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

PubMed Central

Piermarini, Gasper J.

2001-01-01

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

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

PubMed

Czjzek, Mirjam; Ficko-Blean, Elizabeth

2017-01-01

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

A Compact X-Ray System for Macromolecular Crystallography

NASA Technical Reports Server (NTRS)

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

2000-01-01

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

A Compact X-Ray System for Macromolecular Crystallography. 5

NASA Technical Reports Server (NTRS)

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

2000-01-01

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

Time-Resolved Macromolecular Crystallography at Modern X-Ray Sources.

PubMed

Schmidt, Marius

2017-01-01

Time-resolved macromolecular crystallography unifies protein structure determination with chemical kinetics. With the advent of fourth generation X-ray sources the time-resolution can be on the order of 10-40 fs, which opens the ultrafast time scale to structure determination. Fundamental motions and transitions associated with chemical reactions in proteins can now be observed. Moreover, new experimental approaches at synchrotrons allow for the straightforward investigation of all kind of reactions in biological macromolecules. Here, recent developments in the field are reviewed.

Compound refractive X-ray lens

DOEpatents

Nygren, David R.; Cahn, Robert; Cederstrom, Bjorn; Danielsson, Mats; Vestlund, Jonas

2000-01-01

An apparatus and method for focusing X-rays. In one embodiment, his invention is a commercial-grade compound refractive X-ray lens. The commercial-grade compound refractive X-ray lens includes a volume of low-Z material. The volume of low-Z material has a first surface which is adapted to receive X-rays of commercially-applicable power emitted from a commercial-grade X-ray source. The volume of low-Z material also has a second surface from which emerge the X-rays of commercially-applicable power which were received at the first surface. Additionally, the commercial-grade compound refractive X-ray lens includes a plurality of openings which are disposed between the first surface and the second surface. The plurality of openings are oriented such that the X-rays of commercially-applicable power which are received at the first surface, pass through the volume of low-Z material and through the plurality openings. In so doing, the X-rays which emerge from the second surface are refracted to a focal point.

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

PubMed Central

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

2018-01-01

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. PMID:29714177

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

PubMed

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.

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

DOE PAGES

Ke, Tsung-Wei; Brewster, Aaron S.; Yu, Stella X.; ...

2018-04-24

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ke, Tsung-Wei; Brewster, Aaron S.; Yu, Stella X.

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.

100 Years Later: Celebrating the Contributions of X-ray Crystallography to Allergy and Clinical Immunology

PubMed Central

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

2015-01-01

Current knowledge of molecules involved in immunology and allergic disease results from significant contributions of X-ray crystallography, a discipline that just celebrated its 100th anniversary. The histories of allergens and X-ray crystallography are intimately intertwined. The first enzyme structure to be determined was lysozyme, also known as the chicken food allergen Gal d 4. Crystallography determines the exact three-dimensional positions of atoms in molecules. Structures of molecular complexes in the disciplines of immunology and allergy have revealed the atoms involved in molecular interactions and in mechanisms of disease. These complexes include peptides presented by MHC class II molecules, cytokines bound to their receptors, allergen-antibody complexes, and innate immune receptors with their ligands. The information derived from crystallographic studies provides insights into the function of molecules. Allergen function is one of the determinants of environmental exposure, which is essential for IgE sensitization. Proteolytic activity of allergens or their capacity to bind lipopolysaccharides may also contribute to allergenicity. The atomic positions define the molecular surface that is accessible to antibodies. This surface in turn determines antibody specificity and cross-reactivity that are important factors for the selection of allergen panels used for molecular diagnosis and for the interpretation of clinical symptoms. This review celebrates the contributions of X-ray crystallography to clinical immunology and allergy, focusing on new molecular perspectives that influence the diagnosis and treatment of allergic diseases. PMID:26145985

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

PubMed

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

2015-07-01

Current knowledge of molecules involved in immunology and allergic disease results from the significant contributions of x-ray crystallography, a discipline that just celebrated its 100th anniversary. The histories of allergens and x-ray crystallography are intimately intertwined. The first enzyme structure to be determined was lysozyme, also known as the chicken food allergen Gal d 4. Crystallography determines the exact 3-dimensional positions of atoms in molecules. Structures of molecular complexes in the disciplines of immunology and allergy have revealed the atoms involved in molecular interactions and mechanisms of disease. These complexes include peptides presented by MHC class II molecules, cytokines bound to their receptors, allergen-antibody complexes, and innate immune receptors with their ligands. The information derived from crystallographic studies provides insights into the function of molecules. Allergen function is one of the determinants of environmental exposure, which is essential for IgE sensitization. Proteolytic activity of allergens or their capacity to bind LPSs can also contribute to allergenicity. The atomic positions define the molecular surface that is accessible to antibodies. In turn, this surface determines antibody specificity and cross-reactivity, which are important factors for the selection of allergen panels used for molecular diagnosis and the interpretation of clinical symptoms. This review celebrates the contributions of x-ray crystallography to clinical immunology and allergy, focusing on new molecular perspectives that influence the diagnosis and treatment of allergic diseases. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. All rights reserved.

Protein crystallization: Eluding the bottleneck of X-ray crystallography

PubMed Central

Holcomb, Joshua; Spellmon, Nicholas; Zhang, Yingxue; Doughan, Maysaa; Li, Chunying; Yang, Zhe

2017-01-01

To date, X-ray crystallography remains the gold standard for the determination of macromolecular structure and protein substrate interactions. However, the unpredictability of obtaining a protein crystal remains the limiting factor and continues to be the bottleneck in determining protein structures. A vast amount of research has been conducted in order to circumvent this issue with limited success. No single method has proven to guarantee the crystallization of all proteins. However, techniques using antibody fragments, lipids, carrier proteins, and even mutagenesis of crystal contacts have been implemented to increase the odds of obtaining a crystal with adequate diffraction. In addition, we review a new technique using the scaffolding ability of PDZ domains to facilitate nucleation and crystal lattice formation. Although in its infancy, such technology may be a valuable asset and another method in the crystallography toolbox to further the chances of crystallizing problematic proteins. PMID:29051919

X-ray crystallography

NASA Technical Reports Server (NTRS)

2001-01-01

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

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

PubMed

Tsujino, Soichiro; Tomizaki, Takashi

2016-05-06

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

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

NASA Astrophysics Data System (ADS)

Tsujino, Soichiro; Tomizaki, Takashi

2016-05-01

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

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

PubMed Central

Tsujino, Soichiro; Tomizaki, Takashi

2016-01-01

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

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

PubMed

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

2014-07-17

The serendipitous discovery of the spontaneous growth of protein crystals inside cells has opened the field of crystallography to chemically unmodified samples directly available from their natural environment. On the one hand, through in vivo crystallography, protocols for protein crystal preparation can be highly simplified, although the technique suffers from difficulties in sampling, particularly in the extraction of the crystals from the cells partly due to their small sizes. On the other hand, the extremely intense X-ray pulses emerging from X-ray free-electron laser (XFEL) sources, along with the appearance of serial femtosecond crystallography (SFX) is a milestone for radiation damage-free protein structural studies but requires micrometre-size crystals. The combination of SFX with in vivo crystallography has the potential to boost the applicability of these techniques, eventually bringing the field to the point where in vitro sample manipulations will no longer be required, and direct imaging of the crystals from within the cells will be achievable. To fully appreciate the diverse aspects of sample characterization, handling and analysis, SFX experiments at the Japanese SPring-8 angstrom compact free-electron laser were scheduled on various types of in vivo grown crystals. The first experiments have demonstrated the feasibility of the approach and suggest that future in vivo crystallography applications at XFELs will be another alternative to nano-crystallography. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

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

PubMed

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

2016-01-01

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

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

PubMed Central

van Thor, Jasper J.; Madsen, Anders

2015-01-01

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

van Thor, Jasper J.; Madsen, Anders

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

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

DOE PAGES

van Thor, Jasper J.; Madsen, Anders

2015-01-01

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

Using X-Ray Crystallography to Simplify and Accelerate Biologics Drug Development.

PubMed

Brader, Mark L; Baker, Edward N; Dunn, Michael F; Laue, Thomas M; Carpenter, John F

2017-02-01

Every major biopharmaceutical company incorporates a protein crystallography unit that is central to its structure-based drug discovery efforts. Yet these capabilities are rarely leveraged toward the formal higher order structural characterization that is so challenging but integral to large-scale biologics manufacturing. Although the biotech industry laments the shortcomings of its favored biophysical techniques, x-ray crystallography is not even considered for drug development. Why not? We suggest that this is due, at least in part, to outdated thinking (for a recent industry-wide survey, see Gabrielson JP, Weiss IV WF. Technical decision-making with higher order structure data: starting a new dialogue. J Pharm Sci. 2015;104(4):1240-1245). We examine some myths surrounding protein crystallography and highlight the inherent properties of protein crystals (molecular identity, biochemical purity, conformational uniformity, and macromolecular crowding) as having practicable commonalities with today's patient-focused liquid drug products. In the new millennium, protein crystallography has become essentially a routine analytical test. Its application may aid the identification of better candidate molecules that are more amenable to high-concentration processing, formulation, and analysis thereby helping to make biologics drug development quicker, simpler, and cheaper. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

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

PubMed Central

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

2014-01-01

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

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography.

PubMed

Gicquel, Yannig; Schubert, Robin; Kapis, Svetlana; Bourenkov, Gleb; Schneider, Thomas; Perbandt, Markus; Betzel, Christian; Chapman, Henry N; Heymann, Michael

2018-04-24

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 µm grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography

Curved position-sensitive detector for X-ray crystallography

NASA Astrophysics Data System (ADS)

Izumi, T.

1980-11-01

A new curved position-sensitive proportional detector has been constructed for X-ray crystallography. A very hard steel wire 0.2 mm in diameter was used as a single anode wire. It was bent to a radius of 6.5 cm and was suspended elastically in a wide 160° 2θ angular aperture. An amplifier and ADC-per-cathode strip system was made in order to encode the position. The spatial resolution is better than 0.37 mm (fwhm) along the curved anode wire, and this value corresponds to an angular resolution of 0.28° in 2θ. It is shown that a thick hard anode wire is quite suitable for use as a curved position-sensitive detector.

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

NASA Astrophysics Data System (ADS)

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.

Automated identification of functional dynamic networks from X-ray crystallography

PubMed Central

van den Bedem, Henry; Bhabha, Gira; Yang, Kun; Wright, Peter E.; Fraser, James S.

2013-01-01

Protein function often depends on the exchange between conformational substates. Allosteric ligand binding or distal mutations can stabilize specific active site conformations and consequently alter protein function. In addition to comparing independently determined X-ray crystal structures, alternative conformations observed at low levels of electron density have the potential to provide mechanistic insights into conformational dynamics. Here, we report a new multi-conformer contact network algorithm (CONTACT) that identifies networks of conformationally heterogeneous residues directly from high-resolution X-ray crystallography data. Contact networks in Escherichia coli dihydrofolate reductase (ecDHFR) predict the long-range pattern of NMR chemical shift perturbations of an allosteric mutation. A comparison of contact networks in wild type and mutant ecDHFR suggests how mutations that alter optimized networks of coordinated motions can impair catalytic function. Thus, CONTACT-guided mutagenesis will allow the structure-dynamics-function relationship to be exploited in protein engineering and design. PMID:23913260

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

PubMed

Céolin, R; Rietveld, I-B

2016-01-01

After a short review of the controversies surrounding the discovery of crystalline polymorphism in relation to our present day understanding, the methods of how to solve the stability hierarchy of different polymorphs will be briefly discussed. They involve either theoretical calculations, or, more commonly, experimental methods based on classical thermodynamics. The experimental approach is mainly carried out using heat-exchange data associated to the transition of one form into another. It will be demonstrated that work-related data associated to the phase transition should be taken into account and the role of X-ray crystallography therein will be discussed. X-ray crystallography has become increasingly precise and can nowadays provide specific volumes and their differences as a function of temperature, and also as a function of pressure, humidity, and time. Copyright © 2015 Académie Nationale de Pharmacie. Published by Elsevier Masson SAS. All rights reserved.

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

DOE PAGES

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

2015-09-02

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

Femtosecond crystallography with ultrabright electrons and x-rays: capturing chemistry in action.

PubMed

Miller, R J Dwayne

2014-03-07

With the recent advances in ultrabright electron and x-ray sources, it is now possible to extend crystallography to the femtosecond time domain to literally light up atomic motions involved in the primary processes governing structural transitions. This review chronicles the development of brighter and brighter electron and x-ray sources that have enabled atomic resolution to structural dynamics for increasingly complex systems. The primary focus is on achieving sufficient brightness using pump-probe protocols to resolve the far-from-equilibrium motions directing chemical processes that in general lead to irreversible changes in samples. Given the central importance of structural transitions to conceptualizing chemistry, this emerging field has the potential to significantly improve our understanding of chemistry and its connection to driving biological processes.

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

PubMed

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

2014-07-17

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

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

NASA Astrophysics Data System (ADS)

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.

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

PubMed

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.

Fragment-Based Screening by Protein Crystallography: Successes and Pitfalls

PubMed Central

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

2012-01-01

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

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

PubMed

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.

