to characterize the different size fractions in solution, {mu}GISAXS and roughness are measured on the resulting casein films. GISAXS-Patterns are analyzed by simulations providing the dimension and nearest neighbor distances of casein micelles. In the case of fibroin, ordering of nano-fibers formed during the drying process is investigated. The experimental data are analyzed by simulations and compared to SEM, AFM and Raman scattering experiments.

  14. In situ X-ray scattering studies of protein solution droplets drying on micro- and nanopatterned superhydrophobic PMMA surfaces.

    Science.gov (United States)

    Accardo, Angelo; Gentile, Francesco; Mecarini, Federico; De Angelis, Francesco; Burghammer, Manfred; Di Fabrizio, Enzo; Riekel, Christian

    2010-09-21

    Superhydrophobic poly(methyl methacrylate) surfaces with contact angles of ∼170° and high optical and X-ray transparencies have been fabricated through the use of optical lithography and plasma etching. The surfaces contain either a microscale pattern of micropillars or a random nanofibrillar pattern. Nanoscale asperities on top of the micropillars closely resemble Nelumbo nucifera lotus leaves. The evolution of the contact angle of water and lysozyme solution droplets during evaporation was studied on the micro- and nanopatterned surfaces, showing in particular contact-line pinning for the protein solution droplet on the nanopatterned surface. The microstructural evolution of lysozyme solution droplets was studied on both types of surfaces in situ under nearly contact-free conditions by synchrotron radiation microbeam wide-angle and small-angle X-ray scattering revealing the increasing protein concentration and the onset of precipitation. The solid residuals show hollow sphere morphologies. Rastermicrodiffraction of the detached residuals suggests about a 1/3 volume fraction of ≥17 nm lysozyme nanocrystalline domains and about a 2/3 short-range-order volume fraction. About 5-fold larger nanocrystalline domains were observed at the attachment points of the sphere to the substrates, which is attributed to particle growth in a shear flow. Such surfaces represent nearly contact-free sample supports for studies of inorganic and organic solution droplets, which find applications in biochips.

  15. Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of a cyclophilin A-like protein from Piriformospora indica

    International Nuclear Information System (INIS)

    Bhatt, Harshesh; Trivedi, Dipesh Kumar; Pal, Ravi Kant; Johri, Atul Kumar; Tuteja, Narendra; Bhavesh, Neel Sarovar

    2012-01-01

    Cyclophin A like protein from Piriformospora indica involved in salt-stress tolerance was cloned, overexpressed, purified and crystallized. The crystals obtained diffracted X-rays to 2 Å resolution and belonged to space group C222 1 . Cyclophilins are widely distributed both in eukaryotes and prokaryotes and have a primary role as peptidyl-prolyl cis–trans isomerases (PPIases). This study focuses on the cloning, expression, purification and crystallization of a salinity-stress-induced cyclophilin A (CypA) homologue from the symbiotic fungus Piriformospora indica. Crystallization experiments in the presence of 56 mM sodium phosphate monobasic monohydrate, 1.34 M potassium phosphate dibasic pH 8.2 yielded crystals that were suitable for X-ray diffraction analysis. The crystals belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 121.15, b = 144.12, c = 110.63 Å. The crystals diffracted to a resolution limit of 2.0 Å. Analysis of the diffraction data indicated the presence of three molecules of the protein per asymmetric unit (V M = 4.48 Å 3 Da −1 , 72.6% solvent content)

  16. Crystallization and preliminary X-ray diffraction analysis of the haem-binding protein HemS from Yersinia enterocolitica

    International Nuclear Information System (INIS)

    Schneider, Sabine; Paoli, Massimo

    2005-01-01

    The haem binding protein HemS from Y. enterocolitica has been crystallized in complex with its ligand. The crystals diffracted X-rays to 2.6 Å in-house. Bacteria have evolved strategies to acquire iron from their environment. Pathogenic microbes rely on specialized proteins to ‘steal’ haem from their host and use it as an iron source. HemS is the ultimate recipient of a molecular-relay system for haem uptake in Gram-negative species, functioning as the cytosolic carrier of haem. Soluble expression and high-quality diffraction crystals were obtained for HemS from Yersinia enterocolitica. Crystals belong to the orthorhombic space group I222, with unit-cell parameters a = 74.86, b = 77.45, c = 114.09 Å, and diffract X-rays to 2.6 Å spacing in-house. Determination of the structure of the haem–HemS complex will reveal the molecular basis of haem binding

  17. Crystallization and preliminary X-ray diffraction analysis of the haem-binding protein HemS from Yersinia enterocolitica

    Energy Technology Data Exchange (ETDEWEB)

    Schneider, Sabine; Paoli, Massimo, E-mail: max.paoli@nottingham.ac.uk [School of Pharmacy and Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom)

    2005-08-01

    The haem binding protein HemS from Y. enterocolitica has been crystallized in complex with its ligand. The crystals diffracted X-rays to 2.6 Å in-house. Bacteria have evolved strategies to acquire iron from their environment. Pathogenic microbes rely on specialized proteins to ‘steal’ haem from their host and use it as an iron source. HemS is the ultimate recipient of a molecular-relay system for haem uptake in Gram-negative species, functioning as the cytosolic carrier of haem. Soluble expression and high-quality diffraction crystals were obtained for HemS from Yersinia enterocolitica. Crystals belong to the orthorhombic space group I222, with unit-cell parameters a = 74.86, b = 77.45, c = 114.09 Å, and diffract X-rays to 2.6 Å spacing in-house. Determination of the structure of the haem–HemS complex will reveal the molecular basis of haem binding.

  18. Small-angle X-Ray analysis of macromolecular structure: the structure of protein NS2 (NEP) in solution

    Science.gov (United States)

    Shtykova, E. V.; Bogacheva, E. N.; Dadinova, L. A.; Jeffries, C. M.; Fedorova, N. V.; Golovko, A. O.; Baratova, L. A.; Batishchev, O. V.

    2017-11-01

    A complex structural analysis of nuclear export protein NS2 (NEP) of influenza virus A has been performed using bioinformatics predictive methods and small-angle X-ray scattering data. The behavior of NEP molecules in a solution (their aggregation, oligomerization, and dissociation, depending on the buffer composition) has been investigated. It was shown that stable associates are formed even in a conventional aqueous salt solution at physiological pH value. For the first time we have managed to get NEP dimers in solution, to analyze their structure, and to compare the models obtained using the method of the molecular tectonics with the spatial protein structure predicted by us using the bioinformatics methods. The results of the study provide a new insight into the structural features of nuclear export protein NS2 (NEP) of the influenza virus A, which is very important for viral infection development.

  19. Cloning, purification, crystallization and preliminary X-ray analysis of the Burkholderia pseudomallei L1 ribosomal protein

    International Nuclear Information System (INIS)

    Abd Aziz, Abd Ghani; Ruzheinikov, Sergey N.; Sedelnikova, Svetlana E.; Mohamed, Rahmah; Nathan, Sheila; Baker, Patrick J.; Rice, David W.

    2012-01-01

    The L1 ribosomal protein from B. pseudomallei has been overexpressed, purified and crystallized in a form suitable for X-ray analysis. The gene encoding the L1 ribosomal protein from Burkholderia pseudomallei strain D286 has been cloned into the pETBLUE-1 vector system, overexpressed in Escherichia coli and purified. Crystals of the native protein were grown by the hanging-drop vapour-diffusion technique using PEG 3350 as a precipitant and diffracted to beyond 1.65 Å resolution. The crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 53.6, b = 127.1, c = 31.8 Å and with a single molecule in the asymmetric unit

  20. Calculation of X-ray scattering curves and electron distance distribution functions of biological macromolecules in solution using the PROTEIN DATA BANK

    International Nuclear Information System (INIS)

    Mueller, J.J.; Friedrichowicz, E.; Nothnagel, A.; Wunderlich, T.; Ziehlsdorf, E.; Damaschun, G.

    1983-01-01

    The wide angle X-ray scattering curve, the electron distance distribution function and the solvent excluded volume of a macromolecule in solution are calculated from the atomic coordinates contained in the PROTEIN DATA BANK. The structures and the projections of the excluded volumes are depicted using molecule graphic routines. The described computer programs are used to determine the three-dimensional structure of macromolecules in solution from wide angle X-ray scattering data. (author)

  1. X-Ray

    Science.gov (United States)

    ... enema. What you can expect During the X-ray X-rays are performed at doctors' offices, dentists' offices, ... as those using a contrast medium. Your child's X-ray Restraints or other techniques may be used to ...

  2. Abdominal x-ray

    Science.gov (United States)

    Abdominal film; X-ray - abdomen; Flat plate; KUB x-ray ... There is low radiation exposure. X-rays are monitored and regulated to provide the minimum amount of radiation exposure needed to produce the image. Most ...

  3. Chest X-Ray

    Medline Plus

    Full Text Available ... talk with you about chest radiography also known as chest x-rays. Chest x-rays are the ... treatment for a variety of lung conditions such as pneumonia, emphysema and cancer. A chest x-ray ...

  4. X-ray diffraction

    International Nuclear Information System (INIS)

    Einstein, J.R.; Wei, C.H.

    1982-01-01

    We have been interested in structural elucidation by x-ray diffraction of compounds of biological interest. Understanding exactly how atoms are arranged in three-dimensional arrays as molecules can help explain the relationship between structure and functions. The species investigated may vary in size and shape; our recent studies included such diverse substances as antischistosomal drugs, a complex of cadmium with nucleic acid base, nitrate salts of adenine, and proteins

  5. X-ray astronomy

    International Nuclear Information System (INIS)

    Giacconi, R.; Gursky, H.

    1974-01-01

    This text contains ten chapters and three appendices. Following an introduction, chapters two through five deal with observational techniques, mechanisms for the production of x rays in a cosmic setting, the x-ray sky and solar x-ray emission. Chapters six through ten include compact x-ray sources, supernova remnants, the interstellar medium, extragalactic x-ray sources and the cosmic x-ray background. Interactions of x rays with matter, units and conversion factors and a catalog of x-ray sources comprise the three appendices. (U.S.)

  6. Serial femtosecond X-ray diffraction of enveloped virus microcrystals

    Directory of Open Access Journals (Sweden)

    Robert M. Lawrence

    2015-07-01

    Full Text Available Serial femtosecond crystallography (SFX using X-ray free-electron lasers has produced high-resolution, room temperature, time-resolved protein structures. We report preliminary SFX of Sindbis virus, an enveloped icosahedral RNA virus with ∼700 Å diameter. Microcrystals delivered in viscous agarose medium diffracted to ∼40 Å resolution. Small-angle diffuse X-ray scattering overlaid Bragg peaks and analysis suggests this results from molecular transforms of individual particles. Viral proteins undergo structural changes during entry and infection, which could, in principle, be studied with SFX. This is an important step toward determining room temperature structures from virus microcrystals that may enable time-resolved studies of enveloped viruses.

  7. Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: Insights from X-ray crystallography.

    Science.gov (United States)

    Kumaran, Desigan; Adler, Michael; Levit, Matthew; Krebs, Michael; Sweeney, Richard; Swaminathan, Subramanyam

    2015-11-15

    The seven antigenically distinct serotypes (A-G) of botulinum neurotoxin (BoNT) are responsible for the deadly disease botulism. BoNT serotype A (BoNT/A) exerts its lethal action by cleaving the SNARE protein SNAP-25, leading to inhibition of neurotransmitter release, flaccid paralysis and autonomic dysfunction. BoNTs are dichain proteins consisting of a ∼ 100 kDa heavy chain and a ∼ 50 kDa light chain; the former is responsible for neurospecific binding, internalization and translocation, and the latter for cleavage of neuronal SNARE proteins. Because of their extreme toxicity and history of weaponization, the BoNTs are regarded as potential biowarfare/bioterrorism agents. No post-symptomatic therapeutic interventions are available for BoNT intoxication other than intensive care; therefore it is imperative to develop specific antidotes against this neurotoxin. To this end, a cyclic peptide inhibitor (CPI-1) was evaluated in a FRET assay for its ability to inhibit BoNT/A light chain (Balc). CPI was found to be highly potent, exhibiting a Ki of 12.3 nM with full-length Balc448 and 39.2 nM using a truncated crystallizable form of the light chain (Balc424). Cocrystallization studies revealed that in the Balc424-CPI-1 complex, the inhibitor adopts a helical conformation, occupies a high percentage of the active site cavity and interacts in an amphipathic manner with critical active site residues. The data suggest that CPI-1 prevents SNAP-25 from accessing the Balc active site by blocking both the substrate binding path at the surface and the Zn(2+) binding region involved in catalysis. This differs from linear peptide inhibitors described to date which block only the latter. Published by Elsevier Ltd.

  8. Breaking Symmetry in Viral Icosahedral Capsids as Seen through the Lenses of X-ray Crystallography and Cryo-Electron Microscopy

    Directory of Open Access Journals (Sweden)

    Kristin N. Parent

    2018-02-01

    Full Text Available The majority of viruses on Earth form capsids built by multiple copies of one or more types of a coat protein arranged with 532 symmetry, generating an icosahedral shell. This highly repetitive structure is ideal to closely pack identical protein subunits and to enclose the nucleic acid genomes. However, the icosahedral capsid is not merely a passive cage but undergoes dynamic events to promote packaging, maturation and the transfer of the viral genome into the host. These essential processes are often mediated by proteinaceous complexes that interrupt the shell’s icosahedral symmetry, providing a gateway through the capsid. In this review, we take an inventory of molecular structures observed either internally, or at the 5-fold vertices of icosahedral DNA viruses that infect bacteria, archea and eukaryotes. Taking advantage of the recent revolution in cryo-electron microscopy (cryo-EM and building upon a wealth of crystallographic structures of individual components, we review the design principles of non-icosahedral structural components that interrupt icosahedral symmetry and discuss how these macromolecules play vital roles in genome packaging, ejection and host receptor-binding.

  9. Do All X-ray Structures of Protein-Ligand Complexes Represent Functional States? EPOR, a Case Study.

    Science.gov (United States)

    Corbett, Michael S P; Mark, Alan E; Poger, David

    2017-02-28

    Based on differences between the x-ray crystal structures of ligand-bound and unbound forms, the activation of the erythropoietin receptor (EPOR) was initially proposed to involve a cross-action scissorlike motion. However, the validity of the motions involved in the scissorlike model has been recently challenged. Here, atomistic molecular dynamics simulations are used to examine the structure of the extracellular domain of the EPOR dimer in the presence and absence of erythropoietin and a series of agonistic or antagonistic mimetic peptides free in solution. The simulations suggest that in the absence of crystal packing effects, the EPOR chains in the different dimers adopt very similar conformations with no clear distinction between the agonist and antagonist-bound complexes. This questions whether the available x-ray crystal structures of EPOR truly represent active or inactive conformations. The study demonstrates the difficulty in using such structures to infer a mechanism of action, especially in the case of membrane receptors where just part of the structure has been considered in addition to potential confounding effects that arise from the comparison of structures in a crystal as opposed to a membrane environment. The work highlights the danger of assigning functional significance to small differences between structures of proteins bound to different ligands in a crystal environment without consideration of the effects of the crystal lattice and thermal motion. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. The structure of denisovite, a fibrous nanocrystalline polytypic disordered `very complex' silicate, studied by a synergistic multi-disciplinary approach employing methods of electron crystallography and X-ray powder diffraction

    Directory of Open Access Journals (Sweden)

    Ira V. Rozhdestvenskaya

    2017-05-01

    Full Text Available Denisovite is a rare mineral occurring as aggregates of fibres typically 200–500 nm diameter. It was confirmed as a new mineral in 1984, but important facts about its chemical formula, lattice parameters, symmetry and structure have remained incompletely known since then. Recently obtained results from studies using microprobe analysis, X-ray powder diffraction (XRPD, electron crystallography, modelling and Rietveld refinement will be reported. The electron crystallography methods include transmission electron microscopy (TEM, selected-area electron diffraction (SAED, high-angle annular dark-field imaging (HAADF, high-resolution transmission electron microscopy (HRTEM, precession electron diffraction (PED and electron diffraction tomography (EDT. A structural model of denisovite was developed from HAADF images and later completed on the basis of quasi-kinematic EDT data by ab initio structure solution using direct methods and least-squares refinement. The model was confirmed by Rietveld refinement. The lattice parameters are a = 31.024 (1, b = 19.554 (1 and c = 7.1441 (5 Å, β = 95.99 (3°, V = 4310.1 (5 Å3 and space group P12/a1. The structure consists of three topologically distinct dreier silicate chains, viz. two xonotlite-like dreier double chains, [Si6O17]10−, and a tubular loop-branched dreier triple chain, [Si12O30]12−. The silicate chains occur between three walls of edge-sharing (Ca,Na octahedra. The chains of silicate tetrahedra and the octahedra walls extend parallel to the z axis and form a layer parallel to (100. Water molecules and K+ cations are located at the centre of the tubular silicate chain. The latter also occupy positions close to the centres of eight-membered rings in the silicate chains. The silicate chains are geometrically constrained by neighbouring octahedra walls and present an ambiguity with respect to their z position along these walls, with displacements between neighbouring layers being

  11. Shared active site architecture between archaeal PolD and multi-subunit RNA polymerases revealed by X-ray crystallography.

    Science.gov (United States)

    Sauguet, Ludovic; Raia, Pierre; Henneke, Ghislaine; Delarue, Marc

    2016-08-22

    Archaeal replicative DNA polymerase D (PolD) constitute an atypical class of DNA polymerases made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2), both with unknown structures. We have determined the crystal structures of Pyrococcus abyssi DP1 and DP2 at 2.5 and 2.2 Å resolution, respectively, revealing a catalytic core strikingly different from all other known DNA polymerases (DNAPs). Rather, the PolD DP2 catalytic core has the same 'double-psi β-barrel' architecture seen in the RNA polymerase (RNAP) superfamily, which includes multi-subunit transcriptases of all domains of life, homodimeric RNA-silencing pathway RNAPs and atypical viral RNAPs. This finding bridges together, in non-viral world, DNA transcription and DNA replication within the same protein superfamily. This study documents further the complex evolutionary history of the DNA replication apparatus in different domains of life and proposes a classification of all extant DNAPs.

  12. Crystallization and preliminary x-ray crystallography data of the dimer of tetramer s (abcd)2 of extracellular hemoglobin from Glossoscolex paulistus in cyano met form

    International Nuclear Information System (INIS)

    Ferreira, Frederico M.; Oliveira, Paulo S.L. de; Oliva, Glaucius

    1996-01-01

    Full text. The extracellular hemoglobin from Glossoscolex paulistus has a molecular weight near to 3.1 x 10 6 Da and a structure organized in a double-layered hexagonal oligomer. The tertiary complex of dimer of tetramers (abcd) 2 was obtained by chromography in Sephadex G-200, in pH 9.0, as a result of alkaline dissociation. Aiming to obtain a better understanding of the oligomeric structure and specially for the inter subunit interactions the extracellular hemoglobins, we have obtained crystals of dimer of tetramers (abcd) 2 of hemoglobin from Glossoscolex and we are studying the in behavior in different conditions of precipitants and pH's. Our goal is to solve the crystal structure in order to characterize, at atomic level, the subunits contacts, heme environment and differences in residues involved in oxygenation in order to understand in this hemoglobin. The crystallization experiments the protein concentration in the cyanomet form was about 10 mg/ml and the experiments were carried out at 18 0 C. The optimal crystallization condition achieved from factorial assays was 10% (w/v). Polyethylene glycol (PEG) 8,000 and 8%(v/v) ethylene glycol in 100 mM HEPES pH 7.5. The optimization of this condition was carried out with the variation of PEG concentrations from 6% up to 10% (by 1% step) and pH between 7.0 and 8.0. A quite critical p-H-dependence has been observed on crystal nucleation, decreasing from pH 7.0, in which the number of microcrystals in higher, up to pH 8.0, in which crystals did not appear even at higher PEG 8,000 (10% w/v). As several structures of hemoglobin from different sources (vertebrate and invertebrates) are available, we hope to solve their structure of hemoglobin from Glossoscolex paulistus by Molecular Replacement, even though the tetramer organization may be different in the earthworm as compared related to other known tetrameric hemoglobin structures. (author)

  13. Chest X-Ray

    Medline Plus

    Full Text Available ... about chest radiography also known as chest x-rays. Chest x-rays are the most commonly performed x-ray exams and use a very small dose of ... of the inside of the chest. A chest x-ray is used to evaluate the lungs, heart and ...

  14. X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex

    Science.gov (United States)

    Zhou, X. Edward; Gao, Xiang; Barty, Anton; Kang, Yanyong; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W.; Suino-Powell, Kelly M.; Boutet, Sébastien; Williams, Garth J.; Wang, Meitian; Li, Dianfan; Caffrey, Martin; Chapman, Henry N.; Spence, John C. H.; Fromme, Petra; Weierstall, Uwe; Stevens, Raymond C.; Cherezov, Vadim; Melcher, Karsten; Xu, H. Eric

    2016-04-01

    Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes.

  15. Crystallization and preliminary X-ray diffraction analysis of central structure domains from mumps virus F protein

    International Nuclear Information System (INIS)

    Liu, Yueyong; Xu, Yanhui; Zhu, Jieqing; Qiu, Bingsheng; Rao, Zihe; Gao, George F.; Tien, Po

    2005-01-01

    Single crystals of the central structure domains from mumps virus F protein have been obtained by the hanging-drop vapour-diffusion method. A diffraction data set has been collected to 2.2 Å resolution. Fusion of members of the Paramyxoviridae family involves two glycoproteins: the attachment protein and the fusion protein. Changes in the fusion-protein conformation were caused by binding of the attachment protein to the cellular receptor. In the membrane-fusion process, two highly conserved heptad-repeat (HR) regions, HR1 and HR2, are believed to form a stable six-helix coiled-coil bundle. However, no crystal structure has yet been determined for this state in the mumps virus (MuV, a member of the Paramyxoviridae family). In this study, a single-chain protein consisting of two HR regions connected by a flexible amino-acid linker (named 2-Helix) was expressed, purified and crystallized by the hanging-drop vapour-diffusion method. A complete X-ray data set was obtained in-house to 2.2 Å resolution from a single crystal. The crystal belongs to space group C2, with unit-cell parameters a = 161.2, b = 60.8, c = 40.1 Å, β = 98.4°. The crystal structure will help in understanding the molecular mechanism of Paramyxoviridae family membrane fusion

  16. X-ray sky

    International Nuclear Information System (INIS)

    Gruen, M.; Koubsky, P.

    1977-01-01

    The history is described of the discoveries of X-ray sources in the sky. The individual X-ray detectors are described in more detail, i.e., gas counters, scintillation detectors, semiconductor detectors, and the principles of X-ray spectrometry and of radiation collimation aimed at increased resolution are discussed. Currently, over 200 celestial X-ray sources are known. Some were identified as nebulae, in some pulsations were found or the source was identified as a binary star. X-ray bursts of novae were also observed. The X-ray radiation is briefly mentioned of spherical star clusters and of extragalactic X-ray sources. (Oy)

  17. Humidity control and hydrophilic glue coating applied to mounted protein crystals improves X-ray diffraction experiments

    International Nuclear Information System (INIS)

    Baba, Seiki; Hoshino, Takeshi; Ito, Len; Kumasaka, Takashi

    2013-01-01

    A new crystal-mounting method has been developed that involves a combination of controlled humid air and polymer glue for crystal coating. This method is particularly useful when applied to fragile protein crystals that are known to be sensitive to subtle changes in their physicochemical environment. Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection

  18. Humidity control and hydrophilic glue coating applied to mounted protein crystals improves X-ray diffraction experiments

    Energy Technology Data Exchange (ETDEWEB)

    Baba, Seiki; Hoshino, Takeshi; Ito, Len; Kumasaka, Takashi, E-mail: kumasaka@spring8.or.jp [Japan Synchrotron Radiation Research Institute (JASRI/SPring-8), 1-1-1 Kouto, Sayo, Hyogo 679-5198 (Japan)

    2013-09-01

    A new crystal-mounting method has been developed that involves a combination of controlled humid air and polymer glue for crystal coating. This method is particularly useful when applied to fragile protein crystals that are known to be sensitive to subtle changes in their physicochemical environment. Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection.

  19. Towards phasing using high X-ray intensity

    Directory of Open Access Journals (Sweden)

    Lorenzo Galli

    2015-11-01

    Full Text Available X-ray free-electron lasers (XFELs show great promise for macromolecular structure determination from sub-micrometre-sized crystals, using the emerging method of serial femtosecond crystallography. The extreme brightness of the XFEL radiation can multiply ionize most, if not all, atoms in a protein, causing their scattering factors to change during the pulse, with a preferential `bleaching' of heavy atoms. This paper investigates the effects of electronic damage on experimental data collected from a Gd derivative of lysozyme microcrystals at different X-ray intensities, and the degree of ionization of Gd atoms is quantified from phased difference Fourier maps. A pattern sorting scheme is proposed to maximize the ionization contrast and the way in which the local electronic damage can be used for a new experimental phasing method is discussed.

  20. Towards phasing using high X-ray intensity

    International Nuclear Information System (INIS)

    Galli, Lorenzo; Son, Sang-Kil; Barends, Thomas R. M.; White, Thomas A.; Barty, Anton; Botha, Sabine; Boutet, Sebastien; Caleman, Carl; Doak, R. Bruce; Nanao, Max H.; Nass, Karol; Shoeman, Robert L.; Timneanu, Nicusor; Santra, Robin; Schlichting, Ilme; Chapman, Henry N.

    2015-01-01

    X-ray free-electron lasers (XFELs) show great promise for macromolecular structure determination from sub-micrometre-sized crystals, using the emerging method of serial femtosecond crystallography. The extreme brightness of the XFEL radiation can multiply ionize most, if not all, atoms in a protein, causing their scattering factors to change during the pulse, with a preferential 'bleaching' of heavy atoms. This paper investigates the effects of electronic damage on experimental data collected from a Gd derivative of lysozyme microcrystals at different X-ray intensities, and the degree of ionization of Gd atoms is quantified from phased difference Fourier maps. In conclusion, a pattern sorting scheme is proposed to maximize the ionization contrast and the way in which the local electronic damage can be used for a new experimental phasing method is discussed

  1. Rigid body essential X-ray crystallography

    DEFF Research Database (Denmark)

    Bjerrum, Esben Jannik; Biggin, Philip C

    2008-01-01

    The ligand-binding domain (LBD) from the ionotropic glutamate receptor subtype 2 (GluR2) has been shown to adopt a range of ligand-dependent conformational states. These states have been described in terms of the rotation required to fit subdomain (lobe) 2 following superposition of subdomain (lo...

  2. AXSIS: Exploring the frontiers in attosecond X-ray science, imaging and spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Kärtner, F.X., E-mail: franz.kaertner@cfel.de [Center for Free-Electron Laser Science, Hamburg (Germany); Institute for Experimental Physics, University of Hamburg, Hamburg (Germany); The Hamburg Center for Ultrafast Imaging, Hamburg (Germany); DESY, Hamburg (Germany); Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA (United States); Ahr, F. [Center for Free-Electron Laser Science, Hamburg (Germany); Institute for Experimental Physics, University of Hamburg, Hamburg (Germany); DESY, Hamburg (Germany); Max Planck Institute for the Structure and Dynamics of Matter, Hamburg (Germany); Calendron, A.-L. [Center for Free-Electron Laser Science, Hamburg (Germany); Institute for Experimental Physics, University of Hamburg, Hamburg (Germany); The Hamburg Center for Ultrafast Imaging, Hamburg (Germany); DESY, Hamburg (Germany); Çankaya, H. [Center for Free-Electron Laser Science, Hamburg (Germany); The Hamburg Center for Ultrafast Imaging, Hamburg (Germany); DESY, Hamburg (Germany); Carbajo, S. [Center for Free-Electron Laser Science, Hamburg (Germany); Institute for Experimental Physics, University of Hamburg, Hamburg (Germany); DESY, Hamburg (Germany); Chang, G.; Cirmi, G. [Center for Free-Electron Laser Science, Hamburg (Germany); The Hamburg Center for Ultrafast Imaging, Hamburg (Germany); DESY, Hamburg (Germany); Dörner, K. [Center for Free-Electron Laser Science, Hamburg (Germany); DESY, Hamburg (Germany); Dorda, U. [DESY, Hamburg (Germany); Fallahi, A. [Center for Free-Electron Laser Science, Hamburg (Germany); DESY, Hamburg (Germany); Hartin, A. [Center for Free-Electron Laser Science, Hamburg (Germany); Institute for Experimental Physics, University of Hamburg, Hamburg (Germany); DESY, Hamburg (Germany); Hemmer, M. [Center for Free-Electron Laser Science, Hamburg (Germany); DESY, Hamburg (Germany); and others

    2016-09-01

    X-ray crystallography is one of the main methods to determine atomic-resolution 3D images of the whole spectrum of molecules ranging from small inorganic clusters to large protein complexes consisting of hundred-thousands of atoms that constitute the macromolecular machinery of life. Life is not static, and unravelling the structure and dynamics of the most important reactions in chemistry and biology is essential to uncover their mechanism. Many of these reactions, including photosynthesis which drives our biosphere, are light induced and occur on ultrafast timescales. These have been studied with high time resolution primarily by optical spectroscopy, enabled by ultrafast laser technology, but they reduce the vast complexity of the process to a few reaction coordinates. In the AXSIS project at CFEL in Hamburg, funded by the European Research Council, we develop the new method of attosecond serial X-ray crystallography and spectroscopy, to give a full description of ultrafast processes atomically resolved in real space and on the electronic energy landscape, from co-measurement of X-ray and optical spectra, and X-ray diffraction. This technique will revolutionize our understanding of structure and function at the atomic and molecular level and thereby unravel fundamental processes in chemistry and biology like energy conversion processes. For that purpose, we develop a compact, fully coherent, THz-driven attosecond X-ray source based on coherent inverse Compton scattering off a free-electron crystal, to outrun radiation damage effects due to the necessary high X-ray irradiance required to acquire diffraction signals. This highly synergistic project starts from a completely clean slate rather than conforming to the specifications of a large free-electron laser (FEL) user facility, to optimize the entire instrumentation towards fundamental measurements of the mechanism of light absorption and excitation energy transfer. A multidisciplinary team formed by laser

  3. Nanoflow electrospinning serial femtosecond crystallography

    Science.gov (United States)

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

    2012-01-01

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

  4. Synchrotron small-angle x-ray scattering investigation on integral membrane protein light-harvesting complex LH2 from photosynthetic bacterium rhodopseudomonas acidophila

    International Nuclear Information System (INIS)

    Du Luchao; Weng Yuxiang; Hong Xinguo; Xian Dingchang; Kobayashi Katsumi

    2006-01-01

    Structures of membrane protein in solution are different from that in crystal phase. We present the primary results of small angle x-ray scattering (SAXS) resolved topological structures of a light harvesting antenna membrane protein complex LH2 from photosynthetic bacteria Rhodopseudomonas acidophila in detergent solution for the first time. Our results show that the elliptical shape of the LH2 complex in solution clearly deviates from its circular structure in crystal phase determined by x-ray diffraction. This result provides an insight into the structure and function interplay in LH2. (authors)

  5. Quantitative Characterization of Configurational Space Sampled by HIV-1 Nucleocapsid Using Solution NMR, X-ray Scattering and Protein Engineering.

    Science.gov (United States)

    Deshmukh, Lalit; Schwieters, Charles D; Grishaev, Alexander; Clore, G Marius

    2016-06-03

    Nucleic-acid-related events in the HIV-1 replication cycle are mediated by nucleocapsid, a small protein comprising two zinc knuckles connected by a short flexible linker and flanked by disordered termini. Combining experimental NMR residual dipolar couplings, solution X-ray scattering and protein engineering with ensemble simulated annealing, we obtain a quantitative description of the configurational space sampled by the two zinc knuckles, the linker and disordered termini in the absence of nucleic acids. We first compute the conformational ensemble (with an optimal size of three members) of an engineered nucleocapsid construct lacking the N- and C-termini that satisfies the experimental restraints, and then validate this ensemble, as well as characterize the disordered termini, using the experimental data from the full-length nucleocapsid construct. The experimental and computational strategy is generally applicable to multidomain proteins. Differential flexibility within the linker results in asymmetric motion of the zinc knuckles which may explain their functionally distinct roles despite high sequence identity. One of the configurations (populated at a level of ≈40 %) closely resembles that observed in various ligand-bound forms, providing evidence for conformational selection and a mechanistic link between protein dynamics and function. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Molybdenum x-ray absorption studies of the mutant Kp nifV of nitrogenase MO-FE protein

    International Nuclear Information System (INIS)

    Eidsness, M.K.; Smith, B.E.; Flood, A.C.; Garner, C.D.; Cramer, S.P.

    1985-01-01

    The nifV mutant nitrogenase enzyme of Klebsiella pheumoniae exhibits altered substrate reducing activity. This nitrogenase mutant cannot fix N 2 in vivo but can reduce C 2 H 2 to C 2 H 4 . The nifV mutant enzyme differs further from the wild-type enzyme by CO inhibition of its H 2 evolution activity, up to 80%. The NifV - phenotype (NifV - Kp1) has been shown to be associated with the iron-molybdenum cofactor (FeMoco) in the Mo Fe protein which is generally accepted as the site for substrate reduction. An X-Ray absorption study of the Mo site in this mutant may reveal a difference in its FeMoco structure. The authors report here a comparison of Mo X-Ray absorption data from the nitrogenase enzymes of the wild-type and NifV - strains in three different forms: (1) as isolated, (2) dye-oxidized, and (3) fixing enzyme systems. Mo edge structure of NifV - Kp1 and wild-type enzymes are nearly identical. Small shifts to higher energies are observed in the oxidized and fixing states. Mo EXAFS of NifV - Kp1 and wild-type in the ''as isolated'' state appear indistinguishable. Curve fitting results best describe the molybdenum in FeMoco as bound by 4-5 S atoms at 2.36 A ,3 Fe atoms at 2.69 A, and 0-2 O(N) atoms at 2.19 A. The spectral similarity of these results concerning the nifV mutant FeMoco structure is discussed

  7. Multikilovolt Coherent X-Ray Generation for Protein Analysis and Biological Threat Reduction

    National Research Council Canada - National Science Library

    Rhodes, Charles

    2003-01-01

    Efforts to trump bioterrorism can be sharply advanced by the development of new modalities for the rapid measurement and quantitative classification of protein structural and regulatory information. This report (1...

  8. Larval x-ray irradiation influences protein expression in pupae of the Oriental fruit fly, Bactrocera Dorsalis

    Science.gov (United States)

    Third instar larvae were exposed to X-ray treatment of the Oriental fruit fly, Bactrocera dorsalis. Irradiated pupae were collected daily. Biological performance parameters of pupae and adults of larvae treated with X-ray irradiation were evaluated. Standard proteomics procedures such as densitometr...

  9. X-ray structure of the mammalian GIRK2-βγ G-protein complex

    Energy Technology Data Exchange (ETDEWEB)

    Whorton, Matthew R.; MacKinnon, Roderick [Rockefeller

    2013-07-30

    G-protein-gated inward rectifier K+ (GIRK) channels allow neurotransmitters, through G-protein-coupled receptor stimulation, to control cellular electrical excitability. In cardiac and neuronal cells this control regulates heart rate and neural circuit activity, respectively. Here we present the 3.5Å resolution crystal structure of the mammalian GIRK2 channel in complex with βγ G-protein subunits, the central signalling complex that links G-protein-coupled receptor stimulation to K+ channel activity. Short-range atomic and long-range electrostatic interactions stabilize four βγ G-protein subunits at the interfaces between four K+ channel subunits, inducing a pre-open state of the channel. The pre-open state exhibits a conformation that is intermediate between the closed conformation and the open conformation of the constitutively active mutant. The resultant structural picture is compatible with ‘membrane delimited’ activation of GIRK channels by G proteins and the characteristic burst kinetics of channel gating. The structures also permit a conceptual understanding of how the signalling lipid phosphatidylinositol-4,5-bisphosphate (PIP2) and intracellular Na+ ions participate in multi-ligand regulation of GIRK channels.

  10. Purification, crystallization and preliminary X-ray diffraction of fluorescence recovery protein from Synechocystis PCC 6803

    International Nuclear Information System (INIS)

    Liu, Ting; Shuai, Yingli; Zhou, Honggang

    2011-01-01

    Fluorescence recovery protein from Synechocystis PCC 6803 plays a key role in the orange carotenoid protein-related photoprotective mechanism in cyanobacteria. The full-length form and a truncated form were overexpressed, purified and crystallized, and diffraction was observed to 2.75 Å resolution. Fluorescence recovery protein (FRP), which is encoded by the slr1964 gene in Synechocystis PCC 6803, plays a key role in the orange carotenoid protein-related photoprotective mechanism in cyanobacteria. As the crystal structure of FRP may provide information about the biological functions and mechanism of action of the protein, recombinant full-length FRP and a truncated form were overexpressed, purified and crystallized at 291 K using ethylene imine polymer as the precipitant. An FRP data set was collected to a resolution of 2.75 Å at low temperature (100 K). The crystal belonged to space group P4 1 2 1 2, with unit-cell parameters a = b = 61.9, c = 160.7 Å, α = β = γ = 90°. Assuming that the asymmetric unit contains three molecules, the Matthews coefficient was calculated to be 2.1 Å 3 Da −1

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

    International Nuclear Information System (INIS)

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

    1994-01-01

    The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H 2 O/D 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Timmins, P.A. [ILL, Grenoble (France); Pebay-Peyroula, E. [IBS-UJF Grenoble (France)

    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.

  13. Crystallization and preliminary X-ray crystallographic analysis of SMU.412c protein from the caries pathogen Streptococcus mutans

    International Nuclear Information System (INIS)

    Ye, Zhao-Yang; Hou, Qiao-Ming; Li, Lan-Fen; Su, Xiao-Dong

    2009-01-01

    Crystallization of SMU.412c protein from the caries pathogen Streptococcus mutans can easily appear in the condition 2.8 M sodium acetate pH 7.0 and its crystal belongs to space group P4 1 2 1 2. The smu.412c gene encodes a putative histidine triad-like protein (SMU.412c) with 139 residues that is involved in cell-cycle regulation in Streptococcus mutans. The gene was cloned into the expression vector pET28a and subsequently expressed in Escherichia coli strain BL21 (DE3) to give a substantially soluble form of SMU.412c with a His 6 tag at its N-terminus. The recombinant protein was purified to homogeneity in a two-step procedure involving Ni 2+ -chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained using the sitting-drop vapour-diffusion method and diffracted to 1.8 Å resolution on beamline BL6A at Photon Factory, Tsukuba, Japan. The crystal belonged to space group P4 1 2 1 2, with unit-cell parameters a = b = 53.5, c = 141.1 Å

  14. Crystallization and preliminary X-ray analysis of the chemokine-binding protein from orf virus (Poxviridae)

    International Nuclear Information System (INIS)

    Couñago, Rafael Miguez; Fleming, Stephen B.; Mercer, Andrew A.; Krause, Kurt L.

    2010-01-01

    The chemokine-binding protein from orf virus was purified and crystallized. The morphology and diffraction behaviour of these crystals was significantly improved through the use of additives known as Silver Bullets. The parapoxvirus orf virus (ORFV) encodes a chemokine-binding protein (CBP) that functions to downregulate the host’s immune response at the site of infection by blocking the chemokine-induced recruitment of immune cells. In order to shed light on the structural determinants of CBP–chemokine binding, ORFV CBP was crystallized as part of an ongoing structure–function study on this protein. ORFV CBP crystals were obtained by the sitting-drop vapour-diffusion technique using ammonium citrate as a precipitant. The crystal quality was greatly improved through the addition of small-molecule additives to the crystallization mother liquor. ORFV CBP crystals diffracted X-rays to 2.50 Å resolution and belonged to the hexagonal space group P6 1 22 or its enantiomorph P6 5 22, with unit-cell parameters a = b = 75.62, c = 282.49 Å, α = 90, β = 90, γ = 120°

  15. Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers.

    Science.gov (United States)

    Fuller, Franklin D; Gul, Sheraz; Chatterjee, Ruchira; Burgie, E Sethe; Young, Iris D; Lebrette, Hugo; Srinivas, Vivek; Brewster, Aaron S; Michels-Clark, Tara; Clinger, Jonathan A; Andi, Babak; Ibrahim, Mohamed; Pastor, Ernest; de Lichtenberg, Casper; Hussein, Rana; Pollock, Christopher J; Zhang, Miao; Stan, Claudiu A; Kroll, Thomas; Fransson, Thomas; Weninger, Clemens; Kubin, Markus; Aller, Pierre; Lassalle, Louise; Bräuer, Philipp; Miller, Mitchell D; Amin, Muhamed; Koroidov, Sergey; Roessler, Christian G; Allaire, Marc; Sierra, Raymond G; Docker, Peter T; Glownia, James M; Nelson, Silke; Koglin, Jason E; Zhu, Diling; Chollet, Matthieu; Song, Sanghoon; Lemke, Henrik; Liang, Mengning; Sokaras, Dimosthenis; Alonso-Mori, Roberto; Zouni, Athina; Messinger, Johannes; Bergmann, Uwe; Boal, Amie K; Bollinger, J Martin; Krebs, Carsten; Högbom, Martin; Phillips, George N; Vierstra, Richard D; Sauter, Nicholas K; Orville, Allen M; Kern, Jan; Yachandra, Vittal K; Yano, Junko

    2017-04-01

    X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.

  16. Crystallization and preliminary X-ray diffraction analysis of rat protein tyrosine phosphatase η

    Energy Technology Data Exchange (ETDEWEB)

    Matozo, Huita C.; Nascimento, Alessandro S.; Santos, Maria A. M. [Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, CEP 13566-590 São Carlos, SP (Brazil); Iuliano, Rodolfo [Dipartimento di Medicina Sperimentale e Clinica, Facoltà di Medicina e Chirurgia, Università di Catanzaro, 88100 Catanzaro (Italy); Fusco, Alfredo [Dipartimento di Biologia e Patologia Cellulare e Molecolare, c/o Instituto di Endocrinologia ed Oncologia Sperimentale del CNR, Facolta di Medicina e Chirurgia, Università degli Studi di Napoli ‘Federico II’, Via Pansini 5, 80131 Naples (Italy); NOGEC (Naples Oncogenomocs Center)-CEINGE, Biotecnologie Avanzate, Via Comunale Margherita 482, 80145 Naples (Italy); Polikarpov, Igor, E-mail: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, CEP 13566-590 São Carlos, SP (Brazil); Laboratório Nacional de Luz Síncrotron, Campinas, SP (Brazil)

    2006-09-01

    In this study, the catalytic domain of rat protein tyrosine phosphatase η was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. The rat protein tyrosine phosphatase η (rPTPη) is a cysteine-dependent phosphatase which hydrolyzes phosphoester bonds in proteins and other molecules. rPTPη and its human homologue DEP-1 are involved in neoplastic transformations. Thus, expression of the protein is reduced in all oncogene-transformed thyroid cell lines and is absent in highly malignant thyroid cells. Moreover, consistent with the suggested tumour suppression role of PTPη, inhibition of the tumorigenic process occurs after its exogenous reconstitution, suggesting that PTPη might be important for gene therapy of cancers. In this study, the catalytic domain of rPTPη was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.87 Å resolution. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.46, b = 63.07, c = 111.64 Å, and contains one molecule per asymmetric unit.