Accessing protein conformational ensembles using room-temperature X-ray crystallography

PubMed Central

Fraser, James S.; van den Bedem, Henry; Samelson, Avi J.; Lang, P. Therese; Holton, James M.; Echols, Nathaniel; Alber, Tom

2011-01-01

Modern protein crystal structures are based nearly exclusively on X-ray data collected at cryogenic temperatures (generally 100 K). The cooling process is thought to introduce little bias in the functional interpretation of structural results, because cryogenic temperatures minimally perturb the overall protein backbone fold. In contrast, here we show that flash cooling biases previously hidden structural ensembles in protein crystals. By analyzing available data for 30 different proteins using new computational tools for electron-density sampling, model refinement, and molecular packing analysis, we found that crystal cryocooling remodels the conformational distributions of more than 35% of side chains and eliminates packing defects necessary for functional motions. In the signaling switch protein, H-Ras, an allosteric network consistent with fluctuations detected in solution by NMR was uncovered in the room-temperature, but not the cryogenic, electron-density maps. These results expose a bias in structural databases toward smaller, overpacked, and unrealistically unique models. Monitoring room-temperature conformational ensembles by X-ray crystallography can reveal motions crucial for catalysis, ligand binding, and allosteric regulation. PMID:21918110

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

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

PubMed

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

2010-01-01

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Chia-Ying; Olieric, Vincent; Ma, Pikyee

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,more » including the β{sub 2}-adrenoreceptor–G{sub s} protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful in meso protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing in situ X-ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high-throughput in situ measurements. The novel in meso in situ serial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 Å using single-digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high-throughput in situ serial data collection at

X-ray targeted bond or compound destruction

DOEpatents

Pravica, Sr., Michael G.

2016-11-01

This document provides methods, systems, and devices for inducing a decomposition reaction by directing x-rays towards a location including a particular compound. The x-rays can have an irradiation energy that corresponds to a bond distance of a bond in the particular compound in order to break that bond and induce a decomposition of that particular compound. In some cases, the particular compound is a hazardous substance or part of a hazardous substance. In some cases, the particular compound is delivered to a desired location in an organism and x-rays induce a decomposition reaction that creates a therapeutic substance (e.g., a toxin that kills cancer cells) in the location of the organism. In some cases, the particular compound decomposes to produce a reactant in a reactor apparatus (e.g., fuel cell or semiconductor fabricator).

X-ray free electron laser: opportunities for drug discovery.

PubMed

Cheng, Robert K Y; Abela, Rafael; Hennig, Michael

2017-11-08

Past decades have shown the impact of structural information derived from complexes of drug candidates with their protein targets to facilitate the discovery of safe and effective medicines. Despite recent developments in single particle cryo-electron microscopy, X-ray crystallography has been the main method to derive structural information. The unique properties of X-ray free electron laser (XFEL) with unmet peak brilliance and beam focus allow X-ray diffraction data recording and successful structure determination from smaller and weaker diffracting crystals shortening timelines in crystal optimization. To further capitalize on the XFEL advantage, innovations in crystal sample delivery for the X-ray experiment, data collection and processing methods are required. This development was a key contributor to serial crystallography allowing structure determination at room temperature yielding physiologically more relevant structures. Adding the time resolution provided by the femtosecond X-ray pulse will enable monitoring and capturing of dynamic processes of ligand binding and associated conformational changes with great impact to the design of candidate drug compounds. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

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

NASA Astrophysics Data System (ADS)

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.

Hit detection in serial femtosecond crystallography using X-ray spectroscopy of plasma emission.

PubMed

Jönsson, H Olof; Caleman, Carl; Andreasson, Jakob; Tîmneanu, Nicuşor

2017-11-01

Serial femtosecond crystallography is an emerging and promising method for determining protein structures, making use of the ultrafast and bright X-ray pulses from X-ray free-electron lasers. The upcoming X-ray laser sources will produce well above 1000 pulses per second and will pose a new challenge: how to quickly determine successful crystal hits and avoid a high-rate data deluge. Proposed here is a hit-finding scheme based on detecting photons from plasma emission after the sample has been intercepted by the X-ray laser. Plasma emission spectra are simulated for systems exposed to high-intensity femtosecond pulses, for both protein crystals and the liquid carrier systems that are used for sample delivery. The thermal radiation from the glowing plasma gives a strong background in the XUV region that depends on the intensity of the pulse, around the emission lines from light elements (carbon, nitrogen, oxygen). Sample hits can be reliably distinguished from the carrier liquid based on the characteristic emission lines from heavier elements present only in the sample, such as sulfur. For buffer systems with sulfur present, selenomethionine substitution is suggested, where the selenium emission lines could be used both as an indication of a hit and as an aid in phasing and structural reconstruction of the protein.

X-Ray Lasers

ERIC Educational Resources Information Center

Chapline, George; Wood, Lowell

1975-01-01

Outlines the prospects of generating coherent x rays using high-power lasers and indentifies problem areas in their development. Indicates possible applications for coherent x rays in the fields of chemistry, biology, and crystallography. (GS)

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

PubMed

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.

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

PubMed

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

2016-03-10

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mandal, Kalyaneswar; Pentelute, Brad L.; Tereshko, Valentina

2009-04-08

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

X-Ray Crystallography Reagent

NASA Technical Reports Server (NTRS)

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

2003-01-01

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

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

PubMed

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.

Molecular structure in the solid state by X-ray crystallography and SSNMR and in solution by NMR of two 1,4-diazepines

NASA Astrophysics Data System (ADS)

Nieto, Carla I.; Sanz, Dionisia; Claramunt, Rosa M.; Torralba, M. Carmen; Torres, M. Rosario; Alkorta, Ibon; Elguero, José

2018-03-01

The crystals of two 1,4-diazepines prepared from curcuminoid β-diketones and ethylenediamine were studied by X-ray crystallography and NMR. Their tautomerism, intramolecular hydrogen bonds and conformation were determined.

Extending X-Ray Crystallography to Allow the Imaging of Noncrystalline Materials, Cells, and Single Protein Complexes

NASA Astrophysics Data System (ADS)

Miao, Jianwei; Ishikawa, Tetsuya; Shen, Qun; Earnest, Thomas

2008-05-01

In 1999, researchers extended X-ray crystallography to allow the imaging of noncrystalline specimens by measuring the X-ray diffraction pattern of a noncrystalline specimen and then directly phasing it using the oversampling method with iterative algorithms. Since then, the field has evolved moving in three important directions. The first is the 3D structural determination of noncrystalline materials, which includes the localization of the defects and strain field inside nanocrystals, and quantitative 3D imaging of disordered materials such as nanoparticles and biomaterials. The second is the 3D imaging of frozen-hydrated whole cells at a resolution of 10 nm or better. A main thrust is to localize specific multiprotein complexes inside cells. The third is the potential of imaging single large protein complexes using extremely intense and ultrashort X-ray pulses. In this article, we review the principles of this methodology, summarize recent developments in each of the three directions, and illustrate a few examples.

Insight into small molecule binding to the neonatal Fc receptor by X-ray crystallography and 100 kHz magic-angle-spinning NMR

PubMed Central

Macpherson, Alex; Smith-Penzel, Susanne; Basse, Nicolas; Lecomte, Fabien; Deboves, Hervé; Taylor, Richard D.; Norman, Tim; Porter, John; Waters, Lorna C.; Westwood, Marta; Cossins, Ben; Cain, Katharine; White, James; Griffin, Robert; Prosser, Christine; Kelm, Sebastian; Sullivan, Amy H.; Fox, David; Carr, Mark D.; Henry, Alistair; Taylor, Richard; Meier, Beat H.; Oschkinat, Hartmut; Lawson, Alastair D.

2018-01-01

Aiming at the design of an allosteric modulator of the neonatal Fc receptor (FcRn)–Immunoglobulin G (IgG) interaction, we developed a new methodology including NMR fragment screening, X-ray crystallography, and magic-angle-spinning (MAS) NMR at 100 kHz after sedimentation, exploiting very fast spinning of the nondeuterated soluble 42 kDa receptor construct to obtain resolved proton-detected 2D and 3D NMR spectra. FcRn plays a crucial role in regulation of IgG and serum albumin catabolism. It is a clinically validated drug target for the treatment of autoimmune diseases caused by pathogenic antibodies via the inhibition of its interaction with IgG. We herein present the discovery of a small molecule that binds into a conserved cavity of the heterodimeric, extracellular domain composed of an α-chain and β2-microglobulin (β2m) (FcRnECD, 373 residues). X-ray crystallography was used alongside NMR at 100 kHz MAS with sedimented soluble protein to explore possibilities for refining the compound as an allosteric modulator. Proton-detected MAS NMR experiments on fully protonated [13C,15N]-labeled FcRnECD yielded ligand-induced chemical-shift perturbations (CSPs) for residues in the binding pocket and allosteric changes close to the interface of the two receptor heterodimers present in the asymmetric unit as well as potentially in the albumin interaction site. X-ray structures with and without ligand suggest the need for an optimized ligand to displace the α-chain with respect to β2m, both of which participate in the FcRnECD–IgG interaction site. Our investigation establishes a method to characterize structurally small molecule binding to nondeuterated large proteins by NMR, even in their glycosylated form, which may prove highly valuable for structure-based drug discovery campaigns. PMID:29782488

Insight into small molecule binding to the neonatal Fc receptor by X-ray crystallography and 100 kHz magic-angle-spinning NMR.

PubMed

Stöppler, Daniel; Macpherson, Alex; Smith-Penzel, Susanne; Basse, Nicolas; Lecomte, Fabien; Deboves, Hervé; Taylor, Richard D; Norman, Tim; Porter, John; Waters, Lorna C; Westwood, Marta; Cossins, Ben; Cain, Katharine; White, James; Griffin, Robert; Prosser, Christine; Kelm, Sebastian; Sullivan, Amy H; Fox, David; Carr, Mark D; Henry, Alistair; Taylor, Richard; Meier, Beat H; Oschkinat, Hartmut; Lawson, Alastair D

2018-05-01

Aiming at the design of an allosteric modulator of the neonatal Fc receptor (FcRn)-Immunoglobulin G (IgG) interaction, we developed a new methodology including NMR fragment screening, X-ray crystallography, and magic-angle-spinning (MAS) NMR at 100 kHz after sedimentation, exploiting very fast spinning of the nondeuterated soluble 42 kDa receptor construct to obtain resolved proton-detected 2D and 3D NMR spectra. FcRn plays a crucial role in regulation of IgG and serum albumin catabolism. It is a clinically validated drug target for the treatment of autoimmune diseases caused by pathogenic antibodies via the inhibition of its interaction with IgG. We herein present the discovery of a small molecule that binds into a conserved cavity of the heterodimeric, extracellular domain composed of an α-chain and β2-microglobulin (β2m) (FcRnECD, 373 residues). X-ray crystallography was used alongside NMR at 100 kHz MAS with sedimented soluble protein to explore possibilities for refining the compound as an allosteric modulator. Proton-detected MAS NMR experiments on fully protonated [13C,15N]-labeled FcRnECD yielded ligand-induced chemical-shift perturbations (CSPs) for residues in the binding pocket and allosteric changes close to the interface of the two receptor heterodimers present in the asymmetric unit as well as potentially in the albumin interaction site. X-ray structures with and without ligand suggest the need for an optimized ligand to displace the α-chain with respect to β2m, both of which participate in the FcRnECD-IgG interaction site. Our investigation establishes a method to characterize structurally small molecule binding to nondeuterated large proteins by NMR, even in their glycosylated form, which may prove highly valuable for structure-based drug discovery campaigns.

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

PubMed

Mizutani, Kimihiko

2015-01-01

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

X-ray free electron lasers motivate bioanalytical characterization of protein nanocrystals: serial femtosecond crystallography.

PubMed

Bogan, Michael J

2013-04-02

Atomic resolution structures of large biomacromolecular complexes can now be recorded at room temperature from crystals with submicrometer dimensions using intense femtosecond pulses delivered by the world's largest and most powerful X-ray machine, a laser called the Linac Coherent Light Source. Abundant opportunities exist for the bioanalytical sciences to help extend this revolutionary advance in structural biology to the ultimate goal of recording molecular-movies of noncrystalline biomacromolecules. This Feature will introduce the concept of serial femtosecond crystallography to the nonexpert, briefly review progress to date, and highlight some potential contributions from the analytical sciences.

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

PubMed

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.

Study on four polymorphs of bifendate based on X-ray crystallography.

PubMed

Nie, Jinju; Yang, Dezhi; Hu, Kun; Lu, Yang

2016-05-01

Bifendate, a synthetic anti-hepatitis drug, exhibits polycrystalline mode phenomena with 2 polymorphs reported (forms A and B). Single crystals of the known crystalline form B and 3 new crystallosolvates involving bifendate solvated with tetrahydrofuran (C), dioxane (D), and pyridine (E) in a stoichiometric ratio of 1:1 were obtained and characterized by X-ray crystallography, thermal analysis, and Fourier transform infrared (FT-IR) spectroscopy. The differences in molecular conformation, intermolecular interaction and crystal packing arrangement for the four polymorphs were determined and the basis for the polymorphisms was investigated. The rotation of single bonds resulted in different orientations for the biphenyl, methyl ester and methoxyl groups. All guest solvent molecules interacted with the host molecule via an interesting intercalative mode along the [1 0 0] direction in the channel formed by the host molecules through weak aromatic stacking interactions or non-classical hydrogen bonds, of which the volume and planarity played an important role in the intercalation of the host with the guest. The incorporation of solvent-augmented rotation of the C-C bond of the biphenyl group had a striking effect on the host molecular conformation and contributed to the formation of bifendate polymorphs. Moreover, the simulated powder X-ray diffraction (PXRD) patterns for each form were calculated on the basis of the single-crystal data and proved to be unique. The single-crystal structures of the four crystalline forms are reported in this paper.

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

NASA Astrophysics Data System (ADS)

Anfinrud, Philip

2007-03-01

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

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

NASA Astrophysics Data System (ADS)

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

2016-09-01

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

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

PubMed

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.

Protein Crystallography from the Perspective of Technology Developments

PubMed Central

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

2015-01-01

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

X-Ray Crystallography: One Century of Nobel Prizes

ERIC Educational Resources Information Center

Galli, Simona

2014-01-01

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

X-ray spectra and electron structure of A15 compounds of transition metals

NASA Astrophysics Data System (ADS)

Kurmaev, E. Z.; Iarmoshenko, Iu. M.

1988-01-01

Results of an X-ray emission spectroscopy study of the electron structure of A15 compounds are reported. In particular, attention is given to the X-ray spectra of A15 compounds of the A3B type with transition and nontransition elements, effect of alloying on the formation of the electron structure of ternary phases, and effect of atomic ordering in the X-ray spectra of A15 compounds with changes in heat treament and concentration. The X-ray spectra of A15 compounds irradiated by fast neutrons are also examined.

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

PubMed

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.

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

PubMed

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.

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.

PubMed

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.

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.

PubMed

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.

Phosphor Scanner For Imaging X-Ray Diffraction

NASA Technical Reports Server (NTRS)

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.

X-Ray Diffraction of Intermetallic Compounds: A Physical Chemistry Laboratory Experiment

ERIC Educational Resources Information Center

Varberg, Thomas D.; Skakuj, Kacper

2015-01-01

Here we describe an experiment for the undergraduate physical chemistry laboratory in which students synthesize the intermetallic compounds AlNi and AlNi3 and study them by X-ray diffractometry. The compounds are synthesized in a simple one-step reaction occurring in the solid state. Powder X-ray diffractograms are recorded for the two compounds…

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

PubMed Central

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

2014-01-01

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

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

PubMed

Perry, J Jefferson P; Tainer, John A

2013-03-01

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

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.

PubMed

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

Room-temperature serial crystallography at synchrotron X-ray sources using slowly flowing free-standing high-viscosity microstreams.

PubMed

Botha, Sabine; Nass, Karol; Barends, Thomas R M; Kabsch, Wolfgang; Latz, Beatrice; Dworkowski, Florian; Foucar, Lutz; Panepucci, Ezequiel; Wang, Meitian; Shoeman, Robert L; Schlichting, Ilme; Doak, R Bruce

2015-02-01

Recent advances in synchrotron sources, beamline optics and detectors are driving a renaissance in room-temperature data collection. The underlying impetus is the recognition that conformational differences are observed in functionally important regions of structures determined using crystals kept at ambient as opposed to cryogenic temperature during data collection. In addition, room-temperature measurements enable time-resolved studies and eliminate the need to find suitable cryoprotectants. Since radiation damage limits the high-resolution data that can be obtained from a single crystal, especially at room temperature, data are typically collected in a serial fashion using a number of crystals to spread the total dose over the entire ensemble. Several approaches have been developed over the years to efficiently exchange crystals for room-temperature data collection. These include in situ collection in trays, chips and capillary mounts. Here, the use of a slowly flowing microscopic stream for crystal delivery is demonstrated, resulting in extremely high-throughput delivery of crystals into the X-ray beam. This free-stream technology, which was originally developed for serial femtosecond crystallography at X-ray free-electron lasers, is here adapted to serial crystallography at synchrotrons. By embedding the crystals in a high-viscosity carrier stream, high-resolution room-temperature studies can be conducted at atmospheric pressure using the unattenuated X-ray beam, thus permitting the analysis of small or weakly scattering crystals. The high-viscosity extrusion injector is described, as is its use to collect high-resolution serial data from native and heavy-atom-derivatized lysozyme crystals at the Swiss Light Source using less than half a milligram of protein crystals. The room-temperature serial data allow de novo structure determination. The crystal size used in this proof-of-principle experiment was dictated by the available flux density. However, upcoming

Understanding pre-mRNA splicing through crystallography.

PubMed

Espinosa, Sara; Zhang, Lingdi; Li, Xueni; Zhao, Rui

2017-08-01

Crystallography is a powerful tool to determine the atomic structures of proteins and RNAs. X-ray crystallography has been used to determine the structure of many splicing related proteins and RNAs, making major contributions to our understanding of the molecular mechanism and regulation of pre-mRNA splicing. Compared to other structural methods, crystallography has its own advantage in the high-resolution structural information it can provide and the unique biological questions it can answer. In addition, two new crystallographic methods - the serial femtosecond crystallography and 3D electron crystallography - were developed to overcome some of the limitations of traditional X-ray crystallography and broaden the range of biological problems that crystallography can solve. This review discusses the theoretical basis, instrument requirements, troubleshooting, and exciting potential of these crystallographic methods to further our understanding of pre-mRNA splicing, a critical event in gene expression of all eukaryotes. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed

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.

Pink-beam serial crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meents, A.; Wiedorn, M. O.; Srajer, V.

Serial X-ray crystallography allows macromolecular structure determination at both X-ray free electron lasers (XFELs) and, more recently, synchrotron sources. The time resolution for serial synchrotron crystallography experiments has been limited to millisecond timescales with monochromatic beams. The polychromatic, “pink”, beam provides a more than two orders of magnitude increased photon flux and hence allows accessing much shorter timescales in diffraction experiments at synchrotron sources. Here we report the structure determination of two different protein samples by merging pink-beam diffraction patterns from many crystals, each collected with a single 100 ps X-ray pulse exposure per crystal using a setup optimized formore » very low scattering background. In contrast to experiments with monochromatic radiation, data from only 50 crystals were required to obtain complete datasets. The high quality of the diffraction data highlights the potential of this method for studying irreversible reactions at sub-microsecond timescales using high-brightness X-ray facilities.« less

Pink-beam serial crystallography

DOE PAGES

Meents, A.; Wiedorn, M. O.; Srajer, V.; ...

2017-11-03

Serial X-ray crystallography allows macromolecular structure determination at both X-ray free electron lasers (XFELs) and, more recently, synchrotron sources. The time resolution for serial synchrotron crystallography experiments has been limited to millisecond timescales with monochromatic beams. The polychromatic, “pink”, beam provides a more than two orders of magnitude increased photon flux and hence allows accessing much shorter timescales in diffraction experiments at synchrotron sources. Here we report the structure determination of two different protein samples by merging pink-beam diffraction patterns from many crystals, each collected with a single 100 ps X-ray pulse exposure per crystal using a setup optimized formore » very low scattering background. In contrast to experiments with monochromatic radiation, data from only 50 crystals were required to obtain complete datasets. The high quality of the diffraction data highlights the potential of this method for studying irreversible reactions at sub-microsecond timescales using high-brightness X-ray facilities.« less

Neutron Nucleic Acid Crystallography.

PubMed

Chatake, Toshiyuki

2016-01-01

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

High-Resolution Detector For X-Ray Diffraction

NASA Technical Reports Server (NTRS)

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.

X-ray structure determination using low-resolution electron microscopy maps for molecular replacement

DOE PAGES

Jackson, Ryan N.; McCoy, Airlie J.; Terwilliger, Thomas C.; ...

2015-07-30

Structures of multi-subunit macromolecular machines are primarily determined by either electron microscopy (EM) or X-ray crystallography. In many cases, a structure for a complex can be obtained at low resolution (at a coarse level of detail) with EM and at higher resolution (with finer detail) by X-ray crystallography. The integration of these two structural techniques is becoming increasingly important for generating atomic models of macromolecular complexes. A low-resolution EM image can be a powerful tool for obtaining the "phase" information that is missing from an X-ray crystallography experiment, however integration of EM and X-ray diffraction data has been technically challenging.more » Here we show a step-by-step protocol that explains how low-resolution EM maps can be placed in the crystallographic unit cell by molecular replacement, and how initial phases computed from the placed EM density are extended to high resolution by averaging maps over non-crystallographic symmetry. As the resolution gap between EM and Xray crystallography continues to narrow, the use of EM maps to help with X-ray crystal structure determination, as described in this protocol, will become increasingly effective.« less

Small-Angle X-ray Scattering (SAXS) Instrument Performance and Validation Using Silver Nanoparticles

DTIC Science & Technology

2016-12-01

Intercalibration of small-angle X- Ray and neutron-scattering data. Journal of Applied Crystallography . 1988;21:629–638. 7. Zhang F, Ilavsky J, Long GG...Materials Transactions A. 2009;41:1151–1158. 8. Kusz J, Bohm H. Performance of a confocal multilayer X-ray optic. Journal of Applied Crystallography ...Journal of Applied Crystallography . 2004;37:369–380. 10. Orthaber D, Bergmann A, Glatter O. SAXS experiments on absolute scale with Kratky systems using

Small Angle X ray Scattering (SAXS) Instrument Performance and Validation Using Silver Nanoparticles

DTIC Science & Technology

2016-12-01

Intercalibration of small-angle X- Ray and neutron-scattering data. Journal of Applied Crystallography . 1988;21:629–638. 7. Zhang F, Ilavsky J, Long GG...Materials Transactions A. 2009;41:1151–1158. 8. Kusz J, Bohm H. Performance of a confocal multilayer X-ray optic. Journal of Applied Crystallography ...Journal of Applied Crystallography . 2004;37:369–380. 10. Orthaber D, Bergmann A, Glatter O. SAXS experiments on absolute scale with Kratky systems using

Liquid sample delivery techniques for serial femtosecond crystallography

PubMed Central

Weierstall, Uwe

2014-01-01

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

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

PubMed

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.

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

PubMed Central

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

2016-01-01

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. PMID:27386545

Bringing diffuse X-ray scattering into focus

DOE PAGES

Wall, Michael E.; Wolff, Alexander M.; Fraser, James S.

2018-02-16

X-ray crystallography is experiencing a renaissance as a method for probing the protein conformational ensemble. The inherent limitations of Bragg analysis, however, which only reveals the mean structure, have given way to a surge in interest in diffuse scattering, which is caused by structure variations. Diffuse scattering is present in all macromolecular crystallography experiments. Recent studies are shedding light on the origins of diffuse scattering in protein crystallography, and provide clues for leveraging diffuse scattering to model protein motions with atomic detail.

Bringing diffuse X-ray scattering into focus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wall, Michael E.; Wolff, Alexander M.; Fraser, James S.

X-ray crystallography is experiencing a renaissance as a method for probing the protein conformational ensemble. The inherent limitations of Bragg analysis, however, which only reveals the mean structure, have given way to a surge in interest in diffuse scattering, which is caused by structure variations. Diffuse scattering is present in all macromolecular crystallography experiments. Recent studies are shedding light on the origins of diffuse scattering in protein crystallography, and provide clues for leveraging diffuse scattering to model protein motions with atomic detail.

Room temperature neutron crystallography of drug resistant HIV-1 protease uncovers limitations of X-ray structural analysis at 100K

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gerlits, Oksana O.; Keen, David A.; Blakeley, Matthew P.

HIV-1 protease inhibitors are crucial for treatment of HIV-1/AIDS, but their effectiveness is thwarted by rapid emergence of drug resistance. To better understand binding of clinical inhibitors to resistant HIV-1 protease, we used room-temperature joint X-ray/neutron (XN) crystallography to obtain an atomic-resolution structure of the protease triple mutant (V32I/I47V/V82I) in complex with amprenavir. The XN structure reveals a D+ ion located midway between the inner Oδ1 oxygen atoms of the catalytic aspartic acid residues. Comparison of the current XN structure with our previous XN structure of the wild-type HIV-1 protease-amprenavir complex suggests that the three mutations do not significantly altermore » the drug–enzyme interactions. This is in contrast to the observations in previous 100 K X-ray structures of these complexes that indicated loss of interactions by the drug with the triple mutant protease. These findings, thus, uncover limitations of structural analysis of drug binding using X-ray structures obtained at 100 K.« less

Room temperature neutron crystallography of drug resistant HIV-1 protease uncovers limitations of X-ray structural analysis at 100K

DOE PAGES

Gerlits, Oksana O.; Keen, David A.; Blakeley, Matthew P.; ...