  17. Crystallization and preliminary X-ray diffraction analysis of rat protein tyrosine phosphatase η

    International Nuclear Information System (INIS)

    Matozo, Huita C.; Nascimento, Alessandro S.; Santos, Maria A. M.; Iuliano, Rodolfo; Fusco, Alfredo; Polikarpov, Igor

    2006-01-01

    In this study, the catalytic domain of rat protein tyrosine phosphatase η was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. The rat protein tyrosine phosphatase η (rPTPη) is a cysteine-dependent phosphatase which hydrolyzes phosphoester bonds in proteins and other molecules. rPTPη and its human homologue DEP-1 are involved in neoplastic transformations. Thus, expression of the protein is reduced in all oncogene-transformed thyroid cell lines and is absent in highly malignant thyroid cells. Moreover, consistent with the suggested tumour suppression role of PTPη, inhibition of the tumorigenic process occurs after its exogenous reconstitution, suggesting that PTPη might be important for gene therapy of cancers. In this study, the catalytic domain of rPTPη was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.87 Å resolution. The crystal belongs to space group P2 1 2 1 2 1 , with unit-cell parameters a = 46.46, b = 63.07, c = 111.64 Å, and contains one molecule per asymmetric unit

  18. Chemical denaturation of globular proteins at the air/water interface: an x-ray and neutron reflectometry study

    International Nuclear Information System (INIS)

    Perriman, A.W.; Henderson, M.J.; White, J.W.

    2003-01-01

    Full text: X-ray and neutron reflectometry has been used to probe the equilibrium surface structure of hen egg white lysozyme (lysozyme) and bovine β -lactoglobulin (β -lactoglobulin) under denaturing conditions at the air-water interface. This was achieved by performing experiments on 10 mg mL -1 protein solutions containing increasing concentrations of the chemical denaturant guanidinium hydrochloride (G.HCl). For solutions containing no G.HCl, the surface structure of the proteins was represented by a two-layer model with total thicknesses of 48 Angstroms and 38 Angstroms for lysozyme and β -lactoglobulin, respectively. The total volume of a single protein molecule and the associated water molecules was evaluated to be approximately 45 (0.3) nm 3 for lysozyme, and 60 (0.3) nm 3 for β-lactoglobulin. The thickness dimensions and the total volumes compared favourably with the crystal dimensions of 45 x 30 x 30 Angstroms (40.5 nm 3 ),1 and 36 x 36 x 36 Angstroms (47 nm 3 ) 2 for lysozyme and β -lactoglobulin, respectively. This comparison suggests that when no denaturant was present, the structures of lysozyme and β -lactoglobulin were near to their native conformations at the air-water interface. The response to the presence of the chemical denaturant was different for each protein. The surface layer of β-lactoglobulin expanded at very low concentrations (0.2 mol dm -3 ) of G.HCl. In contrast, the lysozyme layer contracted. At higher concentrations, unfolding of both the proteins led to the formation of a third diffuse layer. In general, lysozyme appeared to be less responsive to the chemical denaturant, which is most likely a result of the higher disulfide content of lysozyme. A protocol allowing quantitative thermodynamic analysis of the contribution from the air-water interface to the chemical denaturation of a protein was developed

  19. Crystallization and preliminary X-ray diffraction studies of Drosophila melanogaster Gαo-subunit of heterotrimeric G protein in complex with the RGS domain of CG5036

    International Nuclear Information System (INIS)

    Tishchenko, Svetlana; Gabdulkhakov, Azat; Tin, Uliana; Kostareva, Olga; Lin, Chen; Katanaev, Vladimir L.

    2012-01-01

    D. melanogaster Gαo-subunit and the RGS domain of its interacting partner CG5036 have been overproduced and purified; the crystallization and preliminary X-ray crystallographic analysis of the complex of the two proteins are reported. Regulator of G-protein signalling (RGS) proteins negatively regulate heterotrimeric G-protein signalling through their conserved RGS domains. RGS domains act as GTPase-activating proteins, accelerating the GTP hydrolysis rate of the activated form of Gα-subunits. Although omnipresent in eukaryotes, RGS proteins have not been adequately analysed in non-mammalian organisms. The Drosophila melanogaster Gαo-subunit and the RGS domain of its interacting partner CG5036 have been overproduced and purified; the crystallization of the complex of the two proteins using PEG 4000 as a crystallizing agent and preliminary X-ray crystallographic analysis are reported. Diffraction data were collected to 2.0 Å resolution using a synchrotron-radiation source

  20. Calculation of accurate small angle X-ray scattering curves from coarse-grained protein models

    DEFF Research Database (Denmark)

    Stovgaard, Kasper; Andreetta, Christian; Ferkinghoff-Borg, Jesper

    2010-01-01

    , which is paramount for structure determination based on statistical inference. Results: We present a method for the efficient calculation of accurate SAXS curves based on the Debye formula and a set of scattering form factors for dummy atom representations of amino acids. Such a method avoids......DBN. This resulted in a significant improvement in the decoy recognition performance. In conclusion, the presented method shows great promise for use in statistical inference of protein structures from SAXS data....

  1. Chest X-Ray

    Medline Plus

    Full Text Available ... some concerns about chest x-rays. However, it’s important to consider the likelihood of benefit to your health. While a chest x-ray use a ... posted: How to Obtain and Share ...

  2. Chest X-Ray

    Medline Plus

    Full Text Available ... X-ray Transcript Welcome to Radiology Info dot org! Hello, I’m Dr. Geoffrey Rubin, a radiologist ... about chest x-rays, visit Radiology Info dot org. Thank you for your time! Spotlight Recently posted: ...

  3. X-ray apparatus

    International Nuclear Information System (INIS)

    Sell, L.J.

    1981-01-01

    A diagnostic x-ray device, readily convertible between conventional radiographic and tomographic operating modes, is described. An improved drive system interconnects and drives the x-ray source and the imaging device through coordinated movements for tomography

  4. X-ray - skeleton

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003381.htm X-ray - skeleton To use the sharing features on this ... Degenerative bone conditions Osteomyelitis Risks There is low radiation exposure. X-rays machines are set to provide the smallest ...

  5. High resolution X-ray structures of mouse major urinary protein nasal isoform in complex with pheromones

    Energy Technology Data Exchange (ETDEWEB)

    Perez-Miller, Samantha; Zou, Qin; Novotny, Milos V.; Hurley, Thomas D. (Indiana-Med); (Indiana)

    2010-09-07

    In mice, the major urinary proteins (MUP) play a key role in pheromonal communication by binding and transporting semiochemicals. MUP-IV is the only isoform known to be expressed in the vomeronasal mucosa. In comparison with the MUP isoforms that are abundantly excreted in the urine, MUP-IV is highly specific for the male mouse pheromone 2-sec-butyl-4,5-dihydrothiazole (SBT). To examine the structural basis of this ligand preference, we determined the X-ray crystal structure of MUP-IV bound to three mouse pheromones: SBT, 2,5-dimethylpyrazine, and 2-heptanone. We also obtained the structure of MUP-IV with 2-ethylhexanol bound in the cavity. These four structures show that relative to the major excreted MUP isoforms, three amino acid substitutions within the binding calyx impact ligand coordination. The F103 for A along with F54 for L result in a smaller cavity, potentially creating a more closely packed environment for the ligand. The E118 for G substitution introduces a charged group into a hydrophobic environment. The sidechain of E118 is observed to hydrogen bond to polar groups on all four ligands with nearly the same geometry as seen for the water-mediated hydrogen bond network in the MUP-I and MUP-II crystal structures. These differences in cavity size and interactions between the protein and ligand are likely to contribute to the observed specificity of MUP-IV.

  6. Purification, crystallization and preliminary X-ray diffraction analysis of the human mismatch repair protein MutSβ

    International Nuclear Information System (INIS)

    Tseng, Quincy; Orans, Jillian; Hast, Michael A.; Iyer, Ravi R.; Changela, Anita; Modrich, Paul L.; Beese, Lorena S.

    2011-01-01

    Human MutSβ is a 232 kDa heterodimer (MSH2–MSH3) involved in the lesion-recognition step of mismatch repair. Here, the overexpression, purification, biochemical characterization and cocrystallization of MutSβ with a duplex DNA substrate are reported. MutSβ is a eukaryotic mismatch repair protein that preferentially targets extrahelical unpaired nucleotides and shares partial functional redundancy with MutSα (MSH2–MSH6). Although mismatch recognition by MutSα has been shown to involve a conserved Phe-X-Glu motif, little is known about the lesion-binding mechanism of MutSβ. Combined MSH3/MSH6 deficiency triggers a strong predisposition to cancer in mice and defects in msh2 and msh6 account for roughly half of hereditary nonpolyposis colorectal cancer mutations. These three MutS homologs are also believed to play a role in trinucleotide repeat instability, which is a hallmark of many neurodegenerative disorders. The baculovirus overexpression and purification of recombinant human MutSβ and three truncation mutants are presented here. Binding assays with heteroduplex DNA were carried out for biochemical characterization. Crystallization and preliminary X-ray diffraction analysis of the protein bound to a heteroduplex DNA substrate are also reported

  7. Preliminary X-ray crystallographic analysis of SMU.2055 protein from the caries pathogen Streptococcus mutans

    International Nuclear Information System (INIS)

    Zhao, Wang-Hong; Zhan, Xiu-Rong; Gao, Xiong-Zhuo; Liu, Xiang; Zhang, Yi-Fei; Lin, Jiuxiang; Li, Lan-Fen; Wei, Shi-Cheng; Su, Xio-Dong

    2010-01-01

    The SMU.2055 gene from the major caries pathogen Streptococcus mutans was cloned and native and SeMet-labelled SMU.2055 proteins were expressed at a high level. Diffraction-quality crystals of SeMet-labelled SMU.2055 were obtained using the sitting-drop vapour-diffusion method and diffracted to a resolution of 2.5 Å. The SMU.2055 gene from the major caries pathogen Streptococcus mutans is annotated as a putative acetyltransferase with 163 amino-acid residues. In order to identify its function via structural studies, the SMU.2055 gene was cloned into the expression vector pET28a. Native and SeMet-labelled SMU.2055 proteins with a His 6 tag at the N-terminus were expressed at a high level in Escherichia coli strain BL21 (DE3) and purified to homogeneity by Ni 2+ -chelating affinity chromatography. Diffraction-quality crystals of SeMet-labelled SMU.2055 were obtained using the sitting-drop vapour-diffusion method and diffracted to a resolution of 2.5 Å on beamline BL17A at the Photon Factory, Tsukuba, Japan. The crystals belong to the orthorhombic space group C222 1 , with unit-cell parameters a = 92.0, b = 95.0, c = 192.2 Å. The asymmetric unit contained four molecules, with a solvent content of 57.1%

  8. The action of X-rays on plasmatic proteins (1962); Action des rayonnements X sur les proteines plasmatiques (1962)

    Energy Technology Data Exchange (ETDEWEB)

    Rinaldi, R; Llory, J; Suscillon, M [Commissariat a l' Energie Atomique, Grenoble (France). Centre d' Etudes Nucleaires

    1962-07-01

    The agglomerate property of the human plasma on the erythrocytes is altered by X rays which is seen owing to an, increase of the sedimentation rate of red cells. When using this biological test: reversible agglomeration of the erythrocytes the authors tried to demonstrate the denaturation of the serum albumin under the action of the X rays. Further to this results we see that the doses of about 5. 10{sup 5} roentgens denature completely the macromolecule of serum albumin. (authors) [French] Le pouvoir agglomerant du plasma humain sur les erythrocytes, est modifie par les rayons X, ce qui se traduit par une augmentation de la vitesse de sedimentation des hematies. En utilisant ce test biologique.: agglomeration, reversible des erythrocytes, les auteurs se sont attaches a mettre en evidence la denaturation de la serum albumine humaine sous l'action des rayons X (durs). Les resultats obtenus ont montre que des doses de l'ordre de 5. 10{sup 5} roentgens denaturent completement la macromolecule de serum albumine. (auteurs)

  9. Crystallization and preliminary X-ray analysis of Ebola VP35 interferon inhibitory domain mutant proteins

    International Nuclear Information System (INIS)

    Leung, Daisy W.; Borek, Dominika; Farahbakhsh, Mina; Ramanan, Parameshwaran; Nix, Jay C.; Wang, Tianjiao; Prins, Kathleen C.; Otwinowski, Zbyszek; Honzatko, Richard B.; Helgeson, Luke A.; Basler, Christopher F.; Amarasinghe, Gaya K.

    2010-01-01

    Three mutant forms of Ebola VP35 interferon inhibitory domain were crystallized in three different space groups. VP35 is one of seven structural proteins encoded by the Ebola viral genome and mediates viral replication, nucleocapsid formation and host immune suppression. The C-terminal interferon inhibitory domain (IID) of VP35 is critical for dsRNA binding and interferon inhibition. The wild-type VP35 IID structure revealed several conserved residues that are important for dsRNA binding and interferon antagonism. Here, the expression, purification and crystallization of recombinant Zaire Ebola VP35 IID mutants R312A, K319A/R322A and K339A in space groups P6 1 22, P2 1 2 1 2 1 and P2 1 , respectively, are described. Diffraction data were collected using synchrotron sources at the Advanced Light Source and the Advanced Photon Source

  10. Chest X-Ray

    Medline Plus

    Full Text Available ... I’d like to talk with you about chest radiography also known as chest x-rays. Chest x-rays are the most ... far outweighs any risk. For more information about chest x-rays, visit Radiology Info dot org. Thank you for your time! ...

  11. Chest X-Ray

    Medline Plus

    Full Text Available ... by Image/Video Gallery Your Radiologist Explains Chest X-ray Transcript Welcome to Radiology Info dot org! Hello, ... you about chest radiography also known as chest x-rays. Chest x-rays are the most commonly performed ...

  12. Crystallization and preliminary X-ray crystallographic analysis of human PACSIN 1 protein

    International Nuclear Information System (INIS)

    Bai, Xiaoyun; Meng, Geng; Li, Guoming; Luo, Ming; Zheng, Xiaofeng

    2009-01-01

    A C-terminal truncation construct of human PACSIN 1 (1–344) has been purified and crystallized. Diffraction data were collected to 3.0 Å resolution. PACSIN 1, which is mainly detected in brain tissue, is one of the PACSIN-family proteins involved in endocytosis and recruitment of synaptic vesicles. It binds to dynamin, synaptojanin 1 and N-WASP, and functions in vesicle formation and transport. However, the mechanisms of action of PACSIN 1 in these processes are largely unknown. Here, full-length and five C-terminal truncation constructs of human PACSIN 1 have been successfully expressed and purified in Escherichia coli. PACSIN 1 (1–344) was crystallized and diffracted to a resolution of 3.0 Å. The crystal belonged to space group C2, with unit-cell parameters a = 158.65, b = 87.38, c = 91.76 Å, α = 90.00, β = 113.61, γ = 90.00°. There were two molecules in the asymmetric unit and the solvent content was estimated to be about 70.47%

  13. Crystallization and preliminary X-ray diffraction analysis of the metalloregulatory protein DtxR from Thermoplasma acidophilum

    International Nuclear Information System (INIS)

    Yeo, Hyun Ku; Kang, Jina; Park, Young Woo; Sung, Jung-Suk; Lee, Jae Young

    2012-01-01

    Orthorhombic crystals of DtxR from T. acidophilum have been obtained. X-ray data were collected to 1.8 Å resolution using synchrotron radiation. The diphtheria toxin repressor (DtxR) is a metal-ion-dependent transcriptional regulator which regulates genes encoding proteins involved in metal-ion uptake to maintain metal-ion homeostasis. DtxR from Thermoplasma acidophilum was cloned and overexpressed in Escherichia coli. Crystals of N-terminally His-tagged DtxR were obtained by hanging-drop vapour diffusion and diffracted to 1.8 Å resolution. DtxR was crystallized at 296 K using polyethylene glycol 4000 as a precipitant. The crystals belonged to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 61.14, b = 84.61, c = 46.91 Å, α = β = γ = 90°. The asymmetric unit contained approximately one monomer of DtxR, giving a crystal volume per mass (V M ) of 2.22 Å 3 Da −1 and a solvent content of 44.6%

  14. Flash X-ray

    International Nuclear Information System (INIS)

    Sato, Eiichi

    2003-01-01

    Generation of quasi-monochromatic X-ray by production of weakly ionized line plasma (flash X-ray), high-speed imaging by the X-ray and high-contrast imaging by the characteristic X-ray absorption are described. The equipment for the X-ray is consisted from the high-voltage power supply and condenser, turbo molecular pump, and plasma X-ray tube. The tube has a long linear anticathode to produce the line plasma and flash X-ray at 20 kA current at maximum. X-ray spectrum is measured by the imaging plate equipped in the computed radiography system after diffracted by a LiF single crystal bender. Cu anticathode generates sharp peaks of K X-ray series. The tissue images are presented for vertebra, rabbit ear and heart, and dog heart by X-ray fluoroscopy with Ce anticathode. Generation of K-orbit characteristic X-ray with extremely low bremsstrahung is to be attempted for medical use. (N.I.)

  15. Quantitative characterization of the protein contents of the exocrine pancreatic acinar cell by soft x-ray microscopy and advanced digital imaging methods

    Energy Technology Data Exchange (ETDEWEB)

    Loo, Jr., Billy W. [Univ. of California, Berkeley, CA (United States)

    2000-06-01

    The study of the exocrine pancreatic acinar cell has been central to the development of models of many cellular processes, especially of protein transport and secretion. Traditional methods used to examine this system have provided a wealth of qualitative information from which mechanistic models have been inferred. However they have lacked the ability to make quantitative measurements, particularly of the distribution of protein in the cell, information critical for grounding of models in terms of magnitude and relative significance. This dissertation describes the development and application of new tools that were used to measure the protein content of the major intracellular compartments in the acinar cell, particularly the zymogen granule. Soft x-ray microscopy permits image formation with high resolution and contrast determined by the underlying protein content of tissue rather than staining avidity. A sample preparation method compatible with x-ray microscopy was developed and its properties evaluated. Automatic computerized methods were developed to acquire, calibrate, and analyze large volumes of x-ray microscopic images of exocrine pancreatic tissue sections. Statistics were compiled on the protein density of several organelles, and on the protein density, size, and spatial distribution of tens of thousands of zymogen granules. The results of these measurements, and how they compare to predictions of different models of protein transport, are discussed.

  16. Providing Bright-Hard X-ray Beams from a Lower Energy Light Source

    Science.gov (United States)

    Robin, David

    2002-04-01

    At the Advanced Light Source (ALS) there had been an increasing demand for more high brightness harder X-ray sources in the 7 to 40 KeV range. In response to that demand, the ALS storage ring was modified in August 2001. Three 1.3 Tesla normal conducting bending magnets were removed and replaced with three 5 Tesla superconducting magnets (Superbends). The radiation produced by these Superbends is an order of magnitude higher in photon brightness and flux at 12 keV than the 1.3 Tesla bends, making them excellent sources of harder x-rays for protein crystallography and other harder x-ray applications. At the same time the Superbends do not compromise the performance of the facility in the UV and Soft X-ray regions of the spectrum. The Superbends will eventually feed 12 new x-ray beam lines greatly enhancing the facility's capacity in the hard x-ray region. The Superbend project is the biggest upgrade to the ALS storage ring since the ring was commissioned in 1993. In this paper we present, a history of the project, details of the magnet, installation, commissioning, and resulting performance of the ALS with Superbends.

  17. Neutron protein crystallography hydrogen protons and hydration in bio-macromolecules

    CERN Document Server

    Niimura, Nobuo

    2011-01-01

    This text is dedicated to the emerging field of neutron protein crystallography (NPC). It covers all of the practical aspects of NPC and demonstrates how NPC can explore protein features such as hydrogen bonds, protonation and deprotonation of amino acid residues, and hydration structures.

  18. Optimizing Monocapillary Optics for Synchrotron X-ray Diffraction, Fluorescence Imaging, and Spectroscopy Applications

    International Nuclear Information System (INIS)

    Bilderback, Donald H.; Kazimirov, Alexander; Gillilan, Richard; Cornaby, Sterling; Woll, Arthur; Zha, Chang-Sheng; Huang Rong

    2007-01-01

    A number of synchrotron x-ray applications such as powder diffraction in diamond anvil cells, microbeam protein crystallography, x-ray fluorescence imaging, etc. can benefit from using hollow glass monocapillary optics to improve the flux per square micron on a sample. We currently draw glass tubing into the desired elliptical shape so that only one-bounce under total reflection conditions is needed to bring the x-ray beam to a focus at a 25 to 50 mm distance beyond the capillary tip. For modest focal spot sizes of 10 to 20 microns, we can increase the intensity per square micron by factors of 10 to 1000. We show some of the results obtained at CHESS and Hasylab with capillaries focusing 5 to 40 keV radiation, their properties, and how even better the experimental results could be if more ideal capillaries were fabricated in the future

  19. X-ray astronomy

    International Nuclear Information System (INIS)

    Culhane, J.L.; Sanford, P.W.

    1981-01-01

    X-ray astronomy has been established as a powerful means of observing matter in its most extreme form. The energy liberated by sources discovered in our Galaxy has confirmed that collapsed stars of great density, and with intense gravitational fields, can be studied by making observations in the X-ray part of the electromagnetic spectrum. The astronomical objects which emit detectable X-rays include our own Sun and extend to quasars at the edge of the Universe. This book describes the history, techniques and results obtained in the first twenty-five years of exploration. Space rockets and satellites are essential for carrying the instruments above the Earth's atmosphere where it becomes possible to view the X-rays from stars and nebulae. The subject is covered in chapters, entitled: the birth of X-ray astronomy; the nature of X-radiation; X-rays from the Sun; solar-flare X-rays; X-rays from beyond the solar system; supernovae and their remnants; X-rays from binary stars; white dwarfs and neutron stars; black holes; X-rays from galaxies and quasars; clusters of galaxies; the observatories of the future. (author)

  20. A pixel detector for the protein crystallography beamline at the SLS

    CERN Document Server

    Brönnimann, C; Eikenberry, E F; Fischer, P; Florin, S; Horisberger, R P; Lindner, Manfred; Schmitt, B; Schulze, C

    2002-01-01

    At the Paul Scherrer Institute a new synchrotron light source is currently under construction, the Swiss Light Source (SLS), which will be operational in summer 2001. Among the first beamlines is a high brightness, micro-focusing protein crystallography beamline. It will be equipped with a pixel detector, which has several features of interest for the next generation of protein crystallography detectors. The point spread function and the effect of charge sharing was measured with a prototype detector in a test experiment at the European Synchrotron Radiation Facility in Grenoble. The concepts of the SLS pixel detector is presented as well as test results from radiation hard prototype chips.

  1. Purification, isolation, crystallization, and preliminary X-ray diffraction study of the BTB domain of the centrosomal protein 190 from Drosophila melanogaster

    Science.gov (United States)

    Boyko, K. M.; Nikolaeva, A. Yu.; Kachalova, G. S.; Bonchuk, A. N.; Popov, V. O.

    2017-11-01

    The spatial organization of the genome is controlled by a special class of architectural proteins, including proteins containing BTB domains that are able to dimerize or multimerize. The centrosomal protein 190 is one of such architectural proteins. The purification, crystallization, and preliminary X-ray diffraction study of the BTB domain of the centrosomal protein 190 are reported. The crystallization conditions were found by the vapor-diffusion technique. The crystals diffracted to 1.5 Å resolution and belonged to sp. gr. P3221. The structure was solved by the molecular replacement method. The structure refinement is currently underway.

  2. Characterizing the Secondary Protein Structure of Black Widow Dragline Silk Using Solid-State NMR & X-ray Diffraction

    Science.gov (United States)

    Jenkins, Janelle E.; Sampath, Sujatha; Butler, Emily; Kim, Jihyun; Henning, Robert W.; Holland, Gregory P.; Yarger, Jeffery L.

    2013-01-01

    This study provides a detailed secondary structural characterization of major ampullate dragline silk from Latrodectus hesperus (black widow) spiders. X-ray diffraction results show that the structure of black widow major ampullate silk fibers is comprised of stacked β-sheet nanocrystallites oriented parallel to the fiber axis and an amorphous region with oriented (anisotropic) and isotropic components. The combination of two-dimensional (2D) 13C-13C through-space and through-bond solid-state NMR experiments provide chemical shifts that are used to determine detailed information about amino acid motif secondary structure in black widow spider dragline silk. Individual amino acids are incorporated into different repetitive motifs that make up the majority of this protein-based biopolymer. From the solid-state NMR measurements, we assign distinct secondary conformations to each repetitive amino acid motif and hence to the amino acids that make up the motifs. Specifically, alanine is incorporated in β-sheet (poly(Alan) and poly(Gly-Ala)), 31-helix (poly(Gly-Gly-Xaa), and α-helix (poly(Gln-Gln-Ala-Tyr)) components. Glycine is determined to be in β-sheet (poly(Gly-Ala)) and 31-helical (poly(Gly-Gly-Xaa)) regions, while serine is present in β-sheet (poly(Gly-Ala-Ser)), 31-helix (poly(Gly-Gly-Ser)), and β-turn (poly(Gly-Pro-Ser)) structures. These various motif-specific secondary structural elements are quantitatively correlated to the primary amino acid sequence of major ampullate spidroin 1 and 2 (MaSp1 and MaSp2) and are shown to form a self-consistent model for black widow dragline silk. PMID:24024617

  3. X-ray holography

    International Nuclear Information System (INIS)

    Faigel, G.; Tegze, M.; Belakhovsky, M.; Marchesini, S.; Bortel, G.

    2003-01-01

    In the last decade holographic methods using hard X-rays were developed. They are able to resolve atomic distances, and can give the 3D arrangement of atoms around a selected element. Therefore, hard X-ray holography has potential applications in chemistry, biology and physics. In this article we give a general description of these methods and discuss the developments in the experimental technique. The capabilities of hard X-ray holography are demonstrated by examples

  4. Providing x-rays

    International Nuclear Information System (INIS)

    Mallozzi, P.J.; Epstein, H.M.

    1985-01-01

    This invention provides an apparatus for providing x-rays to an object that may be in an ordinary environment such as air at approximately atmospheric pressure. The apparatus comprises: means (typically a laser beam) for directing energy onto a target to produce x-rays of a selected spectrum and intensity at the target; a fluid-tight enclosure around the target; means for maintaining the pressure in the first enclosure substantially below atmospheric pressure; a fluid-tight second enclosure adjoining the first enclosure, the common wall portion having an opening large enough to permit x-rays to pass through but small enough to allow the pressure reducing means to evacuate gas from the first enclosure at least as fast as it enters through the opening; the second enclosure filled with a gas that is highly transparent to x-rays; the wall of the second enclosure to which the x-rays travel having a portion that is highly transparent to x-rays (usually a beryllium or plastic foil), so that the object to which the x-rays are to be provided may be located outside the second enclosure and adjacent thereto and thus receive the x-rays substantially unimpeded by air or other intervening matter. The apparatus is particularly suited to obtaining EXAFS (extended x-ray fine structure spectroscopy) data on a material

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    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

  6. Crystallization and preliminary X-ray crystallographic studies of the biotin carboxyl carrier protein and biotin protein ligase complex from Pyrococcus horikoshii OT3

    International Nuclear Information System (INIS)

    Bagautdinov, Bagautdin; Matsuura, Yoshinori; Bagautdinova, Svetlana; Kunishima, Naoki

    2007-01-01

    A truncated form of biotin carboxyl carrier protein containing the C-terminal half fragment (BCCPΔN76) and the biotin protein ligase (BPL) with the mutation R48A (BPL*) or the double mutation R48A K111A (BPL**) were successfully cocrystallized in the presence of ATP and biotin. The BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals belong to space group P2 1 and diffract X-rays to 2.7 and 2.0 Å resolution, respectively. Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. To elucidate the exact details of the protein–protein interactions in the biotinylation function, the C-terminal half fragment of BCCP (BCCPΔN76), the R48A mutant of BPL (BPL*) and the R48A K111A double mutant of BPL (BPL**), all of which are from Pyrococcus horikoshii OT3, have been expressed, purified and successfully cocrystallized. Cocrystals of the BPL*–BCCPΔN76 and BPL**–BCCPΔN76 complexes as well as crystals of BPL*, BPL** and BCCPΔN76 were obtained by the oil-microbatch method using PEG 20 000 as a precipitant at 295 K. Complete X-ray diffraction data sets for BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals were collected at 100 K to 2.7 and 2.0 Å resolution, respectively, using synchrotron radiation. They belong to the monoclinic space group P2 1 , with similar unit-cell parameters a = 69.85, b = 63.12, c = 75.64 Å, β = 95.9°. Assuming two subunits of the complex per asymmetric unit gives a V M value of 2.45 Å 3 Da −1 and a solvent content of 50%

  7. Crystallization and preliminary X-ray analysis of PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins

    International Nuclear Information System (INIS)

    Shirokane, Michio; Sawano, Yoriko; Miyazono, Ken-ichi; Nagata, Koji; Tanokura, Masaru

    2007-01-01

    PH1010, a DUF54-family protein from the hyperthermophilic archaeon P. horikoshii OT3, was crystallized and X-ray diffraction data were collected to 1.90 Å resolution. PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins, was expressed, purified and crystallized. Crystallization was performed by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.90 Å resolution using a synchrotron-radiation source. The space group of the crystal was determined to be P2 1 2 1 2 1 , with unit-cell parameters a = 46.9, b = 49.5, c = 132.7 Å. The crystal contained two PH1010 molecules in the asymmetric unit (V M = 2.4 Å 3 Da −1 ) and had a solvent content of 48%

  8. Crystallization and preliminary X-ray analysis of PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins

    Energy Technology Data Exchange (ETDEWEB)

    Shirokane, Michio; Sawano, Yoriko; Miyazono, Ken-ichi; Nagata, Koji; Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)

    2007-06-01

    PH1010, a DUF54-family protein from the hyperthermophilic archaeon P. horikoshii OT3, was crystallized and X-ray diffraction data were collected to 1.90 Å resolution. PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins, was expressed, purified and crystallized. Crystallization was performed by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.90 Å resolution using a synchrotron-radiation source. The space group of the crystal was determined to be P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.9, b = 49.5, c = 132.7 Å. The crystal contained two PH1010 molecules in the asymmetric unit (V{sub M} = 2.4 Å{sup 3} Da{sup −1}) and had a solvent content of 48%.

  9. Imaging Macromolecules with X-ray laser pulses

    CERN Multimedia

    CERN. Geneva

    2017-01-01

    The short wavelength of X-rays allows us to resolve atoms, but in practise for biological materials the achievable resolution is limited by the destruction of the sample by the radiation that forms the image.  For over 100 years, the workaround to this problem of radiation damage has been to average signals from repeating copies of the object arranged in a large crystal.  It is now possible to overcome damage limits by using intense X-ray pulses that vaporise the sample, but which are short enough in duration to freeze any motion of the sample on the atomic scale.  With the advent of X-ray FELs we have been able to confirm this principle, and are now applying it to overcoming a major bottleneck for protein crystallography, which is the need for large well-diffracting crystals.  The intense pulses also open up opportunities to help solve the crystallographic phase problem.  In particular we have found that commonly-occurring disordered crystals that are usually not ...

  10. Structural enzymology using X-ray free electron lasers

    Directory of Open Access Journals (Sweden)

    Christopher Kupitz

    2017-07-01

    Full Text Available Mix-and-inject serial crystallography (MISC is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i room temperature structures at near atomic resolution, (ii time resolution ranging from microseconds to seconds, and (iii convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8 Å and 2.4 Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.

  11. Crystallization of the glycogen-binding domain of the AMP-activated protein kinase β subunit and preliminary X-ray analysis

    Energy Technology Data Exchange (ETDEWEB)

    Polekhina, Galina, E-mail: gpolekhina@svi.edu.au; Feil, Susanne C.; Gupta, Abhilasha [St Vincent’s Institute of Medical Research, 9 Princes Street, Fitzroy, Victoria 3065 (Australia); O’Donnell, Paul [Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville 3010 (Australia); Stapleton, David; Parker, Michael W. [St Vincent’s Institute of Medical Research, 9 Princes Street, Fitzroy, Victoria 3065 (Australia)

    2005-01-01

    The glycogen-binding domain of the AMP-activated kinase β subunit has been crystallized in the presence of β-cyclodextrin. The structure has been determined by single isomorphous replacement and threefold averaging using in-house X-ray data collected from selenomethionine-substituted protein. AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolism in response to energy demand and supply by adjusting the ATP-generating and ATP-consuming pathways. AMPK potentially plays a critical role in diabetes and obesity as it is known to be activated by metforin and rosiglitazone, drugs used for the treatment of type II diabetes. AMPK is a heterotrimer composed of a catalytic α subunit and two regulatory subunits, β and γ. Mutations in the γ subunit are known to cause glycogen accumulation, leading to cardiac arrhythmias. Recently, a functional glycogen-binding domain (GBD) has been identified in the β subunit. Here, the crystallization of GBD in the presence of β-cyclodextrin is reported together with preliminary X-ray data analysis allowing the determination of the structure by single isomorphous replacement and threefold averaging using in-house X-ray data collected from a selenomethionine-substituted protein.

  12. Crystallization of the glycogen-binding domain of the AMP-activated protein kinase β subunit and preliminary X-ray analysis

    International Nuclear Information System (INIS)

    Polekhina, Galina; Feil, Susanne C.; Gupta, Abhilasha; O’Donnell, Paul; Stapleton, David; Parker, Michael W.

    2004-01-01

    The glycogen-binding domain of the AMP-activated kinase β subunit has been crystallized in the presence of β-cyclodextrin. The structure has been determined by single isomorphous replacement and threefold averaging using in-house X-ray data collected from selenomethionine-substituted protein. AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolism in response to energy demand and supply by adjusting the ATP-generating and ATP-consuming pathways. AMPK potentially plays a critical role in diabetes and obesity as it is known to be activated by metforin and rosiglitazone, drugs used for the treatment of type II diabetes. AMPK is a heterotrimer composed of a catalytic α subunit and two regulatory subunits, β and γ. Mutations in the γ subunit are known to cause glycogen accumulation, leading to cardiac arrhythmias. Recently, a functional glycogen-binding domain (GBD) has been identified in the β subunit. Here, the crystallization of GBD in the presence of β-cyclodextrin is reported together with preliminary X-ray data analysis allowing the determination of the structure by single isomorphous replacement and threefold averaging using in-house X-ray data collected from a selenomethionine-substituted protein

  13. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* carbohydrate-binding protein of the human rotavirus strain Wa

    International Nuclear Information System (INIS)

    Kraschnefski, Mark J.; Scott, Stacy A.; Holloway, Gavan; Coulson, Barbara S.; Itzstein, Mark von; Blanchard, Helen

    2005-01-01

    The carbohydrate-binding component (VP8* 64–223 ) of the human Wa rotavirus spike protein has been overexpressed in E. coli, purified and crystallized in two different crystal forms. X-ray diffraction data have been collected that have enabled determination of the Wa VP8* 64–223 structure by molecular replacement. Rotaviruses exhibit host-specificity and the first crystallographic information on a rotavirus strain that infects humans is reported here. Recognition and attachment to host cells, leading to invasion and infection, is critically linked to the function of the outer capsid spike protein of the rotavirus particle. In some strains the VP8* component of the spike protein is implicated in recognition and binding of sialic-acid-containing cell-surface carbohydrates, thereby enabling infection by the virus. The cloning, expression, purification, crystallization and initial X-ray diffraction analysis of the VP8* core from human Wa rotavirus is reported. Two crystal forms (trigonal P3 2 21 and monoclinic P2 1 ) have been obtained and X-ray diffraction data have been collected, enabling determination of the VP8* 64–223 structure by molecular replacement

  14. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* carbohydrate-binding protein of the human rotavirus strain Wa

    Energy Technology Data Exchange (ETDEWEB)

    Kraschnefski, Mark J.; Scott, Stacy A. [Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Queensland 9726 (Australia); Holloway, Gavan; Coulson, Barbara S.; Itzstein, Mark von [Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010 (Australia); Blanchard, Helen, E-mail: h.blanchard@griffith.edu.au [Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Queensland 9726 (Australia)

    2005-11-01

    The carbohydrate-binding component (VP8*{sub 64–223}) of the human Wa rotavirus spike protein has been overexpressed in E. coli, purified and crystallized in two different crystal forms. X-ray diffraction data have been collected that have enabled determination of the Wa VP8*{sub 64–223} structure by molecular replacement. Rotaviruses exhibit host-specificity and the first crystallographic information on a rotavirus strain that infects humans is reported here. Recognition and attachment to host cells, leading to invasion and infection, is critically linked to the function of the outer capsid spike protein of the rotavirus particle. In some strains the VP8* component of the spike protein is implicated in recognition and binding of sialic-acid-containing cell-surface carbohydrates, thereby enabling infection by the virus. The cloning, expression, purification, crystallization and initial X-ray diffraction analysis of the VP8* core from human Wa rotavirus is reported. Two crystal forms (trigonal P3{sub 2}21 and monoclinic P2{sub 1}) have been obtained and X-ray diffraction data have been collected, enabling determination of the VP8*{sub 64–223} structure by molecular replacement.

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

    Directory of Open Access Journals (Sweden)

    Matthew C Clifton

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

  16. X-ray interferometers

    International Nuclear Information System (INIS)

    Franks, A.

    1980-01-01

    An improved type of amplitude-division x-ray interferometer is described. The wavelength at which the interferometer can operate is variable, allowing the instrument to be used to measure x-ray wavelength, and the angle of inclination is variable for sample investigation. (U.K.)

  17. Extremity x-ray

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003461.htm Extremity x-ray To use the sharing features on this page, ... in the body Risks There is low-level radiation exposure. X-rays are monitored and regulated to provide the ...

  18. X-rays utilization

    International Nuclear Information System (INIS)

    Rebigan, F.

    1979-03-01

    The modality of X-ray utilization in different activities and economy is given. One presents firstly quantities and units used in radiation dosimetry and other fields. One gives the generation of X-rays, their properties as well as the elements of radiation protection. The utilization characteristics of these radiations in different fields are finally given. (author)

  19. Chest X-Ray

    Medline Plus

    Full Text Available ... by Image/Video Gallery Your Radiologist Explains Chest X-ray Transcript Welcome to Radiology Info dot org! Hello, ... d like to talk with you about chest radiography also known as chest x-rays. Chest x- ...

  20. Automation of specimen selection and data acquisition for protein electron crystallography

    NARCIS (Netherlands)

    Oostergetel, G.T.; Keegstra, W.; Brisson, A.D R

    A system is presented for semi-automatic specimen selection and data acquisition for protein electron crystallography, based on a slow-scan CCD camera connected to a transmission electron microscope and control from an external computer. Areas of interest on the specimen are localised at low

  1. High-pressure crystallography

    Science.gov (United States)

    Katrusiak, A.

    2008-01-01

    The history and development of high-pressure crystallography are briefly described and examples of structural transformations in compressed compounds are given. The review is focused on the diamond-anvil cell, celebrating its 50th anniversary this year, the principles of its operation and the impact it has had on high-pressure X-ray diffraction.

  2. Crystallization and preliminary X-ray crystallographic analysis of CheW from Thermotoga maritima: a coupling protein of CheA and the chemotaxis receptor

    International Nuclear Information System (INIS)

    Park, SangYoun; Crane, Brian R.

    2011-01-01

    CheW from T. maritima has been crystallized (space group P6 3 , unit-cell parameters a = b = 61.265, c = 361.045 Å). Diffraction data have been collected to 3.1 Å resolution using synchrotron X-ray radiation. The CheW protein plays a key role in bacterial chemotaxis signal transduction by coupling CheA to chemotaxis receptors. CheW from Thermotoga maritima has been overexpressed in Escherichia coli and crystallized at 298 K using ammonium sulfate as a salt precipitant. X-ray diffraction data have been collected to 3.10 Å resolution at 100 K using synchrotron radiation. The crystal belonged to space group P6 3 , with unit-cell parameters a = b = 61.265, c = 361.045 Å. The asymmetric unit may contain four to six CheW molecules

  3. Quantum crystallography: A perspective.

    Science.gov (United States)

    Massa, Lou; Matta, Chérif F

    2018-06-30

    Extraction of the complete quantum mechanics from X-ray scattering data is the ultimate goal of quantum crystallography. This article delivers a perspective for that possibility. It is desirable to have a method for the conversion of X-ray diffraction data into an electron density that reflects the antisymmetry of an N-electron wave function. A formalism for this was developed early on for the determination of a constrained idempotent one-body density matrix. The formalism ensures pure-state N-representability in the single determinant sense. Applications to crystals show that quantum mechanical density matrices of large molecules can be extracted from X-ray scattering data by implementing a fragmentation method termed the kernel energy method (KEM). It is shown how KEM can be used within the context of quantum crystallography to derive quantum mechanical properties of biological molecules (with low data-to-parameters ratio). © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    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. (paper)

  5. Optimizing total reflection X-ray fluorescence for direct trace element quantification in proteins I: Influence of sample homogeneity and reflector type

    Science.gov (United States)

    Wellenreuther, G.; Fittschen, U. E. A.; Achard, M. E. S.; Faust, A.; Kreplin, X.; Meyer-Klaucke, W.

    2008-12-01

    Total reflection X-ray fluorescence (TXRF) is a very promising method for the direct, quick and reliable multi-elemental quantification of trace elements in protein samples. With the introduction of an internal standard consisting of two reference elements, scandium and gallium, a wide range of proteins can be analyzed, regardless of their salt content, buffer composition, additives and amino acid composition. This strategy also enables quantification of matrix effects. Two potential issues associated with drying have been considered in this study: (1) Formation of heterogeneous residues of varying thickness and/or density; and (2) separation of the internal standard and protein during drying (which has to be prevented to allow accurate quantification). These issues were investigated by microbeam X-ray fluorescence (μXRF) with special emphasis on (I) the influence of sample support and (II) the protein / buffer system used. In the first part, a model protein was studied on well established sample supports used in TXRF, PIXE and XRF (Mylar, siliconized quartz, Plexiglas and silicon). In the second part we imaged proteins of different molecular weight, oligomerization state, bound metals and solubility. A partial separation of protein and internal standard was only observed with untreated silicon, suggesting it may not be an adequate support material. Siliconized quartz proved to be the least prone to heterogeneous drying of the sample and yielded the most reliable results.