2017-02-14

HIV-1 protease inhibitors are crucial for treatment of HIV-1/AIDS, but their effectiveness is thwarted by rapid emergence of drug resistance. To better understand binding of clinical inhibitors to resistant HIV-1 protease, we used room-temperature joint X-ray/neutron (XN) crystallography to obtain an atomic-resolution structure of the protease triple mutant (V32I/I47V/V82I) in complex with amprenavir. The XN structure reveals a D+ ion located midway between the inner Oδ1 oxygen atoms of the catalytic aspartic acid residues. Comparison of the current XN structure with our previous XN structure of the wild-type HIV-1 protease-amprenavir complex suggests that the three mutations do not significantly altermore » the drug–enzyme interactions. This is in contrast to the observations in previous 100 K X-ray structures of these complexes that indicated loss of interactions by the drug with the triple mutant protease. These findings, thus, uncover limitations of structural analysis of drug binding using X-ray structures obtained at 100 K.« less

Soft X-ray spectroscopy of transition metal compounds: a theoretical perspective

NASA Astrophysics Data System (ADS)

Bokarev, S. I.; Hilal, R.; Aziz, S. G.; Kühn, O.

2017-01-01

To date, X-ray spectroscopy has become a routine tool that can reveal highly local and element-specific information on the electronic structure of atoms in complex environments. Here, we report on the development of an efficient and versatile theoretical methodology for the treatment of soft X-ray spectra of transition metal compounds based on the multi-configurational self-consistent field electronic structure theory. A special focus is put on the L-edge photon-in/photon-out and photon-in/electron-out processes, i.e. X-ray absorption, resonant inelastic scattering, partial fluorescence yield, and photoelectron spectroscopy, all treated on the same theoretical footing. The investigated systems range from small prototypical coordination compounds and catalysts to aggregates of biomolecules.

Accounting for partiality in serial crystallography using ray-tracing principles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kroon-Batenburg, Loes M. J., E-mail: l.m.j.kroon-batenburg@uu.nl; Schreurs, Antoine M. M.; Ravelli, Raimond B. G.

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

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

NASA Technical Reports Server (NTRS)

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

2005-01-01

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

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

NASA Technical Reports Server (NTRS)

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

2004-01-01

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

The O 2 -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

DOE PAGES

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

2016-12-21

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 4O 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 throughmore » 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 S1 state has been a target for X-ray crystallography for the past 15 years. However, traditional Xray 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

The O 2 -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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 4O 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 throughmore » 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 S1 state has been a target for X-ray crystallography for the past 15 years. However, traditional Xray 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

a-Si:H TFT-silicon hybrid low-energy x-ray detector

DOE PAGES

Shin, Kyung -Wook; Karim, Karim S.

2017-03-15

Direct conversion crystalline silicon X-ray imagers are used for low-energy X-ray photon (4-20 keV) detection in scientific research applications such as protein crystallography. In this paper, we demonstrate a novel pixel architecture that integrates a crystalline silicon X-ray detector with a thin-film transistor amorphous silicon pixel readout circuit. We describe a simplified two-mask process to fabricate a complete imaging array and present preliminary results that show the fabricated pixel to be sensitive to 5.89-keV photons from a low activity Fe-55 gamma source. Furthermore, this paper presented can expedite the development of high spatial resolution, low cost, direct conversion imagers formore » X-ray diffraction and crystallography applications.« less

The future of crystallography in drug discovery

PubMed Central

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

2014-01-01

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

Compound refractive lenses as prefocusing optics for X-ray FEL radiation

DOE PAGES

Heimann, Philip; MacDonald, Michael; Nagler, Bob; ...

2016-01-27

The performance of X-ray free-electron laser beamlines may be limited by the angular aperture. Compound refractive lenses (CRLs) can be employed to prefocus the X-ray beam, thereby increasing the beamline transmission. A prefocusing CRL was implemented in the X-ray transport of the Matter under Extreme Conditions Instrument at the Linac Coherent Light Source. A significant improvement in the beamline transmission was calculated over the 3–10 keV photon energy range. At 5 keV, the relative X-ray intensity was measured and a factor of four increase was seen in the beamline transmission. As a result, the X-ray focus was also determined bymore » the ablation imprint method.« less

Characterization of X-Ray Diffraction System with a Microfocus X-Ray Source and a Polycapillary Optic

NASA Technical Reports Server (NTRS)

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

2000-01-01

We present here an optimized microfocus x-ray source and polycapillary optic system designed for diffraction of small protein crystals. The x-ray beam is formed by a 5.5mm focal length capillary collimator coupled with a 40 micron x-ray source operating at 46Watts. Measurements of the x-ray flux, the divergence and the spectral characteristics of the beam are presented, This optimized system provides a seven fold greater flux than our recently reported configuration [M. Gubarev, et al., J. of Applied Crystallography (2000) 33, in press]. We now make a comparison with a 5kWatts rotating anode generator (Rigaku) coupled with confocal multilayer focusing mirrors (Osmic, CMF12- 38Cu6). The microfocus x-ray source and polycapillary collimator system delivers 60% of the x-ray flux from the rotating anode system. Additional ways to improve our microfocus x-ray system, and thus increase the x-ray flux will be discussed.

Femtosecond X-ray protein nanocrystallography

PubMed Central

Chapman, Henry N.; Fromme, Petra; Barty, Anton; White, Thomas A.; Kirian, Richard A.; Aquila, Andrew; Hunter, Mark S.; Schulz, Joachim; DePonte, Daniel P.; Weierstall, Uwe; Doak, R. Bruce; Maia, Filipe R. N. C.; Martin, Andrew V.; Schlichting, Ilme; Lomb, Lukas; Coppola, Nicola; Shoeman, Robert L.; Epp, Sascha W.; Hartmann, Robert; Rolles, Daniel; Rudenko, Artem; Foucar, Lutz; Kimmel, Nils; Weidenspointner, Georg; Holl, Peter; Liang, Mengning; Barthelmess, Miriam; Caleman, Carl; Boutet, Sébastien; Bogan, Michael J.; Krzywinski, Jacek; Bostedt, Christoph; Bajt, Saša; Gumprecht, Lars; Rudek, Benedikt; Erk, Benjamin; Schmidt, Carlo; Hömke, André; Reich, Christian; Pietschner, Daniel; Strüder, Lothar; Hauser, Günter; Gorke, Hubert; Ullrich, Joachim; Herrmann, Sven; Schaller, Gerhard; Schopper, Florian; Soltau, Heike; Kühnel, Kai-Uwe; Messerschmidt, Marc; Bozek, John D.; Hau-Riege, Stefan P.; Frank, Matthias; Hampton, Christina Y.; Sierra, Raymond G.; Starodub, Dmitri; Williams, Garth J.; Hajdu, Janos; Timneanu, Nicusor; Seibert, M. Marvin; Andreasson, Jakob; Rocker, Andrea; Jönsson, Olof; Svenda, Martin; Stern, Stephan; Nass, Karol; Andritschke, Robert; Schröter, Claus-Dieter; Krasniqi, Faton; Bott, Mario; Schmidt, Kevin E.; Wang, Xiaoyu; Grotjohann, Ingo; Holton, James M.; Barends, Thomas R. M.; Neutze, Richard; Marchesini, Stefano; Fromme, Raimund; Schorb, Sebastian; Rupp, Daniela; Adolph, Marcus; Gorkhover, Tais; Andersson, Inger; Hirsemann, Helmut; Potdevin, Guillaume; Graafsma, Heinz; Nilsson, Björn; Spence, John C. H.

2012-01-01

X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded1-3. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction ‘snapshots’ are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source4. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes5. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (~200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes6. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage. PMID:21293373

Femtosecond X-ray protein nanocrystallography.

PubMed

Chapman, Henry N; Fromme, Petra; Barty, Anton; White, Thomas A; Kirian, Richard A; Aquila, Andrew; Hunter, Mark S; Schulz, Joachim; DePonte, Daniel P; Weierstall, Uwe; Doak, R Bruce; Maia, Filipe R N C; Martin, Andrew V; Schlichting, Ilme; Lomb, Lukas; Coppola, Nicola; Shoeman, Robert L; Epp, Sascha W; Hartmann, Robert; Rolles, Daniel; Rudenko, Artem; Foucar, Lutz; Kimmel, Nils; Weidenspointner, Georg; Holl, Peter; Liang, Mengning; Barthelmess, Miriam; Caleman, Carl; Boutet, Sébastien; Bogan, Michael J; Krzywinski, Jacek; Bostedt, Christoph; Bajt, Saša; Gumprecht, Lars; Rudek, Benedikt; Erk, Benjamin; Schmidt, Carlo; Hömke, André; Reich, Christian; Pietschner, Daniel; Strüder, Lothar; Hauser, Günter; Gorke, Hubert; Ullrich, Joachim; Herrmann, Sven; Schaller, Gerhard; Schopper, Florian; Soltau, Heike; Kühnel, Kai-Uwe; Messerschmidt, Marc; Bozek, John D; Hau-Riege, Stefan P; Frank, Matthias; Hampton, Christina Y; Sierra, Raymond G; Starodub, Dmitri; Williams, Garth J; Hajdu, Janos; Timneanu, Nicusor; Seibert, M Marvin; Andreasson, Jakob; Rocker, Andrea; Jönsson, Olof; Svenda, Martin; Stern, Stephan; Nass, Karol; Andritschke, Robert; Schröter, Claus-Dieter; Krasniqi, Faton; Bott, Mario; Schmidt, Kevin E; Wang, Xiaoyu; Grotjohann, Ingo; Holton, James M; Barends, Thomas R M; Neutze, Richard; Marchesini, Stefano; Fromme, Raimund; Schorb, Sebastian; Rupp, Daniela; Adolph, Marcus; Gorkhover, Tais; Andersson, Inger; Hirsemann, Helmut; Potdevin, Guillaume; Graafsma, Heinz; Nilsson, Björn; Spence, John C H

2011-02-03

X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (∼200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.

High-throughput Crystallography for Structural Genomics

PubMed Central

Joachimiak, Andrzej

2009-01-01

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

Quantum crystallography: A perspective.

PubMed

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.

Crystallography: past and present

NASA Astrophysics Data System (ADS)

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

2007-12-01

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

High resolution imaging and lithography with hard x rays using parabolic compound refractive lenses

NASA Astrophysics Data System (ADS)

Schroer, C. G.; Benner, B.; Günzler, T. F.; Kuhlmann, M.; Zimprich, C.; Lengeler, B.; Rau, C.; Weitkamp, T.; Snigirev, A.; Snigireva, I.; Appenzeller, J.

2002-03-01

Parabolic compound refractive lenses are high quality optical components for hard x rays. They are particularly suited for full field imaging, with applications in microscopy and x-ray lithography. Taking advantage of the large penetration depth of hard x rays, the interior of opaque samples can be imaged with submicrometer resolution. To obtain the three-dimensional structure of a sample, microscopy is combined with tomographic techniques. In a first hard x-ray lithography experiment, parabolic compound refractive lenses have been used to project the reduced image of a lithography mask onto a resist. Future developments are discussed.

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

PubMed Central

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

2009-01-01

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

Structural Basis of Pullulanase Membrane Binding and Secretion Revealed by X-Ray Crystallography, Molecular Dynamics and Biochemical Analysis.

PubMed

East, Alexandra; Mechaly, Ariel E; Huysmans, Gerard H M; Bernarde, Cédric; Tello-Manigne, Diana; Nadeau, Nathalie; Pugsley, Anthony P; Buschiazzo, Alejandro; Alzari, Pedro M; Bond, Peter J; Francetic, Olivera

2016-01-05

The Klebsiella lipoprotein pullulanase (PulA) is exported to the periplasm, triacylated, and anchored via lipids in the inner membrane (IM) prior to its transport to the bacterial surface through a type II secretion system (T2SS). X-Ray crystallography and atomistic molecular dynamics (MD) simulations of PulA in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) model membrane provided an unprecedented molecular view of an N-terminal unstructured tether and the IM lipoprotein retention signal, and revealed novel interactions with the IM via N-terminal immunoglobulin-like domains in PulA. An efficiently secreted nonacylated variant (PulANA) showed similar peripheral membrane association during MD simulations, consistent with the binding of purified PulANA to liposomes. Remarkably, combined X-ray, MD, and functional studies identified a novel subdomain, Ins, inserted in the α-amylase domain, which is required for PulA secretion. Available data support a model in which PulA binding to the IM promotes interactions with the T2SS, possibly via the Ins subdomain. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

PubMed

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

2017-06-01

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

Novel Drift Structures for Silicon and Compound Semiconductor X-Ray and Gamma-Ray Detectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bradley E. Patt; Jan S. Iwanczyk

Recently developed silicon- and compound-semiconductor-based drift detector structures have produced excellent performance for charged particles, X rays, and gamma rays and for low-signal visible light detection. The silicon drift detector (SDD) structures that we discuss relate to direct X-ray detectors and scintillation photon detectors coupled with scintillators for gamma rays. Recent designs include several novel features that ensure very low dark current (both bulk silicon dark current and surface dark current) and hence low noise. In addition, application of thin window technology ensures a very high quantum efficiency entrance window on the drift photodetector.

Complementary uses of small angle X-ray scattering and X-ray crystallography.

PubMed

Pillon, Monica C; Guarné, Alba

2017-11-01

Most proteins function within networks and, therefore, protein interactions are central to protein function. Although stable macromolecular machines have been extensively studied, dynamic protein interactions remain poorly understood. Small-angle X-ray scattering probes the size, shape and dynamics of proteins in solution at low resolution and can be used to study samples in a large range of molecular weights. Therefore, it has emerged as a powerful technique to study the structure and dynamics of biomolecular systems and bridge fragmented information obtained using high-resolution techniques. Here we review how small-angle X-ray scattering can be combined with other structural biology techniques to study protein dynamics. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

XRayView: a teaching aid for X-ray crystallography.