  6. Optimizing total reflection X-ray fluorescence for direct trace element quantification in proteins I: Influence of sample homogeneity and reflector type

    Energy Technology Data Exchange (ETDEWEB)

    Wellenreuther, G. [European Molecular Biology Laboratory, Notkestr. 85, 22603 Hamburg (Germany); Fittschen, U.E.A. [Department of Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg (Germany); Achard, M.E.S.; Faust, A.; Kreplin, X. [European Molecular Biology Laboratory, Notkestr. 85, 22603 Hamburg (Germany); Meyer-Klaucke, W. [European Molecular Biology Laboratory, Notkestr. 85, 22603 Hamburg (Germany)], E-mail: Wolfram@embl-hamburg.de

    2008-12-15

    Total reflection X-ray fluorescence (TXRF) is a very promising method for the direct, quick and reliable multi-elemental quantification of trace elements in protein samples. With the introduction of an internal standard consisting of two reference elements, scandium and gallium, a wide range of proteins can be analyzed, regardless of their salt content, buffer composition, additives and amino acid composition. This strategy also enables quantification of matrix effects. Two potential issues associated with drying have been considered in this study: (1) Formation of heterogeneous residues of varying thickness and/or density; and (2) separation of the internal standard and protein during drying (which has to be prevented to allow accurate quantification). These issues were investigated by microbeam X-ray fluorescence ({mu}XRF) with special emphasis on (I) the influence of sample support and (II) the protein / buffer system used. In the first part, a model protein was studied on well established sample supports used in TXRF, PIXE and XRF (Mylar, siliconized quartz, Plexiglas and silicon). In the second part we imaged proteins of different molecular weight, oligomerization state, bound metals and solubility. A partial separation of protein and internal standard was only observed with untreated silicon, suggesting it may not be an adequate support material. Siliconized quartz proved to be the least prone to heterogeneous drying of the sample and yielded the most reliable results.

  7. X-ray lasers

    CERN Document Server

    Elton, Raymond C

    2012-01-01

    The first in its field, this book is both an introduction to x-ray lasers and a how-to guide for specialists. It provides new entrants and others interested in the field with a comprehensive overview and describes useful examples of analysis and experiments as background and guidance for researchers undertaking new laser designs. In one succinct volume, X-Ray Lasers collects the knowledge and experience gained in two decades of x-ray laser development and conveys the exciting challenges and possibilities still to come._Add on for longer version of blurb_M>The reader is first introduced

  8. X-ray apparatus

    International Nuclear Information System (INIS)

    Bernstein, S.; Stagg, L.; Lambert, T.W.; Griswa, P.J.

    1976-01-01

    A patient support system for X-ray equipment in arteriographic studies of the heart is described in detail. The support system has been designed to overcome many of the practical problems encountered in using previous types of arteriographic X-ray equipment. The support system is capable of horizontal movement and, by a series of shafts attached to the main support system, the X-ray source and image intensifier or detector may be rotated through the same angle. The system is highly flexible and details are given of several possible operational modes. (U.K.)

  9. X-ray detector

    International Nuclear Information System (INIS)

    Whetten, N.R.; Houston, J.M.

    1977-01-01

    An ionization chamber for use in determining the spatial distribution of x-ray photons in tomography systems comprises a plurality of substantially parallel, planar anodes separated by parallel, planar cathodes and enclosed in a gas of high atomic weight at a pressure from approximately 10 atmospheres to approximately 50 atmospheres. The cathode and anode structures comprise metals which are substantially opaque to x-ray radiation and thereby tend to reduce the resolution limiting effects of x-ray fluoresence in the gas. In another embodiment of the invention the anodes comprise parallel conductive bars disposed between two planar cathodes. Guard rings eliminate surface leakage currents between adjacent electrodes. 8 figures

  10. X-ray apparatus

    International Nuclear Information System (INIS)

    Grady, J.K.

    1985-01-01

    X-ray apparatus is described which has a shutter between the X-ray source and the patient. The shutter controls the level of radiation to which the patient is exposed instead of merely discontinuing the electric power supplied to the source. When the shutter is opened a radiation sensor senses the level of X-radiation. When a preset quantity of X-radiation has been measured an exposure control closes the shutter. Instead of using the radiation sensor, the integrated power supplied to the anode of the X-ray source may be measured. (author)

  11. Bone X-Ray (Radiography)

    Medline Plus

    Full Text Available ... Resources Professions Site Index A-Z X-ray (Radiography) - Bone Bone x-ray uses a very small ... X-ray (Radiography)? What is Bone X-ray (Radiography)? An x-ray (radiograph) is a noninvasive medical ...

  12. Bone X-Ray (Radiography)

    Medline Plus

    Full Text Available ... Professions Site Index A-Z X-ray (Radiography) - Bone Bone x-ray uses a very small dose ... limitations of Bone X-ray (Radiography)? What is Bone X-ray (Radiography)? An x-ray (radiograph) is ...

  13. X-ray examination apparatus

    NARCIS (Netherlands)

    2000-01-01

    The invention relates to an X-ray apparatus which includes an adjustable X-ray filter. In order to adjust an intensity profile of the X-ray beam, an X-ray absorbing liquid is transported to filter elements of the X-ray filter. Such transport is susceptible to gravitational forces which lead to an

  14. Bone X-Ray (Radiography)

    Medline Plus

    Full Text Available ... News Physician Resources Professions Site Index A-Z X-ray (Radiography) - Bone Bone x-ray uses a very ... of Bone X-ray (Radiography)? What is Bone X-ray (Radiography)? An x-ray (radiograph) is a noninvasive ...

  15. Bone X-Ray (Radiography)

    Science.gov (United States)

    ... News Physician Resources Professions Site Index A-Z X-ray (Radiography) - Bone Bone x-ray uses a very small ... of Bone X-ray (Radiography)? What is Bone X-ray (Radiography)? An x-ray (radiograph) is a noninvasive ...

  16. Bone X-Ray (Radiography)

    Medline Plus

    Full Text Available ... News Physician Resources Professions Site Index A-Z X-ray (Radiography) - Bone Bone x-ray uses a very small ... of Bone X-ray (Radiography)? What is Bone X-ray (Radiography)? An x-ray (radiograph) is a noninvasive ...

  17. Abdomen X-Ray (Radiography)

    Science.gov (United States)

    ... News Physician Resources Professions Site Index A-Z X-ray (Radiography) - Abdomen Abdominal x-ray uses a very small ... of an abdominal x-ray? What is abdominal x-ray? An x-ray (radiograph) is a noninvasive medical ...

  18. Distributed control of protein crystallography beamline 5.0 using CORBA

    International Nuclear Information System (INIS)

    Timossi, Chris

    1999-01-01

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

  19. Getting the best out of long-wavelength X-rays: de novo chlorine/sulfur SAD phasing of a structural protein from ATV

    DEFF Research Database (Denmark)

    Goulet, Adeline; Vestergaard, Gisle Alberg; Felisberto-Rodrigues, Catarina

    2010-01-01

    The structure of a 14 kDa structural protein from Acidianus two-tailed virus (ATV) was solved by single-wavelength anomalous diffraction (SAD) phasing using X-ray data collected at 2.0 A wavelength. Although the anomalous signal from methionine sulfurs was expected to suffice to solve the structu...... on intrinsic protein light atoms along with associated chloride ions from the solvent. In such cases, data collection at long wavelengths may be a time-efficient alternative to selenomethionine substitution and heavy-atom derivatization....

  20. Chest X-Ray

    Medline Plus

    Full Text Available ... Disorders Video: The Basketball Game: An MRI Story Radiology and You Sponsored by Image/Video Gallery Your Radiologist Explains Chest X-ray Transcript Welcome to Radiology Info dot org! Hello, I’m Dr. Geoffrey ...

  1. Chest X-Ray

    Medline Plus

    Full Text Available ... also be useful to help diagnose and monitor treatment for a variety of lung conditions such as pneumonia, emphysema and cancer. A chest x-ray requires no special preparation. ...

  2. X-ray tubes

    International Nuclear Information System (INIS)

    Young, R.W.

    1979-01-01

    A form of x-ray tube is described which provides satisfactory focussing of the electron beam when the beam extends for several feet from gun to target. Such a tube can be used for computerised tomographic scanning. (UK)

  3. Chest X-Ray

    Medline Plus

    Full Text Available ... breath, persistent cough, fever, chest pain or injury. It may also be useful to help diagnose and ... have some concerns about chest x-rays. However, it’s important to consider the likelihood of benefit to ...

  4. Chest X-Ray

    Medline Plus

    Full Text Available ... An MRI Story Radiology and You Sponsored by Image/Video Gallery Your Radiologist Explains Chest X-ray ... posted: How to Obtain and Share Your Medical Images Movement Disorders Video: The Basketball Game: An MRI ...

  5. Chest X-Ray

    Medline Plus

    Full Text Available ... accurate diagnosis far outweighs any risk. For more information about chest x-rays, visit Radiology Info dot ... Inc. (RSNA). To help ensure current and accurate information, we do not permit copying but encourage linking ...

  6. Chest X-Ray

    Medline Plus

    Full Text Available ... Site Index A-Z Spotlight Recently posted: Pancreatic Cancer The Limitations of Online Dose Calculators Video: The ... of lung conditions such as pneumonia, emphysema and cancer. A chest x-ray requires no special preparation. ...

  7. Chest X-Ray

    Medline Plus

    Full Text Available ... exams and use a very small dose of ionizing radiation to produce pictures of the inside of the ... chest x-ray use a tiny dose of ionizing radiation, the benefit of an accurate diagnosis far outweighs ...

  8. Chest X-Ray

    Medline Plus

    Full Text Available ... However, it’s important to consider the likelihood of benefit to your health. While a chest x-ray use a tiny dose of ionizing radiation, the benefit of an accurate diagnosis far outweighs any risk. ...

  9. Chest X-Ray

    Medline Plus

    Full Text Available ... June is Men's Health Month Recently posted: Pancreatic Cancer The Limitations of Online Dose Calculators Video: The ... of lung conditions such as pneumonia, emphysema and cancer. A chest x-ray requires no special preparation. ...

  10. X-ray sources

    International Nuclear Information System (INIS)

    Masswig, I.

    1986-01-01

    The tkb market survey comparatively evaluates the X-ray sources and replacement tubes for stationary equipment currently available on the German market. It lists the equipment parameters of 235 commercially available X-ray sources and their replacement tubes and gives the criteria for purchase decisions. The survey has been completed with December 1985, and offers good information concerning medical and technical aspects as well as those of safety and maintenance. (orig.) [de

  11. Bone X-Ray (Radiography)

    Medline Plus

    Full Text Available ... of knee x-rays. A portable x-ray machine is a compact apparatus that can be taken ... of the body being examined, an x-ray machine produces a small burst of radiation that passes ...

  12. Bone X-Ray (Radiography)

    Medline Plus

    Full Text Available ... that might interfere with the x-ray images. Women should always inform their physician and x-ray ... Safety page for more information about radiation dose. Women should always inform their physician or x-ray ...

  13. Bone X-Ray (Radiography)

    Medline Plus

    Full Text Available ... ray examination. X-rays usually have no side effects in the typical diagnostic range for this exam. ... x-rays. A Word About Minimizing Radiation Exposure Special care is taken during x-ray examinations to ...

  14. Bone X-Ray (Radiography)

    Medline Plus

    Full Text Available ... drawer under the table holds the x-ray film or image recording plate . Sometimes the x-ray ... extended over the patient while an x-ray film holder or image recording plate is placed beneath ...

  15. Bone X-Ray (Radiography)

    Medline Plus

    Full Text Available ... are the limitations of Bone X-ray (Radiography)? What is Bone X-ray (Radiography)? An x-ray ( ... leg (shin), ankle or foot. top of page What are some common uses of the procedure? A ...

  16. Bone X-Ray (Radiography)

    Medline Plus

    Full Text Available ... current x-ray images for diagnosis and disease management. top of page How is the procedure performed? ... standards used by radiology professionals. Modern x-ray systems have very controlled x-ray beams and dose ...

  17. X-ray diffraction study of Penicillium Vitale catalase in the complex with aminotriazole

    International Nuclear Information System (INIS)

    Borovik, A. A.; Grebenko, A. I.; Melik-Adamyan, V. R.

    2011-01-01

    The three-dimensional structure of the enzyme catalase from Penicillium vitale in a complex with the inhibitor aminotriazole was solved and refined by protein X-ray crystallography methods. An analysis of the three-dimensional structure of the complex showed that the inhibition of the enzyme occurs as a result of the covalent binding of aminotriazole to the amino-acid residue His64 in the active site of the enzyme. An investigation of the three-dimensional structure of the complex resulted in the amino-acid residues being more precisely identified. The binding sites of saccharide residues and calcium ions in the protein molecule were found.

  18. Expression and localization of Smad4 protein in mouse testis after whole-body X-ray irradiation

    International Nuclear Information System (INIS)

    Zhu Huaping; Zhang Yuanqiang; Zhao Jie; Zhao Yong; Ma Jing; Hou Wugang; Qi Yuhong

    2005-01-01

    The work is to determine whether and where Transforming growth factor-betas downstream Signaling molecule Smad4 is expressed in the testes after whole-body X-ray irradiation and shed light on the mechanisms of Transforming growth factor-betas/Smad signal pathway mediates cell fate decisions following X-ray exposure. Five groups of adult BALB/c mice, with ten mice in each group, received whole-body of X-ray at dose levels of 0.1 Gy, 0.5 Gy, 1.0 Gy, 1.5 Gy and 2.0 Gy. They were sacrificed at 16 hour, 1 week, 2 weeks, 3 weeks and 4 weeks after the irradiation. Cellular localization and expression changes were examined by immunohistochemical ABC method. Quantitative analysis of the immunostaining was made by an image analysis system. In the seminiferous tubules, the expression of Smad4 was modulated by irradiation. The immunostaining showed that 16 hour post-irradiation, there was a significant decline in the Leydig cell, and it was dose and time depended. In addition, the immunolocalization showed that Smad4 was not exclusively localized in the cytoplasm of Leydig cells, but also localized in various Stages of spermatogenesis after the exposure, especially in premeiotic spermatogonia and primary spermatocytes. There was just a little expression in the 2.0 Gy group 16 h after the irradiation and the 1.0 Gy and 1.5 Gy groups at 2 weeks after the irradiation. Therefore in the 0.1 Gy to 2.0 Gy groups at 3 weeks after the irradiation, the immunostaining positive cells were significantly increased in spermatogonia and primary spermatocytes. There was no significant change in sertoli cells with different doses and different times after the exposure. The different expression patterns and change by dose and time of Smad4, suggest that TGF-β/Smad signal pathway may affect aspect after X-ray impairment and Smad4 may play an important role during these periods. (authors)

  19. Editorial: Focus on X-ray Beams with High Coherence

    Science.gov (United States)

    Robinson, Ian; Gruebel, Gerhard; Mochrie, Simon

    2010-03-01

    /Pd multilayer films K A Seu, R Su, S Roy, D Parks, E Shipton, E E Fullerton and S D Kevan Holographic and diffractive x-ray imaging using waveguides as quasi-point sources K Giewekemeyer, H Neubauer, S Kalbfleisch, S P Krüger and T Salditt Mapping the conformations of biological assemblies P Schwander, R Fung, G N Phillips Jr and A Ourmazd Imaging the displacement field within epitaxial nanostructures by coherent diffraction: a feasibility study Ana Diaz, Virginie Chamard, Cristian Mocuta, Rogerio Magalhães-Paniago, Julian Stangl, Dina Carbone, Till H Metzger and Günther Bauer The potential for two-dimensional crystallography of membrane proteins at future x-ray free-electron laser sources Cameron M Kewish, Pierre Thibault, Oliver Bunk and Franz Pfeiffer Coherence properties of hard x-ray synchrotron sources and x-ray free-electron lasers I A Vartanyants and A Singer Coherent imaging of biological samples with femtosecond pulses at the free-electron laser FLASH A P Mancuso, Th Gorniak, F Staier, O M Yefanov, R Barth, C Christophis, B Reime, J Gulden, A Singer, M E Pettit, Th Nisius, Th Wilhein, C Gutt, G Grübel, N Guerassimova, R Treusch, J Feldhaus, S Eisebitt, E Weckert, M Grunze, A Rosenhahn and I A Vartanyants

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

    Science.gov (United States)

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

    2015-10-08

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

  1. Protein diffraction experiments with Atlas CCD detector

    Czech Academy of Sciences Publication Activity Database

    Dohnálek, Jan; Kovaľ, Tomáš; Dušek, Michal

    2008-01-01

    Roč. 64, Suppl. - abstracts (2008), C192 ISSN 0108-7673. [Congress of the International Union of Crystallography (IUCr) /21./. 23.08.2008-31.08.2008, Osaka] Institutional research plan: CEZ:AV0Z10100521 Keywords : x-ray data collection * CCD detectors * protein crystallography applications Subject RIV: BM - Solid Matter Physics ; Magnetism

  2. Metalloprotein Crystallography: More than a Structure.

    Science.gov (United States)

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

    2016-04-19

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

  3. Covering complete proteomes with X-ray structures: a current snapshot

    Energy Technology Data Exchange (ETDEWEB)

    Mizianty, Marcin J.; Fan, Xiao; Yan, Jing; Chalmers, Eric; Woloschuk, Christopher [University of Alberta, Edmonton, Alberta T6G 2V4 (Canada); Joachimiak, Andrzej, E-mail: andrzejj@anl.gov [Argonne National Laboratory, Argonne, IL 60439 (United States); Kurgan, Lukasz, E-mail: andrzejj@anl.gov [University of Alberta, Edmonton, Alberta T6G 2V4 (Canada)

    2014-11-01

    The current and the attainable coverage by X-ray structures of proteins and their functions on the scale of the ‘protein universe’ are estimated. A detailed analysis of the coverage across nearly 2000 proteomes from all superkingdoms of life and functional annotations is performed, with particular focus on the human proteome and the family of GPCR proteins. Structural genomics programs have developed and applied structure-determination pipelines to a wide range of protein targets, facilitating the visualization of macromolecular interactions and the understanding of their molecular and biochemical functions. The fundamental question of whether three-dimensional structures of all proteins and all functional annotations can be determined using X-ray crystallography is investigated. A first-of-its-kind large-scale analysis of crystallization propensity for all proteins encoded in 1953 fully sequenced genomes was performed. It is shown that current X-ray crystallographic knowhow combined with homology modeling can provide structures for 25% of modeling families (protein clusters for which structural models can be obtained through homology modeling), with at least one structural model produced for each Gene Ontology functional annotation. The coverage varies between superkingdoms, with 19% for eukaryotes, 35% for bacteria and 49% for archaea, and with those of viruses following the coverage values of their hosts. It is shown that the crystallization propensities of proteomes from the taxonomic superkingdoms are distinct. The use of knowledge-based target selection is shown to substantially increase the ability to produce X-ray structures. It is demonstrated that the human proteome has one of the highest attainable coverage values among eukaryotes, and GPCR membrane proteins suitable for X-ray structure determination were determined.

  4. Covering complete proteomes with X-ray structures: a current snapshot

    International Nuclear Information System (INIS)

    Mizianty, Marcin J.; Fan, Xiao; Yan, Jing; Chalmers, Eric; Woloschuk, Christopher; Joachimiak, Andrzej; Kurgan, Lukasz

    2014-01-01

    The current and the attainable coverage by X-ray structures of proteins and their functions on the scale of the ‘protein universe’ are estimated. A detailed analysis of the coverage across nearly 2000 proteomes from all superkingdoms of life and functional annotations is performed, with particular focus on the human proteome and the family of GPCR proteins. Structural genomics programs have developed and applied structure-determination pipelines to a wide range of protein targets, facilitating the visualization of macromolecular interactions and the understanding of their molecular and biochemical functions. The fundamental question of whether three-dimensional structures of all proteins and all functional annotations can be determined using X-ray crystallography is investigated. A first-of-its-kind large-scale analysis of crystallization propensity for all proteins encoded in 1953 fully sequenced genomes was performed. It is shown that current X-ray crystallographic knowhow combined with homology modeling can provide structures for 25% of modeling families (protein clusters for which structural models can be obtained through homology modeling), with at least one structural model produced for each Gene Ontology functional annotation. The coverage varies between superkingdoms, with 19% for eukaryotes, 35% for bacteria and 49% for archaea, and with those of viruses following the coverage values of their hosts. It is shown that the crystallization propensities of proteomes from the taxonomic superkingdoms are distinct. The use of knowledge-based target selection is shown to substantially increase the ability to produce X-ray structures. It is demonstrated that the human proteome has one of the highest attainable coverage values among eukaryotes, and GPCR membrane proteins suitable for X-ray structure determination were determined

  5. X-ray crystallographic studies of the extracellular domain of the first plant ATP receptor, DORN1, and the orthologous protein from Camelina sativa

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhijie; Chakraborty, Sayan; Xu, Guozhou (NCSU)

    2016-10-26

    Does not respond to nucleotides 1 (DORN1) has recently been identified as the first membrane-integral plant ATP receptor, which is required for ATP-induced calcium response, mitogen-activated protein kinase activation and defense responses inArabidopsis thaliana. In order to understand DORN1-mediated ATP sensing and signal transduction, crystallization and preliminary X-ray studies were conducted on the extracellular domain of DORN1 (atDORN1-ECD) and that of an orthologous protein,Camelina sativalectin receptor kinase I.9 (csLecRK-I.9-ECD or csI.9-ECD). A variety of deglycosylation strategies were employed to optimize the glycosylated recombinant atDORN1-ECD for crystallization. In addition, the glycosylated csI.9-ECD protein was crystallized at 291 K. X-ray diffraction data were collected at 4.6 Å resolution from a single crystal. The crystal belonged to space groupC222 orC2221, with unit-cell parametersa= 94.7,b= 191.5,c= 302.8 Å. These preliminary studies have laid the foundation for structural determination of the DORN1 and I.9 receptor proteins, which will lead to a better understanding of the perception and function of extracellular ATP in plants.

  6. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of universal stress protein F (YnaF) from Salmonella typhimurium

    Energy Technology Data Exchange (ETDEWEB)

    Sagurthi, Someswar Rao; Panigrahi, Rashmi Rekha; Gowda, Giri [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012 (India); Savithri, H. S. [Department of Biochemistry, Indian Institute of Science, Bangalore 560012 (India); Murthy, M. R. N., E-mail: mrn@mbu.iisc.ernet.in [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012 (India)

    2007-11-01

    The cloning, purification and crystallization of YnaF from S. typhimurium are reported along with preliminary X-ray crystallographic studies. The universal stress protein UspF (YnaF) is a small cytoplasmic bacterial protein. The expression of stress proteins is enhanced when cells are exposed to heat shock, nutrition starvation and certain other stress-inducing agents. YnaF promotes cell survival during prolonged exposure to stress and may activate a general mechanism for stress endurance. This manuscript reports preliminary crystallographic studies on YnaF from Salmonella typhimurium. The gene coding for YnaF was cloned and overexpressed and the protein was purified by Ni–NTA affinity chromatography. Purified YnaF was crystallized using vapour-diffusion and microbatch methods. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 37.51, b = 77.18, c = 56.34 Å, β = 101.8°. A data set was collected to 2.5 Å resolution with 94.6% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. Attempts to determine the structure are in progress.

  7. Purification, crystallization and preliminary X-ray diffraction analysis of inner membrane complex (IMC) subcompartment protein 1 (ISP1) from Toxoplasma gondii

    International Nuclear Information System (INIS)

    Tonkin, Michelle L.; Brown, Shannon; Beck, Josh R.; Bradley, Peter J.; Boulanger, Martin J.

    2012-01-01

    To characterize the ISP family of proteins present in apicomplexan parasites, ISP1 from T. gondii was expressed, purified and crystallized. Two crystal forms (cubic and orthorhombic) were analyzed by X-ray diffraction and data were processed to 2.05 and 2.1 Å resolution, respectively. The protozoan parasites of the Apicomplexa phylum are devastating global pathogens. Their success is largely due to phylum-specific proteins found in specialized organelles and cellular structures. The inner membrane complex (IMC) is a unique apicomplexan structure that is essential for motility, invasion and replication. The IMC subcompartment proteins (ISP) have recently been identified in Toxoplasma gondii and shown to be critical for replication, although their specific mechanisms are unknown. Structural characterization of TgISP1 was pursued in order to identify the fold adopted by the ISPs and to generate detailed insight into how this family of proteins functions during replication. An N-terminally truncated form of TgISP1 was purified from Escherichia coli, crystallized and subjected to X-ray diffraction analysis. Two crystal forms of TgISP1 belonging to space groups P4 1 32 or P4 3 32 and P2 1 2 1 2 1 diffracted to 2.05 and 2.1 Å resolution, respectively

  8. Use of dynamic light scattering and small-angle X-ray scattering to characterize new surfactants in solution conditions for membrane-protein crystallization

    Science.gov (United States)

    Dahani, Mohamed; Barret, Laurie-Anne; Raynal, Simon; Jungas, Colette; Pernot, Pétra; Polidori, Ange; Bonneté, Françoise

    2015-01-01

    The structural and interactive properties of two novel hemifluorinated surfactants, F2H9-β-M and F4H5-β-M, the syntheses of which were based on the structure and hydrophobicity of the well known dodecyl-β-maltoside (DD-β-M), are described. The shape of their micellar assemblies was characterized by small-angle X-ray scattering and their intermicellar inter­actions in crystallizing conditions were measured by dynamic light scattering. Such information is essential for surfactant phase-diagram determination and membrane-protein crystallization. PMID:26144228

  9. Purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal fragment of the MvfR protein from Pseudomonas aeruginosa

    International Nuclear Information System (INIS)

    Kefala, Katerina; Kotsifaki, Dina; Providaki, Mary; Kapetaniou, Evangelia G.; Rahme, Lawrence; Kokkinidis, Michael

    2012-01-01

    MvfRC87, a 242-residue C-terminal segment of the LysR-type transcriptional regulator MvfR, was produced in Escherichia coli, purified and crystallized. The LysR-type transcriptional regulator MvfR plays a critical role in Pseudomonas aeruginosa pathogenicity via the transcriptional regulation of multiple quorum-sensing-regulated virulence factors. The protein also controls pathogenic type VI secretion loci. MvfRC87, a 242-residue C-terminal segment of MvfR, was produced in Escherichia coli, purified and crystallized. X-ray diffraction data were collected using synchrotron radiation and crystallographic parameters were determined

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

    DEFF Research Database (Denmark)

    Day, Peter W; Rasmussen, Søren Gøgsig Faarup; Parnot, Charles

    2007-01-01

    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 inf...

  11. SPring-8 Structural Biology Beamlines / Current Status of Public Beamlines for Protein Crystallography at SPring-8

    International Nuclear Information System (INIS)

    Kawamoto, Masahide; Hasegawa, Kazuya; Shimizu, Nobutaka; Sakai, Hisanobu; Shimizu, Tetsuya; Nisawa, Atsushi; Yamamoto, Masaki

    2007-01-01

    SPring-8 has 2 protein crystallography beamlines for public use, BL38B1 (Structural Biology III) and BL41XU (Structural Biology I). The BL38B1 is a bending magnet beamline for routine data collection, and the BL41XU is an undulator beamline specially customized for micro beam and ultra-high resolutional experiment. The designs and the performances of each beamline are presented

  12. X-Ray Absorption with Transmission X-Ray Microscopes

    NARCIS (Netherlands)

    de Groot, F.M.F.

    2016-01-01

    In this section we focus on the use of transmission X-ray microscopy (TXM) to measure the XAS spectra. In the last decade a range of soft X-ray and hard X-ray TXM microscopes have been developed, allowing the measurement of XAS spectra with 10–100 nm resolution. In the hard X-ray range the TXM

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

    Energy Technology Data Exchange (ETDEWEB)

    Coquelle, Nicolas [Université Grenoble Alpes, IBS, 38044 Grenoble (France); CNRS, IBS, 38044 Grenoble (France); CEA, IBS, 38044 Grenoble (France); Brewster, Aaron S. [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Kapp, Ulrike; Shilova, Anastasya; Weinhausen, Britta [European Synchrotron Radiation Facility, BP 220, 38043 Grenoble (France); Burghammer, Manfred, E-mail: burgham@esrf.fr [European Synchrotron Radiation Facility, BP 220, 38043 Grenoble (France); Ghent University, Ghent B-9000 (Belgium); Colletier, Jacques-Philippe, E-mail: burgham@esrf.fr [Université Grenoble Alpes, IBS, 38044 Grenoble (France); CNRS, IBS, 38044 Grenoble (France); CEA, IBS, 38044 Grenoble (France)

    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.

  14. Expression, purification, crystallization and preliminary X-ray analysis of the olfactomedin domain from the sea urchin cell-adhesion protein amassin

    International Nuclear Information System (INIS)

    Hillier, Brian J.; Sundaresan, Vidyasankar; Stout, C. David; Vacquier, Victor D.

    2005-01-01

    The olfactomedin (OLF) domain from the sea urchin cell-adhesion protein amassin has been crystallized. A native data set extending to 2.7 Å has been collected using an in-house X-ray source. A family of animal proteins is emerging which contain a conserved protein motif known as an olfactomedin (OLF) domain. Novel extracellular protein–protein interactions occur through this domain. The OLF-family member amassin, from the sea urchin Strongylocentrotus purpuratus, has previously been identified to mediate a rapid cell-adhesion event resulting in a large aggregation of coelomocytes, the circulating immune cells. In this work, heterologous expression and purification of the OLF domain from amassin was carried out and initial crystallization trials were performed. A native data set has been collected, extending to 2.7 Å under preliminary cryoconditions, using an in-house generator. This work leads the way to the determination of the first structure of an OLF domain

  15. Crystallization and preliminary X-ray diffraction studies of NP24-I, an isoform of a thaumatin-like protein from ripe tomato fruits

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Raka; Chakrabarti, Chandana, E-mail: chandana.chakrabarti@saha.ac.in [Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata 700064 (India)

    2005-08-01

    A thaumatin-like antifungal protein, NP24-I, has been isolated from ripe tomato fruits. It was crystallized by the vapour-diffusion method and data were collected to 2.45 Å. The structure was solved by molecular replacement. NP24 is a 24 kDa (207-amino-acid) antifungal thaumatin-like protein (TLP) found in tomato fruits. An isoform of the protein, NP24-I, is reported to play a possible role in ripening of the fruit in addition to its antifungal properties. The protein has been isolated and purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the tetragonal space group P4{sub 3}, with unit-cell parameters a = b = 61.01, c = 62.90 Å and one molecule per asymmetric unit. X-ray diffraction data were processed to a resolution of 2.45 Å and the structure was solved by molecular replacement.

  16. Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an inositol monophosphatase family protein (SAS2203) from Staphylococcus aureus MSSA476

    International Nuclear Information System (INIS)

    Bhattacharyya, Sudipta; Dutta, Debajyoti; Ghosh, Ananta Kumar; Das, Amit Kumar

    2011-01-01

    The cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an inositol monophosphatase family protein (SAS2203) from S. aureus MSSA476 is reported. The gene product of the sas2203 ORF of Staphylococcus aureus MSSA476 encodes a 30 kDa molecular-weight protein with a high sequence resemblance (29% identity) to tetrameric inositol monophosphatase from Thermotoga maritima. The protein was cloned, expressed, purified to homogeneity and crystallized. Crystals appeared in several conditions and good diffraction-quality crystals were obtained from 0.2 M Li 2 SO 4 , 20% PEG 3350, 0.1 M HEPES pH 7.0 using the sitting-drop vapour-diffusion method. A complete diffraction data set was collected to 2.6 Å resolution using a Rigaku MicroMax-007 HF Cu Kα X-ray generator and a Rigaku R-AXIS IV ++ detector. The diffraction data were consistent with the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 49.98, b = 68.35, c = 143.79 Å, α = β = γ = 90°, and the crystal contained two molecules in the asymmetric unit

  17. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of intracellular growth locus E (IglE) protein from Francisella tularensis subsp. novicida

    International Nuclear Information System (INIS)

    Robb, Craig S.; Nano, Francis E.; Boraston, Alisdair B.

    2010-01-01

    The F. tularensis protein IglE from the FPI, which is a component of the type VI-like secretion system, has been crystallized and preliminary X-ray data have been collected. Tularaemia is an uncommon but potentially dangerous zoonotic disease caused by the bacterium Francisella tularensis. As few as ten bacterial cells are sufficient to cause disease in a healthy human, making this one of the most infectious disease agents known. The virulence of this organism is dependent upon a genetic locus known as the Francisella pathogenicity island (FPI), which encodes components of a secretion system that is related to the type VI secretion system. Here, the cloning, expression, purification and preliminary X-ray diffraction statistics of the FPI-encoded protein IglE are presented. This putative lipoprotein is required for intra-macrophage growth and is thought to be a constituent of the periplasmic portion of the type VI-like protein complex that is responsible for the secretion of critical virulence factors in Francisella

  18. Sulfur K-edge X-ray absorption spectroscopy as an experimental probe for S-nitroso proteins

    International Nuclear Information System (INIS)

    Szilagyi, Robert K.; Schwab, David E.

    2005-01-01

    X-ray absorption spectroscopy at the sulfur K-edge (2.4-2.6 keV) provides a sensitive and specific technique to identify S-nitroso compounds, which have significance in nitric oxide-based cell signaling. Unique spectral features clearly distinguish the S-nitroso-form of a cysteine residue from the sulfhydryl-form or from a methionine thioether. Comparison of the sulfur K-edge spectra of thiolate, thiol, thioether, and S-nitroso thiolate compounds indicates high sensitivity of energy positions and intensities of XAS pre-edge features as determined by the electronic environment of the sulfur absorber. A new experimental setup is being developed for reaching the in vivo concentration range of S-nitroso thiol levels in biological samples

  19. X-ray tube

    International Nuclear Information System (INIS)

    Webley, R.S.

    1975-01-01

    The object of the invention described is to provide an X-ray tube providing a scanned X-ray output which does not require a scanned electron beam. This is obtained by an X-ray tube including an anode which is rotatable about an axis, and a source of a beam of energy, for example an electron beam, arranged to impinge on a surface of the anode to generate X-radiation substantially at the region of incidence on the anode surface. The anode is rotatable about the axis to move the region of incidence over the surface. The anode is so shaped that the rotation causes the region of incidence to move in a predetermined manner relative to fixed parts of the tube so that the generated X-radiation is scanned in a predetermined manner relative to the tube. (UK)

  20. X-ray astronomy

    International Nuclear Information System (INIS)

    Giacconi, R.; Setti, G.

    1980-01-01

    This book contains the lectures, and the most important seminars held at the NATO meeting on X-Ray astronomy in Erice, July 1979. The meeting was an opportune forum to discuss the results of the first 8-months of operation of the X-ray satellite, HEAO-2 (Einstein Observatory) which was launched at the end of 1978. Besides surveying these results, the meeting covered extragalactic astronomy, including the relevant observations obtained in other portions of the electromagnetic spectrum (ultra-violet, optical, infrared and radio). The discussion on galactic X-ray sources essentially covered classical binaries, globular clusters and bursters and its significance to extragalactic sources and to high energy astrophysics was borne in mind. (orig.)

  1. Macromolecular crystallography research at Trombay

    International Nuclear Information System (INIS)

    Kannan, K.K.; Chidamrabam, R.

    1983-01-01

    Neutron diffraction studies of hydrogen positions in small molecules of biological interest at Trombay have provided valuable information that has been used in protein and enzyme structure model-building and in developing hydrogen bond potential functions. The new R-5 reactor is expected to provide higher neutron fluxes and also make possible small-angle neutron scattering studies of large biomolecules and bio-aggregates. In the last few years infrastructure facilities have also been established for macromolecular x-ray crystallography research. Meanwhile, the refinement of carbonic hydrases and lyysozyme structures have been carried out and interesting results obtained on protein dynamics and structure-function relationships. Some interesting presynaptic toxin phospholipases have also taken up for study. (author)

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

    Science.gov (United States)

    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.

  3. Design, synthesis, and protein crystallography of biaryltriazoles as potent tautomerase inhibitors of macrophage migration inhibitory factor.

    Science.gov (United States)

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

    2015-03-04

    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.

  4. Flash x-ray

    International Nuclear Information System (INIS)

    Johnson, Q.; Pellinen, D.

    1976-01-01

    The complementary techniques of flash x-ray radiography (FXR) and flash x-ray diffraction (FXD) provide access to a unique domain in nondestructive materials testing. FXR is useful in studies of macroscopic properties during extremely short time intervals, and FXD, the newer technique, is used in studies of microscopic properties. Although these techniques are similar in many respects, there are some substantial differences. FXD generally requires low-voltage, line-radiation sources and extremely accurate timing; FXR is usually less demanding. Phenomena which can be profitably studied by FXR often can also be studied by FXD to permit a complete materials characterization

  5. X-ray astronomy

    International Nuclear Information System (INIS)

    Narayanan, M.S.

    1976-01-01

    The deployment of detectors outside the deleterious effects of the atmosphere by sending them in space vehicles, has been explained. This has thrown open the entire spectrum of the electromagnetic and particle radiation to direct observations, thus enlarging the vistas of the field of astronomy and astrophysics. The discovery of strong emitters of X-rays such as SCO X-1, NorX-2, transient sources such as Cen X-2, Cen X-4, Cen X-1, Supernova remnants Tan X-1, etc., are reported. The background of the X-ray spectrum as measured during two rocket flights over Thumba, India is presented. (K.B.)

  6. X-ray masks

    International Nuclear Information System (INIS)

    Greenwood, J.C.; Satchell, D.W.

    1984-01-01

    In semiconductor manufacture, where X-ray irradiation is used, a thin silicon membrane can be used as an X-ray mask. This membrane has areas on which are patterns to define the regions to be irradiated. These regions are of antireflection material. With the thin, in the order of 3 microns, membranes used, fragility is a problem. Hence a number of ribs of silicon are formed integral with the membrane, and which are relatively thick, 5 to 10 microns. The ribs may be formed by localised deeper boron deposition followed by a selective etch. (author)

  7. X-ray detector

    International Nuclear Information System (INIS)

    Houston, J.M.; Whetten, N.R.

    1981-01-01

    An ionization chamber for use in determining the spatial distribution of x-ray photons in tomography systems comprises a plurality of substantially parallel, planar anodes separated by parallel, planar cathodes and enclosed in a gas of high atomic weight at a pressure from approximately 10 atmospheres to approximately 50 atmospheres. The cathode and anode structures comprise metals which are substantially opaque to x-ray radiation and thereby tend to reduce the resolution limiting effects of xray fluoresence in the gas. In another embodiment of the invention the anodes comprise parallel conductive bars disposed between two planar cathodes. Guard rings eliminate surface leakage currents between adjacent electrodes

  8. Panoramic Dental X-Ray

    Science.gov (United States)

    ... Physician Resources Professions Site Index A-Z Panoramic Dental X-ray Panoramic dental x-ray uses a very small dose of ... x-ray , is a two-dimensional (2-D) dental x-ray examination that captures the entire mouth ...

  9. Subluminous X-ray binaries

    NARCIS (Netherlands)

    Armas Padilla, M.

    2013-01-01

    The discovery of the first X-ray binary, Scorpius X-1, by Giacconi et al. (1962), marked the birth of X-ray astronomy. Following that discovery, many additional X-ray sources where found with the first generation of X-ray rockets and observatories (e.g., UHURU and Einstein). The short-timescale

  10. X-Ray Exam: Pelvis

    Science.gov (United States)

    ... Staying Safe Videos for Educators Search English Español X-Ray Exam: Pelvis KidsHealth / For Parents / X-Ray Exam: ... Ray Exam: Hip Broken Bones Getting an X-ray (Video) X-Ray (Video) View more Partner Message About Us ...

  11. X-Ray Exam: Forearm

    Science.gov (United States)

    ... Staying Safe Videos for Educators Search English Español X-Ray Exam: Forearm KidsHealth / For Parents / X-Ray Exam: ... Muscles, and Joints Broken Bones Getting an X-ray (Video) X-Ray (Video) View more Partner Message About Us ...

  12. X-Ray Exam: Foot

    Science.gov (United States)

    ... Staying Safe Videos for Educators Search English Español X-Ray Exam: Foot KidsHealth / For Parents / X-Ray Exam: ... Muscles, and Joints Broken Bones Getting an X-ray (Video) X-Ray (Video) View more Partner Message About Us ...

  13. X-Ray Exam: Wrist

    Science.gov (United States)

    ... Staying Safe Videos for Educators Search English Español X-Ray Exam: Wrist KidsHealth / For Parents / X-Ray Exam: ... Muscles, and Joints Broken Bones Getting an X-ray (Video) X-Ray (Video) View more Partner Message About Us ...

  14. Thoracic spine x-ray

    Science.gov (United States)

    Vertebral radiography; X-ray - spine; Thoracic x-ray; Spine x-ray; Thoracic spine films; Back films ... There is low radiation exposure. X-rays are monitored and regulated to provide the minimum amount of radiation exposure needed to produce the image. Most ...

  15. X-Ray Exam: Finger

    Science.gov (United States)

    ... Staying Safe Videos for Educators Search English Español X-Ray Exam: Finger KidsHealth / For Parents / X-Ray Exam: ... Muscles, and Joints Broken Bones Getting an X-ray (Video) X-Ray (Video) View more Partner Message About Us ...

  16. 10 years of protein crystallography at AR-NW12A beamline

    Science.gov (United States)

    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.

  17. CRL X-ray tube

    International Nuclear Information System (INIS)

    Kolchevsky, N.N.; Petrov, P.V.

    2015-01-01

    A novel types of X-ray tubes with refractive lenses are proposed. CRL-R X-ray tube consists of Compound Refractive Lens- CRL and Reflection X-ray tube. CRL acts as X-ray window. CRL-T X-ray consists of CRL and Transmission X-ray tube. CRL acts as target for electron beam. CRL refractive lens acts as filter, collimator, waveguide and focusing lens. Properties and construction of the CRL X-ray tube are discussed. (authors)

  18. X rays and condensed matter

    International Nuclear Information System (INIS)

    Daillant, J.

    1997-01-01

    After a historical review of the discovery and study of X rays, the various interaction processes between X rays and matter are described: Thomson scattering, Compton scattering, X-photon absorption through photoelectric effect, and magnetic scattering. X ray sources such as the European Synchrotron Radiation Facility (ESRF) are described. The various X-ray applications are presented: imagery such as X tomography, X microscopy, phase contrast; X-ray photoelectron spectroscopy and X-ray absorption spectroscopy; X-ray scattering and diffraction techniques

  19. X-ray beam generator

    International Nuclear Information System (INIS)

    Koller, T.J.; Randmer, J.A.

    1977-01-01

    A method of minimizing the preferential angular absorption of the divergent beam from an X-ray generator is described. The generator consists of an X-ray shielded housing with an X-ray transmissive window symmetrically placed in radial alignment with a focal spot area on a sloped target surface of an X-ray tube in the housing. The X-ray tube may be of the stationary anode type or of the rotating anode type. (U.K.)

  20. Chest X-Ray

    Medline Plus

    Full Text Available Toggle navigation Test/Treatment Patient Type Screening/Wellness Disease/Condition Safety En Español More Info Images/Videos ... x-ray is used to evaluate the lungs, heart and chest wall and may be used to ...

  1. X-ray sources

    International Nuclear Information System (INIS)

    Bonse, U.