PubMed

Phillips, G N

1995-10-01

A software package, XRayView, has been developed that uses interactive computer graphics to introduce basic concepts of x-ray diffraction by crystals, including the reciprocal lattice, the Ewald sphere construction, Laue cones, the wavelength dependence of the reciprocal lattice, primitive and centered lattices and systematic extinctions, rotation photography. Laue photography, space group determination and Laue group symmetry, and the alignment of crystals by examination of reciprocal space. XRayView is designed with "user-friendliness" in mind, using pull-down menus to control the program. Many of the experiences of using real x-ray diffraction equipment to examine crystalline diffraction can be simulated. Exercises are available on-line to guide the users through many typical x-ray diffraction experiments.

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

NASA Astrophysics Data System (ADS)

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

Where is crystallography going?

PubMed Central

Ashton, Alun W.; Sorensen, Thomas

2018-01-01

Macromolecular crystallography (MX) has been a motor for biology for over half a century and this continues apace. A series of revolutions, including the production of recombinant proteins and cryo-crystallography, have meant that MX has repeatedly reinvented itself to dramatically increase its reach. Over the last 30 years synchrotron radiation has nucleated a succession of advances, ranging from detectors to optics and automation. These advances, in turn, open up opportunities. For instance, a further order of magnitude could perhaps be gained in signal to noise for general synchrotron experiments. In addition, X-ray free-electron lasers offer to capture fragments of reciprocal space without radiation damage, and open up the subpicosecond regime of protein dynamics and activity. But electrons have recently stolen the limelight: so is X-ray crystallography in rude health, or will imaging methods, especially single-particle electron microscopy, render it obsolete for the most interesting biology, whilst electron diffraction enables structure determination from even the smallest crystals? We will lay out some information to help you decide. PMID:29533241

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

PubMed

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.

Micro-crystallography comes of age

PubMed Central

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

2012-01-01

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

Crystallography with online optical and X-ray absorption spectroscopies demonstrates an ordered mechanism in copper nitrite reductase.

PubMed

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

2008-04-25

Nitrite reductases are key enzymes that perform the first committed step in the denitrification process and reduce nitrite to nitric oxide. In copper nitrite reductases, an electron is delivered from the type 1 copper (T1Cu) centre to the type 2 copper (T2Cu) centre where catalysis occurs. Despite significant structural and mechanistic studies, it remains controversial whether the substrates, nitrite, electron and proton are utilised in an ordered or random manner. We have used crystallography, together with online X-ray absorption spectroscopy and optical spectroscopy, to show that X-rays rapidly and selectively photoreduce the T1Cu centre, but that the T2Cu centre does not photoreduce directly over a typical crystallographic data collection time. Furthermore, internal electron transfer between the T1Cu and T2Cu centres does not occur, and the T2Cu centre remains oxidised. These data unambiguously demonstrate an 'ordered' mechanism in which electron transfer is gated by binding of nitrite to the T2Cu. Furthermore, the use of online multiple spectroscopic techniques shows their value in assessing radiation-induced redox changes at different metal sites and demonstrates the importance of ensuring the correct status of redox centres in a crystal structure determination. Here, optical spectroscopy has shown a very high sensitivity for detecting the change in T1Cu redox state, while X-ray absorption spectroscopy has reported on the redox status of the T2Cu site, as this centre has no detectable optical absorption.

Architectural plasticity of AMPK revealed by electron microscopy and X-ray crystallography

PubMed Central

Ouyang, Yan; Zhu, Li; Li, Yifang; Guo, Miaomiao; Liu, Yang; Cheng, Jin; Zhao, Jing; Wu, Yi

2016-01-01

Mammalian AMP-activated protein kinase (AMPK) acts as an important sensor of cellular energy homeostasis related with AMP/ADP to ATP ratio. The overall architecture of AMPK has been determined in either homotrimer or monomer form by electron microscopy (EM) and X-ray crystallography successively. Accordingly proposed models have consistently revealed a key role of the α subunit linker in sensing adenosine nucleoside binding on the γ subunit and mediating allosteric regulation of kinase domain (KD) activity, whereas there are vital differences in orienting N-terminus of α subunit and locating carbohydrate-binding module (CBM) of β subunit. Given that Mg2+, an indispensable cofactor of AMPK was present in the EM sample preparation buffer however absent when forming crystals, here we carried out further reconstructions without Mg2+ to expectably inspect if this ion may contribute to this difference. However, no essential alteration has been found in this study compared to our early work. Further analyses indicate that the intra-molecular movement of the KD and CBM are most likely due to the flexible linkage of the disordered linkers with the rest portion as well as a contribution from the plasticity in the inter-molecular assembly mode, which might ulteriorly reveal an architectural complication of AMPK. PMID:27063142

NMR and X-ray structural characterization and conformational aspects of fluorinated (5Z)-3-benzil-5-arylidenofuran-2(5H)-ones

NASA Astrophysics Data System (ADS)

Teixeira, R. R.; Barbosa, L. C. A.; Kabeshov, M. A.; Maltha, C. R. A.; Corrêa, R. S.; Doriguetto, A. C.

2014-10-01

Herein we describe structural insights of (5Z)-3-benzyl-5-(2-fluorobenzylidene)furan-2(5H)-one (6) and (5Z)-3-benzyl-5-(pentafluorobenzylidene)furan-2(5H)-one (7), γ-alkylidenebutenolides analogues of the natural products nostoclides. Their structures were investigated by NMR spectroscopy and X-ray crystallography. The stereochemistry of the exocyclic double bond of these fluorinated compounds was determined to be Z by NMR analysis and confirmed by X-ray data. Compounds 6 and 7 crystallized in the monoclinic crystal system P21/c group. A comparison between structural features of (6) and (7) and nostoclide derivatives previously published by us is described.

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

PubMed

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.

Asymmetry in serial femtosecond crystallography data.

PubMed

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

2017-03-01

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

Crystal and Vibrational Structure of Energetic 3,5-dinitro 1,3,5-oxadiazinane (DOD) by Single Crystal X-ray Diffractometry and Raman Spectroscopy

DTIC Science & Technology

2018-03-19

calculations using a temperature of 298 K. 15. SUBJECT TERMS 3,5-dinitro-1,3,5-oxadiazinane (DOD), X-ray crystallography , Raman, energetic material...X-ray analysis. 2.2 Characterization X-ray Crystallography . DOD crystals were characterized with a SuperNova, Dualflex, EosS2 diffractometer using

Micro-crystallography comes of age.

PubMed

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

2012-10-01

The latest revolution in macromolecular crystallography was incited by the development of dedicated, user friendly, micro-crystallography beam lines. Brilliant X-ray beams of diameter 20 μm or less, now available at most synchrotron sources, enable structure determination from samples that previously were inaccessible. Relative to traditional crystallography, crystals with one or more small dimensions have diffraction patterns with vastly improved signal-to-noise when recorded with an appropriately matched beam size. Structures can be solved from isolated, well diffracting regions within inhomogeneous samples. This review summarizes the technological requirements and approaches to producing micro-beams and how they continue to change the practice of crystallography. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

PubMed Central

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

2015-01-01

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

Chemical Crystallography at the Advanced Light Source

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCormick, Laura; Giordano, Nico; Teat, Simon

Chemical crystallography at synchrotrons was pioneered at the Daresbury SRS station 9.8. The chemical crystallography beamlines at the Advanced Light Source seek to follow that example, with orders of magnitude more flux than a lab source, and various in situ experiments. This article thus attempts to answer why a chemist would require synchrotron X-rays, to describe the techniques available at the ALS chemical crystallography beamlines, and place the current facilities in a historical context.

Chemical Crystallography at the Advanced Light Source

DOE PAGES

McCormick, Laura; Giordano, Nico; Teat, Simon; ...

2017-12-18

Chemical crystallography at synchrotrons was pioneered at the Daresbury SRS station 9.8. The chemical crystallography beamlines at the Advanced Light Source seek to follow that example, with orders of magnitude more flux than a lab source, and various in situ experiments. This article thus attempts to answer why a chemist would require synchrotron X-rays, to describe the techniques available at the ALS chemical crystallography beamlines, and place the current facilities in a historical context.

A combined solid-state NMR and X-ray crystallography study of the bromide ion environments in triphenylphosphonium bromides.

PubMed

Burgess, Kevin M N; Korobkov, Ilia; Bryce, David L

2012-04-27

Multinuclear ((31)P and (79/81)Br), multifield (9.4, 11.75, and 21.1 T) solid-state nuclear magnetic resonance experiments are performed for seven phosphonium bromides bearing the triphenylphosphonium cation, a molecular scaffold found in many applications in chemistry. This is undertaken to fully characterise their bromine electric field gradient (EFG) tensors, as well as the chemical shift (CS) tensors of both the halogen and the phosphorus nuclei, providing a rare and novel insight into the local electronic environments surrounding them. New crystal structures, obtained from single-crystal X-ray diffraction, are reported for six compounds to aid in the interpretation of the NMR data. Among them is a new structure of BrPPh(4), because the previously reported one was inconsistent with our magnetic resonance data, thereby demonstrating how NMR data of non-standard nuclei can correct or improve X-ray diffraction data. Our results indicate that, despite sizable quadrupolar interactions, (79/81)Br magnetic resonance spectroscopy is a powerful characterisation tool that allows for the differentiation between chemically similar bromine sites, as shown through the range in the characteristic NMR parameters. (35/37)Cl solid-state NMR data, obtained for an analogous phosphonium chloride sample, provide insight into the relationship between unit cell volume, nuclear quadrupolar coupling constants, and Sternheimer antishielding factors. The experimental findings are complemented by gauge-including projector-augmented wave (GIPAW) DFT calculations, which substantiate our experimentally determined strong dependence of the largest component of the bromine CS tensor, δ(11), on the shortest Br-P distance in the crystal structure, a finding that has possible application in the field of NMR crystallography. This trend is explained in terms of Ramsey's theory on paramagnetic shielding. Overall, this work demonstrates how careful NMR studies of underexploited exotic nuclides, such

Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase

PubMed Central

Sigfridsson, Kajsa G. V.; Chernev, Petko; Leidel, Nils; Popović-Bijelić, Ana; Gräslund, Astrid; Haumann, Michael

2013-01-01

Prototypic dinuclear metal cofactors with varying metallation constitute a class of O2-activating catalysts in numerous enzymes such as ribonucleotide reductase. Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by x-ray photoreduction (XRP). We studied XPR of Fe(III)Fe(III) and Mn(III)Fe(III) sites in the R2 subunit of Chlamydia trachomatis ribonucleotide reductase using x-ray absorption spectroscopy. Rapid and biphasic x-ray photoreduction kinetics at 20 and 80 K for both cofactor types suggested sequential formation of (III,II) and (II,II) species and similar redox potentials of iron and manganese sites. Comparing with typical x-ray doses in crystallography implies that (II,II) states are reached in <1 s in such studies. First-sphere metal coordination and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the XPR-induced (II,II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II,II) cofactors, which deviate from the native structures functional in O2 activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques. PMID:23400774

Rapid X-ray photoreduction of dimetal-oxygen cofactors in ribonucleotide reductase.

PubMed

Sigfridsson, Kajsa G V; Chernev, Petko; Leidel, Nils; Popovic-Bijelic, Ana; Gräslund, Astrid; Haumann, Michael

2013-04-05

Prototypic dinuclear metal cofactors with varying metallation constitute a class of O2-activating catalysts in numerous enzymes such as ribonucleotide reductase. Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by x-ray photoreduction (XRP). We studied XPR of Fe(III)Fe(III) and Mn(III)Fe(III) sites in the R2 subunit of Chlamydia trachomatis ribonucleotide reductase using x-ray absorption spectroscopy. Rapid and biphasic x-ray photoreduction kinetics at 20 and 80 K for both cofactor types suggested sequential formation of (III,II) and (II,II) species and similar redox potentials of iron and manganese sites. Comparing with typical x-ray doses in crystallography implies that (II,II) states are reached in <1 s in such studies. First-sphere metal coordination and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the XPR-induced (II,II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II,II) cofactors, which deviate from the native structures functional in O2 activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques.

Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography*

PubMed Central

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N.; Nishida, Clinton R.; de Montellano, Paul R. Ortiz

2015-01-01

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states. PMID:25670859

Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

DOE PAGES

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; ...

2015-02-10

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop withmore » various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.« less

Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop withmore » various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.« less

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

PubMed

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.

X-ray evaluation of SEM technique for determining the crystallography of echinoid skeletons.

PubMed

Dillaman, R M; Hart, H V

1981-01-01

Coronal plates of the sea urchin Strongylocentrotus purpuratus have a microstructure typified by smooth textured trabeculae. When plates were decorated with calcite crystals each rhombohedron had the same orientation regardless of its location on the plate. When the sample was oriented so that the three edges of the rhombohedron formed equal 120 degrees angles and the three crystal faces appeared to form equal angles with the plane of the photograph, the c-axis of the plate paralleled the electron beam and the three a-axes were 30 degrees counterclockwise from the edges. These a-axes were then related to a plate edge recorded in a low magnification micrograph. Directions of the a-axes of each plate were also measured using a back-reflection Laue x-ray diffraction camera. A comparison of a-axes measured by the two techniques showed an average difference of 3 degrees, indicating that decorated crystal grew in crystallographic continuity with the plate. Assuming this relationship remains constant, the decoration technique appears to be an accurate and efficient method for evaluating the crystallography of echinoid skeletal units. Analysis of a polar plot of a-axes for 11 plates indicated that the a-axes were not randomly oriented; however, definitive relationships must await more extensive investigations.