    1979-11-01

    The author describes several possibilities for the production of X-radiation. Especially he discusses the use of bremsstrahlung at electron impact on solid targets and the synchrotron radiation. He presents some equations for the calculation of X-ray intensities. Especially the X-radiation from the DORIS storage ring is discussed. (HSI)

  2. Medical x-ray

    International Nuclear Information System (INIS)

    Abd Aziz Mhd Ramli; Gui Ah Auu; Husaini Salleh; Idris Besar; Mohd Ashhar Khalid; Muhammad Jamal Md Isa; Shaharuddin Mohd; Siti Najila Mohd Janib; Mohamed Ali Abdul Khader; Mahalatchimi Dave; Mohd Fazly Abdul Rahim; Ng Chee Moon; Ram Piari; Teoh Hoon Heng; Lee Peter

    2004-01-01

    This book describes the fundamental subject about medical radiography. It is a multidisciplinary field that requires cross professional input from scientists, engineers and medical doctors. However, it is presented in simple language to suit different levels of readers from x-ray operators and radiographers to physists, general practitioners and radiology specialists.The book is written in accordance to the requirements of the standard syllabus approved by the Ministry of Health Malaysia for the training of medical x-ray operator and general practitioners. In general, the content is not only designed to provide relevant and essential subject for related professionals in medical radiological services such as x-ray operator, radiographer and radiologists, but also to address those in associated radiological services including nurses, medical technologists and physicists.The book is organized and arranged sequentially into 3 parts for easy reference: Radiation safety; X-ray equipment and associated facilities; Radiography practices. With proper grasping of all these parts, the radiological services could be provided with confident and the highest professional standard. Thus, medical imaging with highest quality that can provide useful diagnostic information at minimum doses and at cost effective could be assured

  3. Chest X-Ray

    Medline Plus

    Full Text Available ... change into a gown. You may have some concerns about chest x-rays. However, it’s important to ... You Sponsored by About Us | Contact Us | FAQ | Privacy | Terms of Use | Links | Site Map Copyright © 2018 ...

  4. Structural dissection of human metapneumovirus phosphoprotein using small angle x-ray scattering.

    Science.gov (United States)

    Renner, Max; Paesen, Guido C; Grison, Claire M; Granier, Sébastien; Grimes, Jonathan M; Leyrat, Cédric

    2017-11-01

    The phosphoprotein (P) is the main and essential cofactor of the RNA polymerase (L) of non-segmented, negative-strand RNA viruses. P positions the viral polymerase onto its nucleoprotein-RNA template and acts as a chaperone of the nucleoprotein (N), thereby preventing nonspecific encapsidation of cellular RNAs. The phosphoprotein of human metapneumovirus (HMPV) forms homotetramers composed of a stable oligomerization domain (P core ) flanked by large intrinsically disordered regions (IDRs). Here we combined x-ray crystallography of P core with small angle x-ray scattering (SAXS)-based ensemble modeling of the full-length P protein and several of its fragments to provide a structural description of P that captures its dynamic character, and highlights the presence of varyingly stable structural elements within the IDRs. We discuss the implications of the structural properties of HMPV P for the assembly and functioning of the viral transcription/replication machinery.

  5. X-ray structure determination and deuteration of nattokinase

    International Nuclear Information System (INIS)

    Yanagisawa, Yasuhide; Chatake, Toshiyuki; Naito, Sawa; Ohsugi, Tadanori; Yatagai, Chieko; Sumi, Hiroyuki; Kawaguchi, Akio; Chiba-Kamosida, Kaori; Ogawa, Megumi; Adachi, Tatsumi; Morimoto, Yukio

    2013-01-01

    X-ray structure determination and deuteration of nattokinase were performed to facilitate neutron crystallographic analysis. Nattokinase (NK) is a strong fibrinolytic enzyme, which is produced in abundance by Bacillus subtilis natto. Although NK is a member of the subtilisin family, it displays different substrate specificity when compared with other subtilisins. The results of molecular simulations predict that hydrogen arrangements around Ser221 at the active site probably account for the substrate specificity of NK. Therefore, neutron crystallographic analysis should provide valuable information that reveals the enzymatic mechanism of NK. In this report, the X-ray structure of the non-hydrogen form of undeuterated NK was determined, and the preparation of deuterated NK was successfully achieved. The non-hydrogen NK structure was determined at 1.74 Å resolution. The three-dimensional structures of NK and subtilisin E from Bacillus subtilis DB104 are near identical. Deuteration of NK was carried out by cultivating Bacillus subtilis natto in deuterated medium. The D 2 O resistant strain of Bacillus subtilis natto was obtained by successive cultivation rounds, in which the concentration of D 2 O in the medium was gradually increased. NK was purified from the culture medium and its activity was confirmed by the fibrin plate method. The results lay the framework for neutron protein crystallography analysis

  6. X-ray structure determination and deuteration of nattokinase

    Energy Technology Data Exchange (ETDEWEB)

    Yanagisawa, Yasuhide [Chiba Institute of Science, 15-8 Shiomi-cho, Cho-shi, Chiba 288-025 (Japan); Chatake, Toshiyuki [Kyoto University, Asashironishi 2, Kumatori, Osaka 590-0494 (Japan); Naito, Sawa; Ohsugi, Tadanori; Yatagai, Chieko; Sumi, Hiroyuki [Kurashiki University of Science and the Arts, 2640 Nishinoura, Tsurajima-cho, Kurashiki, Okayama 712-8505 (Japan); Kawaguchi, Akio [Kyoto University, Asashironishi 2, Kumatori, Osaka 590-0494 (Japan); Chiba-Kamosida, Kaori [Nippon Advanced Technology Co. Ltd, J-PARC, 2-4 Shirane Shirakata, Tokai, Ibaraki 319-1195 (Japan); Ogawa, Megumi; Adachi, Tatsumi [Chiba Institute of Science, 15-8 Shiomi-cho, Cho-shi, Chiba 288-025 (Japan); Morimoto, Yukio [Kyoto University, Asashironishi 2, Kumatori, Osaka 590-0494 (Japan)

    2013-11-01

    X-ray structure determination and deuteration of nattokinase were performed to facilitate neutron crystallographic analysis. Nattokinase (NK) is a strong fibrinolytic enzyme, which is produced in abundance by Bacillus subtilis natto. Although NK is a member of the subtilisin family, it displays different substrate specificity when compared with other subtilisins. The results of molecular simulations predict that hydrogen arrangements around Ser221 at the active site probably account for the substrate specificity of NK. Therefore, neutron crystallographic analysis should provide valuable information that reveals the enzymatic mechanism of NK. In this report, the X-ray structure of the non-hydrogen form of undeuterated NK was determined, and the preparation of deuterated NK was successfully achieved. The non-hydrogen NK structure was determined at 1.74 Å resolution. The three-dimensional structures of NK and subtilisin E from Bacillus subtilis DB104 are near identical. Deuteration of NK was carried out by cultivating Bacillus subtilis natto in deuterated medium. The D{sub 2}O resistant strain of Bacillus subtilis natto was obtained by successive cultivation rounds, in which the concentration of D{sub 2}O in the medium was gradually increased. NK was purified from the culture medium and its activity was confirmed by the fibrin plate method. The results lay the framework for neutron protein crystallography analysis.

  7. Synthesis, X-ray crystallography, spectroscopic characterization and spectroscopic/electrochemical evidence of formation of phenoxy free radical in active center analogs of galactose oxidase - [Cu(Salgly)H₂O] and [Cu(Salphenylalanine)H₂O].

    Science.gov (United States)

    Das, Biva; Medhi, Okhil K

    2013-03-01

    The formation of phenolate free radical is the factor of high turnover for catalytic activity of galactose oxidase (GO) compared to that by inorganic complexes. A new active center analog of GO, [Cu(II)(Salphenylalanine)H(2)O] have been synthesized and its single crystal X-ray analysis was done. In aqueous surfactant micellar solution chemical oxidation as well as electrochemical oxidation of structural models of galactose oxidase - [Cu(II)Salgly·H(2)O] and [Cu(II)(Salphenylalanine)·H(2)O], have been found to generate free radical originating at the phenolate group. Formation of the free radical have been proved by electron paramagnetic resonance spectroscopy, electronic spectroscopy and electrochemistry. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Racemic DNA Crystallography

    OpenAIRE

    Mandal , Pradeep K.; Collie , Gavin W.; Kauffmann , Brice; Huc , Ivan

    2014-01-01

    International audience; Racemates increase the chances of crystallization by allowing molecular contacts to be formed in a greater number of ways. With the advent of protein synthesis, the production of protein racemates and racemic-protein crystallography are now possible. Curiously, racemic DNA crystallography had not been investigated despite the commercial availability of Land D-deoxyribo-oligonucleotides. Here, we report a study into racemic DNA crystallography showing the strong propens...

  9. Total reflection X-ray fluorescence measurements of S and P in proteins using a vacuum chamber specially designed for low Z elements

    International Nuclear Information System (INIS)

    Rauwolf, M.; Vanhoof, C.; Tirez, K.; Maes, E.; Ingerle, D.; Wobrauschek, P.; Streli, C.

    2014-01-01

    As the ratio of phosphorus and sulfur in proteins allows the determination of the phosphorylation degree in proteins, the absolute determination of phosphorus and sulfur in organic samples is of growing interest. While it takes some effort to quantify phosphorus and sulfur with inductively coupled quadrupole plasma mass spectrometry (ICP-QMS), total reflection X-ray fluorescence analysis (TXRF) allows easy quantification. In the presented work, the low Z TXRF spectrometer at the Atominstitut was used to analyze phosphorus and sulfur in proteins. Although the preparation of the protein samples proved to be more difficult than originally expected, it could be shown that TXRF is well suited for the determination of P and S in proteins. The obtained lower limits of detection (LLD) for P and S in proteins were extrapolated for 1000s and were 34 pg and 19 pg, respectively. The importance of height scans for each sample to exclude heterogeneities was demonstrated. - Highlights: • Low Z TXRF spectrometry was used to analyze phosphorus and sulfur in proteins. • TXRF is well suited for the determination of P and S in proteins. • Good detection limits for P (34 pg) and S (19 pg) were achieved. • Due to the detection limits, we propose that TXRF is a suitable method to analyze protein fractions

  10. Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein from Staphylococcus aureus peptidoglycan.

    Science.gov (United States)

    Vieira, Diana; Figueiredo, Teresa A; Verma, Anil; Sobral, Rita G; Ludovice, Ana M; de Lencastre, Hermínia; Trincao, Jose

    2014-05-01

    Amidation of peptidoglycan is an essential feature in Staphylococcus aureus that is necessary for resistance to β-lactams and lysozyme. GatD, a 27 kDa type I glutamine amidotransferase-like protein, together with MurT ligase, catalyses the amidation reaction of the glutamic acid residues of the peptidoglycan of S. aureus. The native and the selenomethionine-derivative proteins were crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol, sodium acetate and calcium acetate. The crystals obtained diffracted beyond 1.85 and 2.25 Å, respectively, and belonged to space group P212121. X-ray diffraction data sets were collected at Diamond Light Source (on beamlines I02 and I04) and were used to obtain initial phases.

  11. Purification, crystallization and preliminary X-ray diffraction analysis of enoyl-acyl carrier protein reductase (FabK) from Streptococcus mutans strain UA159

    International Nuclear Information System (INIS)

    Kim, Tae-O; Im, Dong-Won; Jung, Ha Yun; Kwon, Seong Jung; Heo, Yong-Seok

    2012-01-01

    Enoyl-acyl carrier protein reductase (FabK) from S. mutans strain UA159 was cloned, overexpressed, purified and crystallized. X-ray diffraction data were collected to a resolution of 2.40 Å. A triclosan-resistant flavoprotein termed FabK is the sole enoyl-acyl carrier protein reductase in Streptococcus pneumoniae and Streptococcus mutans. In this study, FabK from S. mutans strain UA159 was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.40 Å resolution using a synchrotron-radiation source. The crystal belonged to space group P6 2 , with unit-cell parameters a = b = 105.79, c = 44.15 Å. The asymmetric unit contained one molecule, with a corresponding V M of 2.05 Å 3 Da −1 and a solvent content of 39.9%

  12. Metal ion controlled self-assembly of a chemically reengineered protein drug studied by small-angle X-ray scattering

    DEFF Research Database (Denmark)

    Jesper, Nygaard; Munch, Henrik K.; Thulstrup, Peter W.

    2012-01-01

    . A small-angle X-ray scattering analysis of the bipyridine-modified insulin system confirmed an organization into a novel well-ordered structure based on insulin trimers, as induced by the addition of Fe(II). In contrast, unmodified monomeric insulin formed larger and more randomly structured assemblies......Precise control of the oligomeric state of proteins is of central importance for biological function and for the properties of biopharmaceutical drugs. Here, the self-assembly of 2,2′-bipyridine conjugated monomeric insulin analogues, induced through coordination to divalent metal ions, was studied....... This protein drug system was designed to form non-native homo-oligomers through selective coordination of two divalent metal ions, Fe(II) and Zn(II), respectively. The insulin type chosen for this study is a variant designed for a reduced tendency toward native dimer formation at physiological concentrations...

  13. Crystallization and preliminary X-ray analysis of stationary phase survival protein E (SurE) from Xylella fastidiosa in two crystal forms

    International Nuclear Information System (INIS)

    Reis, Marcelo Augusto dos; Saraiva, Antonio Marcos; Santos, Marcelo Leite dos; Souza, Anete Pereira de; Aparicio, Ricardo

    2012-01-01

    The crystallization and preliminary analysis of two crystal forms of survival protein E from X. fastidiosa are reported. The bacterium Xylella fastidiosa is a phytopathogenic organism that causes citrus variegated chlorosis, a disease which attacks economically important crops, mainly oranges. In this communication, the crystallization and preliminary X-ray crystallographic analysis of XfSurE, a survival protein E from X. fastidiosa, are reported. Data were collected for two crystal forms, I and II, to 1.93 and 2.9 Å resolution, respectively. Crystal form I belonged to space group C2, with unit-cell parameters a = 172.36, b = 84.18, c = 87.24 Å, α = γ = 90, β = 96.59°, whereas crystal form II belonged to space group C2, with unit-cell parameters a = 88.05, b = 81.26, c = 72.84 Å, α = γ = 90, β = 94.76°

  14. X-ray structural analysis of Plasmodium falciparum enoyl acyl carrier protein reductase as a pathway toward the optimization of triclosan antimalarial efficacy.

    Science.gov (United States)

    Freundlich, Joel S; Wang, Feng; Tsai, Han-Chun; Kuo, Mack; Shieh, Hong-Ming; Anderson, John W; Nkrumah, Louis J; Valderramos, Juan-Carlos; Yu, Min; Kumar, T R Santha; Valderramos, Stephanie G; Jacobs, William R; Schiehser, Guy A; Jacobus, David P; Fidock, David A; Sacchettini, James C

    2007-08-31

    The x-ray crystal structures of five triclosan analogs, in addition to that of the isoniazid-NAD adduct, are described in relation to their integral role in the design of potent inhibitors of the malarial enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of the novel 5-substituted analogs exhibit low micromolar potency against in vitro cultures of drug-resistant and drug-sensitive strains of the P. falciparum parasite and inhibit purified PfENR enzyme with IC50 values of <200 nM. This study has significantly expanded the knowledge base with regard to the structure-activity relationship of triclosan while affording gains against cultured parasites and purified PfENR enzyme. In contrast to a recent report in the literature, these results demonstrate the ability to improve the in vitro potency of triclosan significantly by replacing the suboptimal 5-chloro group with larger hydrophobic moieties. The biological and x-ray crystallographic data thus demonstrate the flexibility of the active site and point to future rounds of optimization to improve compound potency against purified enzyme and intracellular Plasmodium parasites.

  15. Severe Acute Respiratory Syndrome-Coronavirus Papain-Like Novel Protease Inhibitors: Design, Synthesis, Protein-Ligand X-ray Structure and Biological Evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Arun K.; Takayama, Jun; Rao, Kalapala Venkateswar; Ratia, Kiira; Chaudhuri, Rima; Mulhearn, Debbie C.; Lee, Hyun; Nichols, Daniel B.; Baliji, Surendranath; Baker, Susan C.; Johnson, Michael E.; Mesecar, Andrew D. (Purdue); (UC); (UIC)

    2012-02-21

    The design, synthesis, X-ray crystal structure, molecular modeling, and biological evaluation of a series of new generation SARS-CoV PLpro inhibitors are described. A new lead compound 3 (6577871) was identified via high-throughput screening of a diverse chemical library. Subsequently, we carried out lead optimization and structure-activity studies to provide a series of improved inhibitors that show potent PLpro inhibition and antiviral activity against SARS-CoV infected Vero E6 cells. Interestingly, the (S)-Me inhibitor 15h (enzyme IC{sub 50} = 0.56 {mu}M; antiviral EC{sub 50} = 9.1 {mu}M) and the corresponding (R)-Me 15g (IC{sub 50} = 0.32 {mu}M; antiviral EC{sub 50} = 9.1 {mu}M) are the most potent compounds in this series, with nearly equivalent enzymatic inhibition and antiviral activity. A protein-ligand X-ray structure of 15g-bound SARS-CoV PLpro and a corresponding model of 15h docked to PLpro provide intriguing molecular insight into the ligand-binding site interactions.

  16. Determination of the separate lipid and protein profile structures derived from the total membrane profile structure or isolated sarcoplasmic reticulum via x-ray and neutron diffraction

    International Nuclear Information System (INIS)

    Herbette, L.; Blasie, J.K.

    1984-01-01

    Sarcoplasmic reticulum (SR) membranes were prepared to contain biosynthetically deuterated SR phospholipids utilizing specific and general phospholipid exchange proteins (PLEP). Functional measurements and freeze fracture on SR dispersions and x-ray diffraction of hydrated oriented membrane multilayers revealed that the exchanged SR membranes were very similar to unexchanged SR membranes. Low resolution (28-A) neutron diffraction studies utilizing SR membranes exchanged with either protonated or perdeuterated SR phospholipids allowed direct determination of the lipid profile within the isolated SR membrane at two different unit cell repeat distances. These lipid profile structures were found to be highly asymmetric regarding the conformation of the fatty acid chain extents and compositional distribution of phospholipid molecules in the inner vs. outer monolayer of the SR membrane bilayer. The relatively high resolution (11-A) electron-density profile from x-ray diffraction was decomposed by utilizing the asymmetry in the number of phospholipid molecules residing in the inner vs. outer monolayer of the SR lipid bilayer as obtained from the neutron diffraction study. To our knowledge, this represents the first direct determination of a lipid bilayer profile structure within an isolated membrane system

  17. Role of protein synthesis in the repair of sublethal x-ray damage in a mutant Chinese hamster ovary cell line

    International Nuclear Information System (INIS)

    Yezzi, M.J.

    1985-04-01

    A temperature-sensitive mutant for protein synthesis, CHO-TSH1, has been compared to the wild-type cell, CHO-sC1, in single- and split-radiation-dose schemes. When the exponentially growing TS mutant and the wild-type cells were treated at 40 0 C for up to 2 hrs prior to graded doses of x rays, the survival curves were identical and were the same as those obtained without heat treatment. If the cultures were incubated at 40 0 C for 2 hrs before a first dose and maintained at 40 0 C during a 2 hr dose fractionation interval, repair of radiation damage was reduced in the mutant compared to the wild type. These observations implied that a pool of proteins was involved in the repair of sublethal x-ray damage. However, if repair was measured by the alkaline-unwinding technique under the same time and temperature schemes, no difference in the kientics of DNA strand rejoining was observed. Misrepair processes may permit restoration of DNA strand integrity but not allow functional repair. The effect of diminished repair under conditions of inhibition of protein synthesis was found to be cell-cycle dependent in survival studies with synchronized mutant cell populations. Repair was found to be almost completely eliminated if the temperature sequence described above was applied in the middle of the DNA synthetic phase. Treatment of cell populations in the middle of G 1 -phase yielded repair inhibition comparable to that observed with the asynchronous cells. Splitdose experiments were done using pre-incubation with cycloheximide to chemically inhibit protein synthesis. WT cells and TS cells were treated with cycloheximide at 35 0 C for 2 hrs before a first dose and during a 2 hr dose fractionation interval. 23 figs., 7 tabs

  18. Role of protein synthesis in the repair of sublethal x-ray damage in a mutant Chinese hamster ovary cell line

    Energy Technology Data Exchange (ETDEWEB)

    Yezzi, M.J.

    1985-04-01

    A temperature-sensitive mutant for protein synthesis, CHO-TSH1, has been compared to the wild-type cell, CHO-sC1, in single- and split-radiation-dose schemes. When the exponentially growing TS mutant and the wild-type cells were treated at 40/sub 0/C for up to 2 hrs prior to graded doses of x rays, the survival curves were identical and were the same as those obtained without heat treatment. If the cultures were incubated at 40/sup 0/C for 2 hrs before a first dose and maintained at 40/sup 0/C during a 2 hr dose fractionation interval, repair of radiation damage was reduced in the mutant compared to the wild type. These observations implied that a pool of proteins was involved in the repair of sublethal x-ray damage. However, if repair was measured by the alkaline-unwinding technique under the same time and temperature schemes, no difference in the kientics of DNA strand rejoining was observed. Misrepair processes may permit restoration of DNA strand integrity but not allow functional repair. The effect of diminished repair under conditions of inhibition of protein synthesis was found to be cell-cycle dependent in survival studies with synchronized mutant cell populations. Repair was found to be almost completely eliminated if the temperature sequence described above was applied in the middle of the DNA synthetic phase. Treatment of cell populations in the middle of G/sub 1/-phase yielded repair inhibition comparable to that observed with the asynchronous cells. Splitdose experiments were done using pre-incubation with cycloheximide to chemically inhibit protein synthesis. WT cells and TS cells were treated with cycloheximide at 35/sup 0/C for 2 hrs before a first dose and during a 2 hr dose fractionation interval. 23 figs., 7 tabs.

  19. Protein crystallography beamline (PX-BL21); its utilization and research highlights

    International Nuclear Information System (INIS)

    Kumar, Ashwani; Ghosh, Biplab; Singh, Rahul; Makde, Ravindra; Sharma, Surinder M.