Graphene-based microfluidics for serial crystallography.

PubMed

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

2016-08-02

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

Sub-25-nm laboratory x-ray microscopy using a compound Fresnel zone plate.

PubMed

von Hofsten, Olov; Bertilson, Michael; Reinspach, Julia; Holmberg, Anders; Hertz, Hans M; Vogt, Ulrich

2009-09-01

Improving the resolution in x-ray microscopes is of high priority to enable future applications in nanoscience. However, high-resolution zone-plate optics often have low efficiency, which makes implementation in laboratory microscopes difficult. We present a laboratory x-ray microscope based on a compound zone plate. The compound zone plate utilizes multiple diffraction orders to achieve high resolution while maintaining reasonable efficiency. We analyze the illumination conditions necessary for this type of optics in order to suppress stray light and demonstrate microscopic imaging resolving 25 nm features.

Synthesis, spectroscopic investigations (X-ray, NMR and TD-DFT), antimicrobial activity and molecular docking of 2,6-bis(hydroxy(phenyl)methyl)cyclohexanone.

PubMed

Barakat, Assem; Ghabbour, Hazem A; Al-Majid, Abdullah Mohammed; Soliman, Saied M; Ali, M; Mabkhot, Yahia Nasser; Shaik, Mohammed Rafi; Fun, Hoong-Kun

2015-07-21

The synthesis of 2,6-bis(hydroxy(phenyl)methyl)cyclohexanone 1 is described. The molecular structure of the title compound 1 was confirmed by NMR, FT-IR, MS, CHN microanalysis, and X-ray crystallography. The molecular structure was also investigated by a set of computational studies and found to be in good agreement with the experimental data obtained from the various spectrophotometric techniques. The antimicrobial activity and molecular docking of the synthesized compound was investigated.

X-rays in the Cryo-EM Era: Structural Biology’s Dynamic Future

PubMed Central

Shoemaker, Susannah C.; Ando, Nozomi

2018-01-01

Over the past several years, single-particle cryo-electron microscopy (cryo-EM) has emerged as a leading method for elucidating macromolecular structures at near-atomic resolution, rivaling even the established technique of X-ray crystallography. Cryo-EM is now able to probe proteins as small as hemoglobin (64 kDa), while avoiding the crystallization bottleneck entirely. The remarkable success of cryo-EM has called into question the continuing relevance of X-ray methods, particularly crystallography. To say that the future of structural biology is either cryo-EM or crystallography, however, would be misguided. Crystallography remains better suited to yield precise atomic coordinates of macromolecules under a few hundred kDa in size, while the ability to probe larger, potentially more disordered assemblies is a distinct advantage of cryo-EM. Likewise, crystallography is better equipped to provide high-resolution dynamic information as a function of time, temperature, pressure, and other perturbations, whereas cryo-EM offers increasing insight into conformational and energy landscapes, particularly as algorithms to deconvolute conformational heterogeneity become more advanced. Ultimately, the future of both techniques depends on how their individual strengths are utilized to tackle questions on the frontiers of structural biology. Structure determination is just one piece of a much larger puzzle: a central challenge of modern structural biology is to relate structural information to biological function. In this perspective, we share insight from several leaders in the field and examine the unique and complementary ways in which X-ray methods and cryo-EM can shape the future of structural biology. PMID:29227642

Soft X-ray photoemission study of Co2(Cr1-xFex)Ga Heusler compounds

NASA Astrophysics Data System (ADS)

Tsunekawa, Masanori; Hattori, Yoshiro; Sekiyama, Akira; Fujiwara, Hidenori; Suga, Shigemasa; Muro, Takayuki; Kanomata, Takeshi; Imada, Shin

2015-08-01

We have performed soft X-ray photoemission spectroscopy (SXPES) and X-ray absorption spectroscopy (XAS) of the Co-based Heusler compounds Co2(Cr1-xFex)Ga (x = 0.0, 0.4, and 1.0) in order to study their electronic structures. Band-structure calculation was carried out and compared with the experimental results. SXPES spectra show hν-dependence, revealing the contributions of the Co, Cr, and Fe 3d electronic states in the valence band. The band width observed by the SXPES seems to be narrower than that predicted by the band-structure calculation. XAS spectra depend strongly on the the value of x in Co2(Cr1-xFex)Ga. The electron correlation effects are found to be stronger as x changes from 0.0 to 1.0.

Accurate determination of segmented X-ray detector geometry

PubMed Central

Yefanov, Oleksandr; Mariani, Valerio; Gati, Cornelius; White, Thomas A.; Chapman, Henry N.; Barty, Anton

2015-01-01

Recent advances in X-ray detector technology have resulted in the introduction of segmented detectors composed of many small detector modules tiled together to cover a large detection area. Due to mechanical tolerances and the desire to be able to change the module layout to suit the needs of different experiments, the pixels on each module might not align perfectly on a regular grid. Several detectors are designed to permit detector sub-regions (or modules) to be moved relative to each other for different experiments. Accurate determination of the location of detector elements relative to the beam-sample interaction point is critical for many types of experiment, including X-ray crystallography, coherent diffractive imaging (CDI), small angle X-ray scattering (SAXS) and spectroscopy. For detectors with moveable modules, the relative positions of pixels are no longer fixed, necessitating the development of a simple procedure to calibrate detector geometry after reconfiguration. We describe a simple and robust method for determining the geometry of segmented X-ray detectors using measurements obtained by serial crystallography. By comparing the location of observed Bragg peaks to the spot locations predicted from the crystal indexing procedure, the position, rotation and distance of each module relative to the interaction region can be refined. We show that the refined detector geometry greatly improves the results of experiments. PMID:26561117

Accurate determination of segmented X-ray detector geometry

DOE PAGES

Yefanov, Oleksandr; Mariani, Valerio; Gati, Cornelius; ...

2015-10-22

Recent advances in X-ray detector technology have resulted in the introduction of segmented detectors composed of many small detector modules tiled together to cover a large detection area. Due to mechanical tolerances and the desire to be able to change the module layout to suit the needs of different experiments, the pixels on each module might not align perfectly on a regular grid. Several detectors are designed to permit detector sub-regions (or modules) to be moved relative to each other for different experiments. Accurate determination of the location of detector elements relative to the beam-sample interaction point is critical formore » many types of experiment, including X-ray crystallography, coherent diffractive imaging (CDI), small angle X-ray scattering (SAXS) and spectroscopy. For detectors with moveable modules, the relative positions of pixels are no longer fixed, necessitating the development of a simple procedure to calibrate detector geometry after reconfiguration. We describe a simple and robust method for determining the geometry of segmented X-ray detectors using measurements obtained by serial crystallography. By comparing the location of observed Bragg peaks to the spot locations predicted from the crystal indexing procedure, the position, rotation and distance of each module relative to the interaction region can be refined. Furthermore, we show that the refined detector geometry greatly improves the results of experiments.« less

Analysis of cytochrome P450 CYP119 ligand-dependent conformational dynamics by two-dimensional NMR and X-ray crystallography.

PubMed

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N; Nishida, Clinton R; de Montellano, Paul R Ortiz

2015-04-17

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional (1)H,(15)N HSQC chemical shift perturbation mapping of (15)N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Synchrotron X-ray powder diffraction data of LASSBio-1515: A new N-acylhydrazone derivative compound

NASA Astrophysics Data System (ADS)

Costa, F. N.; Braz, D.; Ferreira, F. F.; da Silva, T. F.; Barreiro, E. J.; Lima, L. M.; Colaço, M. V.; Kuplich, L.; Barroso, R. C.

2014-02-01

In this work, synchrotron X-ray powder diffraction data allowed for a successful indexing of LASSBio-1515 compound, candidate to analgesic and anti-inflammatory activity. X-ray powder diffraction data collected in transmission and high-throughput geometries were used to analyze this compound. The X-ray wavelength of the synchrotron radiation used in this study was determined to be λ=1.55054 Å. LASSBio-1515 was found to be monoclinic with space group P21/c and unit cell parameters a=11.26255(16) Å, b=12.59785(16) Å, c=8.8540(1) Å, β=90.5972(7)° and V=1256.17(3) Å3.

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

PubMed

Kärtner, F X; Ahr, F; Calendron, A-L; Çankaya, H; Carbajo, S; Chang, G; Cirmi, G; Dörner, K; Dorda, U; Fallahi, A; Hartin, A; Hemmer, M; Hobbs, R; Hua, Y; Huang, W R; Letrun, R; Matlis, N; Mazalova, V; Mücke, O D; Nanni, E; Putnam, W; Ravi, K; Reichert, F; Sarrou, I; Wu, X; Yahaghi, A; Ye, H; Zapata, L; Zhang, D; Zhou, C; Miller, R J D; Berggren, K K; Graafsma, H; Meents, A; Assmann, R W; Chapman, H N; Fromme, P

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 atto-second 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

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

PubMed Central

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 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. PMID:23793151

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

PubMed

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.

Accounting for partiality in serial crystallography using ray-tracing principles

PubMed Central

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

2015-01-01

Serial crystallography generates ‘still’ diffraction data sets that are composed of single diffraction images obtained from a large number of crystals arbitrarily oriented in the X-ray beam. Estimation of the reflection partialities, which accounts for the expected observed fractions of diffraction intensities, has so far been problematic. In this paper, a method is derived for modelling the partialities by making use of the ray-tracing diffraction-integration method EVAL. The method estimates partialities based on crystal mosaicity, beam divergence, wavelength dispersion, crystal size and the interference function, accounting for crystallite size. It is shown that modelling of each reflection by a distribution of interference-function weighted rays yields a ‘still’ Lorentz factor. Still data are compared with a conventional rotation data set collected from a single lysozyme crystal. Overall, the presented still integration method improves the data quality markedly. The R factor of the still data compared with the rotation data decreases from 26% using a Monte Carlo approach to 12% after applying the Lorentz correction, to 5.3% when estimating partialities by EVAL and finally to 4.7% after post-refinement. The merging R int factor of the still data improves from 105 to 56% but remains high. This suggests that the accuracy of the model parameters could be further improved. However, with a multiplicity of around 40 and an R int of ∼50% the merged still data approximate the quality of the rotation data. The presented integration method suitably accounts for the partiality of the observed intensities in still diffraction data, which is a critical step to improve data quality in serial crystallography. PMID:26327370

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

PubMed

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

2015-09-01

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

Novel drift structures for silicon and compound semiconductor X-ray and gamma-ray detectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patt, B.E.; Iwanczyk, J.S.

Recently developed silicon- and compound-semiconductor-based drift detector structures have produced excellent performance for charged particles, X-rays, and gamma rays and for low-signal visible light detection. The silicon drift detector (SDD) structures that the authors discuss relate to direct X-ray detectors and scintillation photon detectors coupled with scintillators for gamma rays. Recent designs include several novel features that ensure very low dark current and hence low noise. In addition, application of thin window technology ensures a very high quantum efficiency entrance window on the drift photodetector. The main features of the silicon drift structures for X rays and light detection aremore » very small anode capacitance independent of the overall detector size, low noise, and high throughput. To take advantage of the small detector capacitance, the first stage of the electronics needs to be integrated into the detector anode. In the gamma-ray application, factors other than electronic noise dominate, and there is no need to integrate the electronics into the anode. Thus, a different drift structure is needed in conjunction with a high-Z material. The main features in this case are large active detector volume and electron-only induced signal.« less

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

DOE PAGES

Wu, Wenting; Nogly, Przemyslaw; Rheinberger, Jan; ...

2015-06-27

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 in this study, 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 crystallizationmore » 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.« less

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

PubMed Central

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

2015-01-01

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

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

DOE PAGES

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

2015-03-17

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

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

DOE PAGES

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

2015-03-17

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

Microfocus/Polycapillary-Optic Crystallographic X-Ray System

NASA Technical Reports Server (NTRS)

Joy, Marshall; Gubarev, Mikhail; Ciszak, Ewa

2005-01-01

A system that generates an intense, nearly collimated, nearly monochromatic, small-diameter x-ray beam has been developed for use in macromolecular crystallography. A conventional x-ray system for macromolecular crystallography includes a rotating-anode x-ray source, which is massive (.500 kg), large (approximately 2 by 2 by 1 m), and power-hungry (between 2 and 18 kW). In contrast, the present system generates a beam of the required brightness from a microfocus source, which is small and light enough to be mounted on a laboratory bench, and operates at a power level of only tens of watts. The figure schematically depicts the system as configured for observing x-ray diffraction from a macromolecular crystal. In addition to the microfocus x-ray source, the system includes a polycapillary optic . a monolithic block (typically a bundle of fused glass tubes) that contains thousands of straight or gently curved capillary channels, along which x-rays propagate with multiple reflections. This particular polycapillary optic is configured to act as a collimator; the x-ray beam that emerges from its output face consists of quasi-parallel subbeams with a small angular divergence and a diameter comparable to the size of a crystal to be studied. The gap between the microfocus x-ray source and the input face of the polycapillary optic is chosen consistently with the focal length of the polycapillary optic and the need to maximize the solid angle subtended by the optic in order to maximize the collimated x-ray flux. The spectrum from the source contains a significant component of Cu K (photon energy is 8.08 keV) radiation. The beam is monochromatized (for Cu K ) by a nickel filter 10 m thick. In a test, this system was operated at a power of 40 W (current of 897 A at an accelerating potential of 45 kV), with an anode x-ray spot size of 41+/-2 microns. Also tested, in order to provide a standard for comparison, was a commercial rotating-anode x-ray crystallographic system with a

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.

PubMed

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.

[Study of selegiline and related compounds with x-ray diffraction].

PubMed

Simon, K; Böcskei, Z; Török, Z

1992-09-01

Selegiline and its parent compounds were studied by X-ray diffraction. It was established that the racemates of primary and secondary amines (p-fluoro-amphetamine, methamphetamine, p-fluoro-methamphetamine) hydrochloride do not form racemic compounds but crystalline as conglomerates, at the same time tertiary amines like selegiline and p-fluoro-selegiline hydrochlorides do. The crystalline structure of five enantiomeric hydrochlorides were determined, the CPhe-C-C-N torsion angle is anti-periplanar in all cases but in p-fluoro-amphetamine where it is gauche.

Identifying anthropogenic uranium compounds using soft X-ray near-edge absorption spectroscopy

NASA Astrophysics Data System (ADS)

Ward, Jesse D.; Bowden, Mark; Tom Resch, C.; Eiden, Gregory C.; Pemmaraju, C. D.; Prendergast, David; Duffin, Andrew M.

2017-01-01

Uranium ores mined for industrial use are typically acid-leached to produce yellowcake and then converted into uranium halides for enrichment and purification. These anthropogenic chemical forms of uranium are distinct from their mineral counterparts. The purpose of this study is to use soft X-ray absorption spectroscopy to characterize several common anthropogenic uranium compounds important to the nuclear fuel cycle. Chemical analyses of these compounds are important for process and environmental monitoring. X-ray absorption techniques have several advantages in this regard, including element-specificity, chemical sensitivity, and high spectral resolution. Oxygen K-edge spectra were collected for uranyl nitrate, uranyl fluoride, and uranyl chloride, and fluorine K-edge spectra were collected for uranyl fluoride and uranium tetrafluoride. Interpretation of the data is aided by comparisons to calculated spectra. The effect of hydration state on the sample, a potential complication in interpreting oxygen K-edge spectra, is discussed. These compounds have unique spectral signatures that can be used to identify unknown samples.

Identifying anthropogenic uranium compounds using soft X-ray near-edge absorption spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ward, Jesse D.; Bowden, Mark; Tom Resch, C.

2017-01-01

Uranium ores mined for industrial use are typically acid-leached to produce yellowcake and then converted into uranium halides for enrichment and purification. These anthropogenic chemical forms of uranium are distinct from their mineral counterparts. The purpose of this study is to use soft X-ray absorption spectroscopy to characterize several common anthropogenic uranium compounds important to the nuclear fuel cycle. Non-destructive chemical analyses of these compounds is important for process and environmental monitoring and X-ray absorption techniques have several advantages in this regard, including element-specificity, chemical sensitivity, and high spectral resolution. Oxygen K-edge spectra were collected for uranyl nitrate, uranyl fluoride,more » and uranyl chloride, and fluorine K-edge spectra were collected for uranyl fluoride and uranium tetrafluoride. Interpretation of the data is aided by comparisons to calculated spectra. These compounds have unique spectral signatures that can be used to identify unknown samples.« less

Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature.

PubMed

Ishigami, Izumi; Zatsepin, Nadia A; Hikita, Masahide; Conrad, Chelsie E; Nelson, Garrett; Coe, Jesse D; Basu, Shibom; Grant, Thomas D; Seaberg, Matthew H; Sierra, Raymond G; Hunter, Mark S; Fromme, Petra; Fromme, Raimund; Yeh, Syun-Ru; Rousseau, Denis L

2017-07-25

Cytochrome c oxidase (C c O), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine C c O (bC c O) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a 3 iron atom, with a bent Fe-C-O angle of ∼142°. In contrast, in the synchrotron structure, the Fe-CO bond is cleaved; CO relocates to a new site near Cu B , which, in turn, moves closer to the heme a 3 iron by ∼0.38 Å. Structural comparison reveals that ligand binding to the heme a 3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a 3 , thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.

Recent advances in racemic protein crystallography.

PubMed

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.

On the state of crystallography at the dawn of the electron microscopy revolution.

PubMed

Higgins, Matthew K; Lea, Susan M

2017-10-01

While protein crystallography has, for many years, been the most used method for structural analysis of macromolecular complexes, remarkable recent advances in high-resolution electron cryo-microscopy led to suggestions that 'the revolution will not be crystallised'. Here we highlight the current success rate, speed and ease of modern crystallographic structure determination and some recent triumphs of both 'classical' crystallography and the use of X-ray free electron lasers. We also outline fundamental differences between structure determination using X-ray crystallography and electron microscopy. We suggest that crystallography will continue to co-exist with electron microscopy as part of an integrated array of methods, allowing structural biologists to focus on fundamental biological questions rather than being constrained by the methods available. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Biochemical, spectroscopic and X-ray structural analysis of deuterated multicopper oxidase CueO prepared from a new expression construct for neutron crystallography.

PubMed

Akter, Mahfuza; Inoue, Chika; Komori, Hirofumi; Matsuda, Nana; Sakurai, Takeshi; Kataoka, Kunishige; Higuchi, Yoshiki; Shibata, Naoki

2016-10-01

Multicopper oxidases oxidize various phenolic and nonphenolic compounds by using molecular oxygen as an electron acceptor to produce water. A multicopper oxidase protein, CueO, from Escherichia coli is involved in copper homeostasis in the bacterial cell. Although X-ray crystallographic studies have been conducted, the reduction mechanism of oxygen and the proton-transfer pathway remain unclear owing to the difficulty in identifying H atoms from X-ray diffraction data alone. To elucidate the reaction mechanism using neutron crystallography, a preparation system for obtaining large, high-quality single crystals of deuterated CueO was developed. Tiny crystals were obtained from the deuterated CueO initially prepared from the original construct. The X-ray crystal structure of the deuterated CueO showed that the protein contained an incompletely truncated signal sequence at the N-terminus, which resulted in the heterogeneity of the protein sample for crystallization. Here, a new CueO expression system that had an HRV3C cleavage site just after the signal sequence was constructed. Deuterated CueO from the new construct was expressed in cells cultured in deuterated algae-extract medium and the signal sequence was completely eliminated by HRV3C protease. The deuteration level of the purified protein was estimated by MALDI-TOF mass spectrometry to be at least 83.2% compared with nondeuterated protein. Nondeuterated CueO crystallized in space group P2 1 , with unit-cell parameters a = 49.51, b = 88.79, c = 53.95 Å, β = 94.24°, and deuterated CueO crystallized in space group P2 1 2 1 2 1 , with unit-cell parameters a = 49.91, b = 106.92, c = 262.89 Å. The crystallographic parameters for the crystals of the new construct were different from those previously reported for nondeuterated crystals. The nondeuterated and deuterated CueO from the new construct had similar UV-Vis spectra, enzymatic activities and overall structure and geometry of the ligands of the Cu atoms

Metalloprotein Crystallography: More than a Structure.

PubMed

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

2016-04-19

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

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

PubMed

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.

Johann Deisenhofer, Crystallography, and Proteins

Science.gov Websites

research using X-ray crystallography to elucidate for the first time the three-dimensional structure of a large membrane-bound protein molecule. This structure helped explain the process of photosynthesis, by a protein structure determination that relied on complementary features of two different beam lines

Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sutton, Kristin A.; Black, Paul J.; Mercer, Kermit R.

2013-12-01

Electron paramagnetic resonance (EPR) and online UV–visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage, to confirm a multi-track radiation-damage process and to develop a model of that process. Electron paramagnetic resonance (EPR) and online UV–visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage. Online UV–visible spectroscopy showed that upon X-irradiation, disulfide radicalization appeared to saturate at an absorbed dose of approximately 0.5–0.8 MGy, in contrast to the saturating dose of ∼0.2 MGy observed using EPR at much lower dose rates. Themore » observations suggest that a multi-track model involving product formation owing to the interaction of two separate tracks is a valid model for radiation damage in protein crystals. The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied. The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized. Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure.« less

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

DOEpatents

Atac, Muzaffer; McKay, Timothy

1999-01-01

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

Virtual Screening and X-ray Crystallography for Human Kallikrein 6 Inhibitors with an Amidinothiophene P1 Group.

PubMed

Liang, Guyan; Chen, Xin; Aldous, Suzanne; Pu, Su-Fen; Mehdi, Shujaath; Powers, Elaine; Giovanni, Andrew; Kongsamut, Sathapana; Xia, Tianhui; Zhang, Ying; Wang, Rachel; Gao, Zhongli; Merriman, Gregory; McLean, Larry R; Morize, Isabelle

2012-02-09

A series of compounds with an amidinothiophene P1 group and a pyrrolidinone-sulphonamide scaffold linker was identified as potent inhibitors of human kallikrein 6 by structure-based virtual screening based on the union accessible binding space of serine proteases. As the first series of potent nonmechanism-based hK6 inhibitors, they may be used as tool compounds for target validation. An X-ray structure of a representative compound complexed with hK6, resolved at a resolution of 1.88 Å, revealed that the amidinothiophene moiety bound in the S1 pocket and the pyrrolidinone-sulphonamide linker projected the aromatic tail into the S' pocket.

Microscope using an x-ray tube and a bubble compound refractive lens

NASA Astrophysics Data System (ADS)

Piestrup, M. A.; Gary, C. K.; Park, H.; Harris, J. L.; Cremer, J. T.; Pantell, R. H.; Dudchik, Y. I.; Kolchevsky, N. N.; Komarov, F. F.

2005-03-01

We present x-ray images of grid meshes and biological material obtained using an unfiltered x-ray tube and a compound refractive lens composed of microbubbles embedded in epoxy inside a glass capillary. Images obtained using this apparatus are compared with those using a synchrotron source and the same lens. We find that the field of view is larger than that obtained using the synchrotron source, whereas the contrast and resolution are reduced. Geometrical distortion around the edges of the field of view is also reduced. The experiments demonstrate the usefulness of the apparatus in a modest laboratory setting.

Sub-500  nm hard x ray focusing by compound long kinoform lenses.

PubMed

Liao, Keliang; Liu, Jing; Liang, Hao; Wu, Xuehui; Zhang, Kai; Yuan, Qingxi; Yi, Futing; Sheng, Weifan

2016-01-01

The focusing performance of polymethyl methacrylate compound long kinoform lenses with 70 μm aperture and 19.5 mm focal length was characterized with 8 keV x rays using the knife-edge scan method at the 4W1A transmission x-ray microscope beamline of Beijing Synchrotron Radiation Facility. The experiment result shows a best FWHM focus size of 440 nm with 31% diffraction efficiency.

IP3-mediated gating mechanism of the IP3 receptor revealed by mutagenesis and X-ray crystallography.

PubMed

Hamada, Kozo; Miyatake, Hideyuki; Terauchi, Akiko; Mikoshiba, Katsuhiko

2017-05-02

The inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R) is an IP 3 -gated ion channel that releases calcium ions (Ca 2+ ) from the endoplasmic reticulum. The IP 3 -binding sites in the large cytosolic domain are distant from the Ca 2+ conducting pore, and the allosteric mechanism of how IP 3 opens the Ca 2+ channel remains elusive. Here, we identify a long-range gating mechanism uncovered by channel mutagenesis and X-ray crystallography of the large cytosolic domain of mouse type 1 IP 3 R in the absence and presence of IP 3 Analyses of two distinct space group crystals uncovered an IP 3 -dependent global translocation of the curvature α-helical domain interfacing with the cytosolic and channel domains. Mutagenesis of the IP 3 R channel revealed an essential role of a leaflet structure in the α-helical domain. These results suggest that the curvature α-helical domain relays IP 3 -controlled global conformational dynamics to the channel through the leaflet, conferring long-range allosteric coupling from IP 3 binding to the Ca 2+ channel.