    2016-01-01

    The protein crystallography beamline (PX-BL21) is sourced on 1.5 T bending magnet of 2.5 GeV Indus-2 synchrotron. This beamline has been designed to perform monochromatic and anomalous diffraction experiments on single crystals of biological macromolecules such as protein, DNA and their complexes. PX beamline also has a state-of-art ancillary biochemical laboratory to prepare single crystals of biological macromolecules. Since the commissioning of the beamline, it has been utilized by more than 70% of research groups working in the area of protein crystallography in India. About 30 crystal structures of proteins, determined using this beamline, have been deposited in Protein Data Bank (PDB). Some of these structures have been determined using experimental phasing, such as the single wavelength anomalous diffraction (SAD) experiments. The energy tunability of the synchrotron have been exploited to carry our various SAD experiments: Selenium-SAD, Zinc-SAD and Manganese-SAD and Sulphar-SAD. In the present talk, the key results from the PX-BL21 beamline will be discussed. (author)

  20. Protein crowding in solution, frozen and freeze-dried states: small-angle neutron and X-ray scattering study of lysozyme/sorbitol/water systems

    Science.gov (United States)

    Krueger, Susan; Khodadadi, Sheila; Clark, Nicholas; McAuley, Arnold; Cristiglio, Viviana; Theyencheri, Narayanan; Curtis, Joseph; Shalaev, Evgenyi

    2015-03-01

    For effective preservation, proteins are often stored as frozen solutions or in glassy states using a freeze-drying process. However, aggregation is often observed after freeze-thaw or reconstitution of freeze-dried powder and the stability of the protein is no longer assured. In this study, small-angle neutron and X-ray scattering (SANS and SAXS) have been used to investigate changes in protein-protein interaction distances of a model protein/cryoprotectant system of lysozyme/sorbitol/water, under representative pharmaceutical processing conditions. The results demonstrate the utility of SAXS and SANS methods to monitor protein crowding at different stages of freezing and drying. The SANS measurements of solution samples showed at least one protein interaction peak corresponding to an interaction distance of ~ 90 Å. In the frozen state, two protein interaction peaks were observed by SANS with corresponding interaction distances at 40 Å as well as 90 Å. On the other hand, both SAXS and SANS data for freeze-dried samples showed three peaks, suggesting interaction distances ranging from ~ 15 Å to 170 Å. Possible interpretations of these interaction peaks will be discussed, as well as the role of sorbitol as a cryoprotectant during the freezing and drying process.

  1. CCD [charge-coupled device] sensors in synchrotron x-ray detectors

    International Nuclear Information System (INIS)

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

    1987-01-01

    The intense photon flux from advanced synchrotron light sources, such as the 7-GeV synchrotron being designed at Argonne, require integrating-type detectors. Charge-coupled devices (CCDs) are well suited as synchrotron x-ray detectors. When irradiated indirectly via a phosphor followed by reducing optics, diffraction patterns of 100 cm 2 can be imaged on a 2 cm 2 CCD. With a conversion efficiency of ∼1 CCD electron/x-ray photon, a peak saturation capacity of >10 6 x rays can be obtained. A programmable CCD controller operating at a clock frequency of 20 MHz has been developed. The readout rate is 5 x 10 6 pixels/s and the shift rate in the parallel registers is 10 6 lines/s. The test detector was evaluated in two experiments. In protein crystallography diffraction patterns have been obtained from a lysozyme crystal using a conventional rotating anode x-ray generator. Based on these results we expect to obtain at a synchrotron diffraction images at the rate of ∼1 frame/s or a complete 3-dimensional data set from a single crystal in ∼2 min. 16 refs., 16 figs., 2 tabs

  2. Discovery of x-rays and its impact on the development of physics

    International Nuclear Information System (INIS)

    Sirdeshmukh, D.B.

    1996-01-01

    Roentgen's discovery of x-rays is discussed. Roentgen's discovery was important not only for the property of transmission of x-rays, which immediately resulted in the new field of radiology, but also because it led to several experiments which had a great impact on the development of physics. Laue's discovery of x-ray diffraction in turn led to the development of x-ray crystallography and x-ray spectroscopy. The contributions of Barkla, Moseley and Siegbahn are discussed. The experiments of Duane and Hunt on continuous x-rays and Compton on scattering of x-rays lent support to the photon nature of radiation. In recent times, the line-widths of x-ray emission lines have given information about the band structure of solids. (author). 21 refs., 6 figs

  3. X ray Production. Chapter 5

    Energy Technology Data Exchange (ETDEWEB)

    Nowotny, R. [Medical University of Vienna, Vienna (Austria)

    2014-09-15

    The differential absorption of X rays in tissues and organs, owing to their atomic composition, is the basis for the various imaging methods used in diagnostic radiology. The principles in the production of X rays have remained the same since their discovery. However, much refinement has gone into the design of X ray tubes to achieve the performance required for today’s radiological examinations. In this chapter, an outline of the principles of X ray production and a characterization of the radiation output of X ray tubes will be given. The basic processes producing X rays are dealt with in Section 1.4.

  4. X-ray filter for x-ray powder diffraction

    Science.gov (United States)

    Sinsheimer, John Jay; Conley, Raymond P.; Bouet, Nathalie C. D.; Dooryhee, Eric; Ghose, Sanjit

    2018-01-23

    Technologies are described for apparatus, methods and systems effective for filtering. The filters may comprise a first plate. The first plate may include an x-ray absorbing material and walls defining first slits. The first slits may include arc shaped openings through the first plate. The walls of the first plate may be configured to absorb at least some of first x-rays when the first x-rays are incident on the x-ray absorbing material, and to output second x-rays. The filters may comprise a second plate spaced from the first plate. The second plate may include the x-ray absorbing material and walls defining second slits. The second slits may include arc shaped openings through the second plate. The walls of the second plate may be configured to absorb at least some of second x-rays and to output third x-rays.

  5. X-ray refractometer

    International Nuclear Information System (INIS)

    Tur'yanskij, A.G.; Pirshin, I.V.

    2001-01-01

    Paper introduces a new circuit of X-ray refractometer to study angular and spectral features of refracted radiation within hard X-ray range. Refractometer incorporates two goniometers, two crystal-analyzers and three radiation detectors. The maximum distance between radiation source focal point and a receiving slit of the second goniometer is equal to 1.4 m. For the first time one obtained refraction patterns of fine-film specimens including C/Si stressed structure. Paper describes a new technique of refractometry via specimen oscillation at fixed position of a detecting device. Paper presents the measurement results of oscillation refraction patterns for specimens of melted quartz and ZnSe single crystal [ru

  6. X-ray radiotherapy

    International Nuclear Information System (INIS)

    Tronc, D.

    1995-01-01

    Full text: The most common form of radio therapy is X-ray therapy, where a beam of photons or their parent electrons break down hydrogen bonds within the body's cells and remove certain DNA information necessary for cell multiplication. This process can eradicate malignant cells leading to complete recovery, to the remission of some cancers, or at least to a degree of pain relief. The radiotherapy instrument is usually an electron linac, and the electrons are used either directly in 'electrotherapy' for some 10% of patients, or the electrons bombard a conversion target creating a broad beam of high energy photons or 'penetration X-rays'. The simplest machine consists of several accelerating sections at around 3 GHz, accelerating electrons to 6 MeV; a cooled tungsten target is used to produce a 4 Gray/min X-ray field which can be collimated into a rectangular shape at the patient position. This tiny linac is mounted inside a rotating isocentric gantry above the patient who must remain perfectly still. Several convergent beams can also be used to increase the delivered dose. More sophisticated accelerators operate at up to 18 MeV to increase penetration depths and decrease skin exposure. Alternatively, electrotherapy can be used with different energies for lower and variable penetration depths - approximately 0.5 cm per MeV. In this way surface tissue may be treated without affecting deeper and more critical anatomical regions. This type of linac, 1 to 2 metres long, is mounted parallel to the patient with a bending magnet to direct the beam to the radiotherapy system, which includes the target, thick movable collimator jaws, a beam field equalizer, dose rate and optical field simulation and energy controls. There are over 2000 acceleratorbased X-ray treatment units worldwide. Western countries have up to two units per million population, whereas in developing countries such as Bangladesh, the density is only one per 100 million. Several

  7. X-ray generator

    Energy Technology Data Exchange (ETDEWEB)

    Zucarias, A; Shepherd, J W

    1982-09-08

    An X-ray tube has a tubular envelope with a cathode for directing an electron beam onto a focal spot area of a spaced anode target to generate X-rays. The target is mounted for axial rotation on one end of a rotor disposed in an end portion of the envelope and encircled by a stator of an alternating current induction motor. An annular shield of high permeability magnetic material extends transversely between the electron beam and the stator of the induction motor for shunting stray or fringe electromagnetic fields established by the stator away from the electron beam to avoid consequent lateral deflections of the electron and corresponding lateral movements of the focal spot area.

  8. X-ray microtomography

    International Nuclear Information System (INIS)

    Dunsmuir, J.H.; Ferguson, S.R.; D'Amico, K.L.; Stokes, J.P.

    1991-01-01

    In this paper the authors describe the application of a new high-resolution X-ray tomographic microscope to the study of porous media. The microscope was designed to exploit the properties of a synchrotron X-ray source to perform three dimensional tomography on millimeter sized objects with micron resolution and has been used in materials science studies with both synchrotron and conventional and synchrotron sources will be compared. In this work the authors have applied the microscope to measure the three dimensional structure of fused bead packs and berea sandstones with micron resolution and have performed preliminary studies of flow in these media with the microscope operated in a digital subtraction radiography mode. Computer graphics techniques have been applied to the data to visually display the structure of the pore body system. Tomographic imaging after flow experiments should detect the structure of the oil-water interface in the pore network and this work is ongoing

  9. X-ray apparatus

    International Nuclear Information System (INIS)

    Tomita, Chuji.

    1980-01-01

    A principal object of the present invention is to provide an X-ray apparatus which is such that the distance between the surface of the patient's table and the floor on which the apparatus is installed is sufficiently small in the horizontal position of the patient's table of the roentgenographical pedestal and that the rotation of the pedestal from the horizontal position to a tilted position and further to the vertical position of the table can be carried out smoothly. (auth)

  10. X-ray Ordinance

    International Nuclear Information System (INIS)

    Kramer, R.; Zerlett, G.

    1983-01-01

    This commentary, presented as volume 2 of the Deutsches Strahlenschutzrecht (German legislation on radiation protection) deals with the legal provisions of the ordinance on the protection against harmful effects of X-radiation (X-ray Ordinance - RoeV), of March 1, 1973 (announced in BGBl.I, page 173), as amended by the ordinance on the protection against harmful effects of ionizing radiation, of October 13, 1976 (announced in BGBl. I, page 2905). Thus volume 2 completes the task started with volume 1, namely to present a comprehensive view and account of the body of laws governing radiation protection, a task which was thought useful as developments in the FRG led to regulations being split up into the X-ray Ordinance, and the Radiation Protection Ordinance. In order to present a well-balanced commentary on the X-ray Ordinance, it was necessary to discuss the provisions both from the legal and the medical point of view. This edition takes into account the Fourth Public Notice of the BMA (Fed. Min. of Labour and Social Affairs) concerning the implementation of the X-ray Ordinance of January 4, 1982, as well as court decisions and literature published in this field, until September 1982. In addition, the judgment of the Federal Constitutional Court, dated October 19, 1982, concerning the voidness of the law on government liability, and two decisions by the Federal High Court, dated November 23, 1982, concerning the right to have insight into medical reports - of great significance in practice - have been considered. This commentary therefore is up to date with current developments. (orig.) [de

  11. Femtosecond response of polyatomic molecules to ultra-intense hard X-rays.

    Science.gov (United States)

    Rudenko, A; Inhester, L; Hanasaki, K; Li, X; Robatjazi, S J; Erk, B; Boll, R; Toyota, K; Hao, Y; Vendrell, O; Bomme, C; Savelyev, E; Rudek, B; Foucar, L; Southworth, S H; Lehmann, C S; Kraessig, B; Marchenko, T; Simon, M; Ueda, K; Ferguson, K R; Bucher, M; Gorkhover, T; Carron, S; Alonso-Mori, R; Koglin, J E; Correa, J; Williams, G J; Boutet, S; Young, L; Bostedt, C; Son, S-K; Santra, R; Rolles, D

    2017-06-01

    X-ray free-electron lasers enable the investigation of the structure and dynamics of diverse systems, including atoms, molecules, nanocrystals and single bioparticles, under extreme conditions. Many imaging applications that target biological systems and complex materials use hard X-ray pulses with extremely high peak intensities (exceeding 10 20 watts per square centimetre). However, fundamental investigations have focused mainly on the individual response of atoms and small molecules using soft X-rays with much lower intensities. Studies with intense X-ray pulses have shown that irradiated atoms reach a very high degree of ionization, owing to multiphoton absorption, which in a heteronuclear molecular system occurs predominantly locally on a heavy atom (provided that the absorption cross-section of the heavy atom is considerably larger than those of its neighbours) and is followed by efficient redistribution of the induced charge. In serial femtosecond crystallography of biological objects-an application of X-ray free-electron lasers that greatly enhances our ability to determine protein structure-the ionization of heavy atoms increases the local radiation damage that is seen in the diffraction patterns of these objects and has been suggested as a way of phasing the diffraction data. On the basis of experiments using either soft or less-intense hard X-rays, it is thought that the induced charge and associated radiation damage of atoms in polyatomic molecules can be inferred from the charge that is induced in an isolated atom under otherwise comparable irradiation conditions. Here we show that the femtosecond response of small polyatomic molecules that contain one heavy atom to ultra-intense (with intensities approaching 10 20 watts per square centimetre), hard (with photon energies of 8.3 kiloelectronvolts) X-ray pulses is qualitatively different: our experimental and modelling results establish that, under these conditions, the ionization of a molecule is

  12. Producing x-rays

    International Nuclear Information System (INIS)

    Mallozzi, P.J.; Epstein, H.M.; Jung, R.G.; Applebaum, D.C.; Fairand, B.P.; Gallagher, W.J.

    1977-01-01

    A method of producing x-rays by directing radiant energy from a laser onto a target is described. Conversion efficiency of at least about 3 percent is obtained by providing the radiant energy in a low-power precursor pulse of approximately uniform effective intensity focused onto the surface of the target for about 1 to 30 nanoseconds so as to generate an expanding unconfined coronal plasma having less than normal solid density throughout and comprising a low-density (underdense) region wherein the plasma frequency is less than the laser radiation frequency and a higher-density (overdense) region wherein the plasma frequency is greater than the laser radiation frequency and, about 1 to 30 nanoseconds after the precursor pulse strikes the target, a higher-power main pulse focused onto the plasma for about 10 -3 to 30 nanoseconds and having such power density and total energy that the radiant energy is absorbed in the underdense region and conducted into the overdense region to heat it and thus to produce x-rays therefrom with the plasma remaining substantially below normal solid density and thus facilitating the substantial emission of x-rays in the form of spectral lines arising from nonequilibrium ionization states

  13. Improved selectivity for Pb(II) by sulfur, selenium and tellurium analogues of 1,8-anthraquinone-18-crown-5: synthesis, spectroscopy, X-ray crystallography and computational studies.

    Science.gov (United States)

    Mariappan, Kadarkaraisamy; Alaparthi, Madhubabu; Hoffman, Mariah; Rama, Myriam Alcantar; Balasubramanian, Vinothini; John, Danielle M; Sykes, Andrew G

    2015-07-14

    We report here a series of heteroatom-substituted macrocycles containing an anthraquinone moiety as a fluorescent signaling unit and a cyclic polyheteroether chain as the receptor. Sulfur, selenium, and tellurium derivatives of 1,8-anthraquinone-18-crown-5 (1) were synthesized by reacting sodium sulfide (Na2S), sodium selenide (Na2Se) and sodium telluride (Na2Te) with 1,8-bis(2-bromoethylethyleneoxy)anthracene-9,10-dione in a 1 : 1 ratio. The optical properties of the new compounds are examined and the sulfur and selenium analogues produce an intense green emission enhancement upon association with Pb(II) in acetonitrile. Selectivity for Pb(II) is markedly improved as compared to the oxygen analogue 1 which was also competitive for Ca(II) ion. UV-Visible and luminescence titrations reveal that 2 and 3 form 1 : 1 complexes with Pb(II), confirmed by single-crystal X-ray studies where Pb(II) is complexed within the macrocycle through coordinate covalent bonds to neighboring carbonyl, ether and heteroether donor atoms. Cyclic voltammetry of 2-8 showed classical, irreversible oxidation potentials for sulfur, selenium and tellurium heteroethers in addition to two one-electron reductions for the anthraquinone carbonyl groups. DFT calculations were also conducted on 1, 2, 3, 6, 6 + Pb(II) and 6 + Mg(II) to determine the trend in energies of the HOMO and the LUMO levels along the series.

  14. Neutron beam-line shield design for the protein crystallography instrument at the Lujan Center

    International Nuclear Information System (INIS)

    Russell, G.J.; Pitcher, E.J.; Muhrer, G.; Ferguson, P.D.

    2001-01-01

    We have developed a very useful methodology for calculating absolute total (neutron plus gamma-ray) dose equivalent rates for use in the design of neutron beam line shields at a spallation neutron source. We have applied this technique to the design of beam line shields for several new materials science instruments being built at the Manuel Lujan Jr. Neutron Scattering Center. These instruments have a variety of collimation systems and different beam line shielding issues. We show here some specific beam line shield designs for the Protein Crystallography Instrument. (author)

  15. Driving force behind adsorption-induced protein unfolding: a time-resolved X-ray reflectivity study on lysozyme adsorbed at an air/water interface.

    Science.gov (United States)

    Yano, Yohko F; Uruga, Tomoya; Tanida, Hajime; Toyokawa, Hidenori; Terada, Yasuko; Takagaki, Masafumi; Yamada, Hironari

    2009-01-06

    Time-resolved X-ray reflectivity measurements for lysozyme (LSZ) adsorbed at an air/water interface were performed to study the mechanism of adsorption-induced protein unfolding. The time dependence of the density profile at the air/water interface revealed that the molecular conformation changed significantly during adsorption. Taking into account previous work using Fourier transform infrared (FTIR) spectroscopy, we propose that the LSZ molecules initially adsorbed on the air/water interface have a flat unfolded structure, forming antiparallel beta-sheets as a result of hydrophobic interactions with the gas phase. In contrast, as adsorption continues, a second layer forms in which the molecules have a very loose structure having random coils as a result of hydrophilic interactions with the hydrophilic groups that protrude from the first layer.

  16. Crystallization and preliminary X-ray analysis of a U2AF65 variant in complex with a polypyrimidine-tract analogue by use of protein engineering

    International Nuclear Information System (INIS)

    Sickmier, E. Allen; Frato, Katherine E.; Kielkopf, Clara L.

    2006-01-01

    A complex of the essential splicing factor U2AF 65 and a deoxyuridine oligonucleotide has been crystallized by modification of an interdomain linker. The large subunit of the essential pre-mRNA splicing factor U2 auxiliary factor (U2AF 65 ) binds the polypyrimidine tract near the 3′ splice site of pre-mRNA introns and directs the association of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) of the spliceosome with the pre-mRNA. Protein engineering, in which the flexible linker region connecting tandem RNA-recognition motifs (RRMs) within the U2AF 65 RNA-binding domain was partially deleted, allowed successful crystallization of the protein–nucleic acid complex. Cocrystals of a U2AF 65 variant with a deoxyuridine dodecamer diffract X-rays to 2.9 Å resolution and contain one complex per asymmetric unit

  17. Influence of x-rays and UV-light on the presence of oncogene proteins in spleen cells of leukemic mice

    International Nuclear Information System (INIS)

    Popovic Hadzija, M.; Poljak Blazi, M.

    1996-01-01

    Proto-oncogenes are involved in growth, defferentiation and proliferation of normal cells, and in process of neoplastic transformation. In genome of normal cells, exist also tumor-suppressor genes, which contribute to cancer when they are inactivated. Those genes are target for carcinogenesis provoked by radiation. However, species specific genetic factors are important in determing which, if any, gene will be transformed by radiation. It is possible to presume that oncogenes are involved in the development of radioresistant phenotype of ML. Because of that, we examined the presence of c-myc protein in ML cells during the growth of ML and after the irradiation of these cells. Also, we examined the presence of tumor-suppressor protein p53, because inactivation or loss of p53 gene is in connection with transformation of cells. ML is strain specific for RFM mice. Spleen cells were tested 9 (nonterminal phase NTP) or 12 days (terminal phase TP) after inoculation of ML. Cells from NTP were also irradiate with x-rays or UV-light. C-myc protein expresse 74.98% spleen cells of healthy RFM mice. Wild type of p53 protein was detected in 60% of these cells, but mp53 was found in only 5.3% of cells. These results could be explained by the role of c-myc and p53 proteins in regulation of biologic processes. A few spleen cells of NTP expressed c-myc (15%) and mp53 (9.6%) proteins. But, in the same phase higher expressions of wp53 protein (30.5%) was found. On the other hand, the number of c-myc positive cells in TP of leukemia explanation lies in connection of c-myc protein and process of programmed cell death (apoptosis). During growth of ML the number of mp53 positive cells increased (to 47.8%), but wp53 positive cells decreased (to13.4%9). Both types of irradiation provoked strong activation of cellular c-myc gene in ML cells of NTP. We found about 95% c-myc positive cells after x-rays and 93% after UV-light

  18. Bone X-Ray (Radiography)

    Medline Plus

    Full Text Available ... the body. X-rays are the oldest and most frequently used form of medical imaging. A bone ... bones. top of page How should I prepare? Most bone x-rays require no special preparation. You ...

  19. Bone X-Ray (Radiography)

    Medline Plus

    Full Text Available ... evaluation. National and international radiology protection organizations continually review and update the technique standards used by radiology professionals. Modern x-ray systems have very controlled x-ray beams and dose ...

  20. Bone X-Ray (Radiography)

    Medline Plus

    Full Text Available ... can be taken to the patient in a hospital bed or the emergency room. The x-ray ... position possible that still ensures x-ray image quality. top of page Who interprets the results and ...