IP3-mediated gating mechanism of the IP3 receptor revealed by mutagenesis and X-ray crystallography

PubMed Central

Hamada, Kozo; Miyatake, Hideyuki; Terauchi, Akiko; Mikoshiba, Katsuhiko

2017-01-01

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is an IP3-gated ion channel that releases calcium ions (Ca2+) from the endoplasmic reticulum. The IP3-binding sites in the large cytosolic domain are distant from the Ca2+ conducting pore, and the allosteric mechanism of how IP3 opens the Ca2+ channel remains elusive. Here, we identify a long-range gating mechanism uncovered by channel mutagenesis and X-ray crystallography of the large cytosolic domain of mouse type 1 IP3R in the absence and presence of IP3. Analyses of two distinct space group crystals uncovered an IP3-dependent global translocation of the curvature α-helical domain interfacing with the cytosolic and channel domains. Mutagenesis of the IP3R channel revealed an essential role of a leaflet structure in the α-helical domain. These results suggest that the curvature α-helical domain relays IP3-controlled global conformational dynamics to the channel through the leaflet, conferring long-range allosteric coupling from IP3 binding to the Ca2+ channel. PMID:28416699

Macromolecular crystallography beamline X25 at the NSLS

PubMed Central

Héroux, Annie; Allaire, Marc; Buono, Richard; Cowan, Matthew L.; Dvorak, Joseph; Flaks, Leon; LaMarra, Steven; Myers, Stuart F.; Orville, Allen M.; Robinson, Howard H.; Roessler, Christian G.; Schneider, Dieter K.; Shea-McCarthy, Grace; Skinner, John M.; Skinner, Michael; Soares, Alexei S.; Sweet, Robert M.; Berman, Lonny E.

2014-01-01

Beamline X25 at the NSLS is one of the five beamlines dedicated to macromolecular crystallography operated by the Brookhaven National Laboratory Macromolecular Crystallography Research Resource group. This mini-gap insertion-device beamline has seen constant upgrades for the last seven years in order to achieve mini-beam capability down to 20 µm × 20 µm. All major components beginning with the radiation source, and continuing along the beamline and its experimental hutch, have changed to produce a state-of-the-art facility for the scientific community. PMID:24763654

How cryo‐electron microscopy and X‐ray crystallography complement each other

PubMed Central

Wang, Jia‐Wei

2016-01-01

Abstract With the ability to resolve structures of macromolecules at atomic resolution, X‐ray crystallography has been the most powerful tool in modern structural biology. At the same time, recent technical improvements have triggered a resolution revolution in the single particle cryo‐EM method. While the two methods are different in many respects, from sample preparation to structure determination, they both have the power to solve macromolecular structures at atomic resolution. It is important to understand the unique advantages and caveats of the two methods in solving structures and to appreciate the complementary nature of the two methods in structural biology. In this review we provide some examples, and discuss how X‐ray crystallography and cryo‐EM can be combined in deciphering structures of macromolecules for our full understanding of their biological mechanisms. PMID:27543495

Hard X-ray full field microscopy and magnifying microtomography using compound refractive lenses

NASA Astrophysics Data System (ADS)

Schroer, Christian G.; Günzler, Til Florian; Benner, Boris; Kuhlmann, Marion; Tümmler, Johannes; Lengeler, Bruno; Rau, Christoph; Weitkamp, Timm; Snigirev, Anatoly; Snigireva, Irina

2001-07-01

For hard X-rays, parabolic compound refractive lenses (PCRLs) are genuine imaging devices like glass lenses for visible light. Based on these new lenses, a hard X-ray full field microscope has been constructed that is ideally suited to image the interior of opaque samples with a minimum of sample preparation. As a result of a large depth of field, CRL micrographs are sharp projection images of most samples. To obtain 3D information about a sample, tomographic techniques are combined with magnified imaging.

Compound semiconductor detectors for X-ray astronomy: Spectroscopic measurements and material characterization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bavdaz, M.; Kraft, S.; Peacock, A.

1998-12-31

The use of some specific compound semiconductors in the fabrication of high energy X-ray detectors shows significant potential for X-ray astrophysics space missions. The authors are currently investigating three high purity crystals--CdZnTe, GaAs and TlBr--as the basis for future hard X-ray detectors (above 10 keV). In this paper the authors present the first results on CdZnTe and GaAs based detectors and evaluate the factors currently still constraining the performance. Energy resolutions (FWHM) of 0.9 keV and 1.1 keV at 14 keV and 60 keV, respectively, have been obtained with an epitaxial GaAs detector, while 0.7 keV and 1.5 keV FWHMmore » were measured at the same energies with a CdZnTe detector. Based on these results it is clear, that the next generation of X-ray astrophysics missions now in the planning phase may well consider extending the photon energy range up to {approximately} 100 keV by use of efficient detectors with reasonable spectroscopic capabilities.« less

Deformable complex network for refining low-resolution X-ray structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Chong; Wang, Qinghua; Ma, Jianpeng, E-mail: jpma@bcm.edu

2015-10-27

A new refinement algorithm called the deformable complex network that combines a novel angular network-based restraint with a deformable elastic network model in the target function has been developed to aid in structural refinement in macromolecular X-ray crystallography. In macromolecular X-ray crystallography, building more accurate atomic models based on lower resolution experimental diffraction data remains a great challenge. Previous studies have used a deformable elastic network (DEN) model to aid in low-resolution structural refinement. In this study, the development of a new refinement algorithm called the deformable complex network (DCN) is reported that combines a novel angular network-based restraint withmore » the DEN model in the target function. Testing of DCN on a wide range of low-resolution structures demonstrated that it constantly leads to significantly improved structural models as judged by multiple refinement criteria, thus representing a new effective refinement tool for low-resolution structural determination.« less

Resolution of x-ray parabolic compound refractive diamond lens defined at the home laboratory

NASA Astrophysics Data System (ADS)

Polyakov, S. N.; Zholudev, S. I.; Gasilov, S. V.; Martyushov, S. Yu.; Denisov, V. N.; Terentiev, S. A.; Blank, V. D.

2017-05-01

Here we demonstrate performance of an original lab system designed for testing of X-ray parabolic compound refractive lenses (CRL) manufactured from a high-quality single-crystalline synthetic diamond grown by the high-pressure hightemperature technique. The basic parameters of a diamond CRL comprised from 28 plano-concave lenses such as the focal length of 634 mm, transmissivity of 0.36, field of view of 1 mm and resolution of 6 µm have been determined. Usually such measurements are performed on synchrotron radiation facilities. In this work characterization of CRL was performed by means of instruments and components that are available for laboratories such as the Rigaku 9kW rotating anode X-ray generator, the PANalytical parallel beam X-ray mirror, a 6 m long optical bench, high precision multi-axis goniometers, high resolution X-ray emulsion films, and ultra-fast high-sensitive X-ray area detector PIXel3D. Developed setup was used to find differences between experimental and design parameters, which is very important for the improvement of CRLs manufacturing technology.

A Bright Future for Serial Femtosecond Crystallography with XFELs.

PubMed

Johansson, Linda C; Stauch, Benjamin; Ishchenko, Andrii; Cherezov, Vadim

2017-09-01

X-ray free electron lasers (XFELs) have the potential to revolutionize macromolecular structural biology due to the unique combination of spatial coherence, extreme peak brilliance, and short duration of X-ray pulses. A recently emerged serial femtosecond (fs) crystallography (SFX) approach using XFEL radiation overcomes some of the biggest hurdles of traditional crystallography related to radiation damage through the diffraction-before-destruction principle. Intense fs XFEL pulses enable high-resolution room-temperature structure determination of difficult-to-crystallize biological macromolecules, while simultaneously opening a new era of time-resolved structural studies. Here, we review the latest developments in instrumentation, sample delivery, data analysis, crystallization methods, and applications of SFX to important biological questions, and conclude with brief insights into the bright future of structural biology using XFELs. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

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.

Determination of X-ray flux using silicon pin diodes

PubMed Central

Owen, Robin L.; Holton, James M.; Schulze-Briese, Clemens; Garman, Elspeth F.

2009-01-01

Accurate measurement of photon flux from an X-ray source, a parameter required to calculate the dose absorbed by the sample, is not yet routinely available at macromolecular crystallography beamlines. The development of a model for determining the photon flux incident on pin diodes is described here, and has been tested on the macromolecular crystallography beamlines at both the Swiss Light Source, Villigen, Switzerland, and the Advanced Light Source, Berkeley, USA, at energies between 4 and 18 keV. These experiments have shown that a simple model based on energy deposition in silicon is sufficient for determining the flux incident on high-quality silicon pin diodes. The derivation and validation of this model is presented, and a web-based tool for the use of the macromolecular crystallography and wider synchrotron community is introduced. PMID:19240326

Difficult macromolecular structures determined using X-ray diffraction techniques.

PubMed

Hernández-Santoyo, Alejandra

2012-07-01

Macromolecular crystallography has been, for the last few decades, the main source of structural information of biological macromolecular systems and it is one of the most powerful techniques for the analysis of enzyme mechanisms and macromolecular interactions at the atomic level. In addition, it is also an extremely powerful tool for drug design. Recent technological and methodological developments in macromolecular X-ray crystallography have allowed solving structures that until recently were considered difficult or even impossible, such as structures at atomic or subatomic resolution or large macromolecular complexes and assemblies at low resolution. These developments have also helped to solve the 3D-structure of macromolecules from twin crystals. Recently, this technique complemented with cryo-electron microscopy and neutron crystallography has provided the structure of large macromolecular machines with great precision allowing understanding of the mechanisms of their function.

High-speed fixed-target serial virus crystallography

PubMed Central

Roedig, Philip; Ginn, Helen M.; Pakendorf, Tim; Sutton, Geoff; Harlos, Karl; Walter, Thomas S.; Meyer, Jan; Fischer, Pontus; Duman, Ramona; Vartiainen, Ismo; Reime, Bernd; Warmer, Martin; Brewster, Aaron S.; Young, Iris D.; Michels-Clark, Tara; Sauter, Nicholas K.; Kotecha, Abhay; Kelly, James; Rowlands, David J.; Sikorsky, Marcin; Nelson, Silke; Damiani, Daniel S.; Alonso-Mori, Roberto; Ren, Jingshan; Fry, Elizabeth E.; David, Christian; Stuart, David I.; Wagner, Armin; Meents, Alke

2017-01-01

We report a method for serial X-ray crystallography at X-ray free electron lasers (XFELs), which allows for full use of the current 120 Hz repetition rate of the Linear Coherent Light Source (LCLS). Using a micro-patterned silicon chip in combination with the high-speed Roadrunner goniometer for sample delivery we were able to determine the crystal structures of a picornavirus, bovine enterovirus 2 (BEV2), and the cytoplasmic polyhedrosis virus type 18 polyhedrin. Total data collection times were less than 14 and 10 minutes, respectively. Our method requires only micrograms of sample and will therefore broaden the applicability of serial femtosecond crystallography to challenging projects for which only limited sample amounts are available. By synchronizing the sample exchange to the XFEL repetition rate, our method allows for the most efficient use of the limited beamtime available at XFELs and should enable a substantial increase in sample throughput at these facilities. PMID:28628129

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

PubMed

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

2016-07-01

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

Protein crystallography beamline BL2S1 at the Aichi synchrotron

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, Nobuhisa; Nagae, Takayuki; Yamada, Yusuke

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

Protein crystallography beamline BL2S1 at the Aichi synchrotron.

PubMed

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

2017-01-01

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

Discovery and development of x-ray diffraction

NASA Astrophysics Data System (ADS)

Jeong, Yeuncheol; Yin, Ming; Datta, Timir

2013-03-01

In 1912 Max Laue at University of Munich reasoned x-rays to be short wavelength electromagnetic waves and figured interference would occur when scattered off crystals. Arnold Sommerfeld, W. Wien, Ewald and others, raised objections to Laue's idea, but soon Walter Friedrich succeeded in recording x-ray interference patterns off copper sulfate crystals. But the Laue-Ewald's 3-dimensional formula predicted excess spots. Fewer spots were observed. William Lawrence Bragg then 22 year old studying at Cambridge University heard the Munich results from father William Henry Brag, physics professor at Univ of Leeds. Lawrence figured the spots are 2-d interference of x-ray wavelets reflecting off successive atomic planes and derived a simple eponymous equation, the Bragg equation d*sin(theta) = n*lamda. 1913 onward the Braggs dominated the crystallography. Max Laue was awarded the physics Nobel in 1914 and the Braggs shared the same in 1915. Starting with Rontgen's first ever prize in 1901, the importance of x-ray techniques is evident from the four out of a total 16 physics Nobels between 1901-1917. We will outline the historical back ground and importance of x-ray diffraction giving rise to techniques that even in 2013, remain work horses in laboratories all over the globe.

From lows to highs: using low-resolution models to phase X-ray data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stuart, David I.; Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot; Abrescia, Nicola G. A., E-mail: nabrescia@cicbiogune.es

2013-11-01

An unusual example of how virus structure determination pushes the limits of the molecular replacement method is presented. The study of virus structures has contributed to methodological advances in structural biology that are generally applicable (molecular replacement and noncrystallographic symmetry are just two of the best known examples). Moreover, structural virology has been instrumental in forging the more general concept of exploiting phase information derived from multiple structural techniques. This hybridization of structural methods, primarily electron microscopy (EM) and X-ray crystallography, but also small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy, is central to integrative structural biology. Here,more » the interplay of X-ray crystallography and EM is illustrated through the example of the structural determination of the marine lipid-containing bacteriophage PM2. Molecular replacement starting from an ∼13 Å cryo-EM reconstruction, followed by cycling density averaging, phase extension and solvent flattening, gave the X-ray structure of the intact virus at 7 Å resolution This in turn served as a bridge to phase, to 2.5 Å resolution, data from twinned crystals of the major coat protein (P2), ultimately yielding a quasi-atomic model of the particle, which provided significant insights into virus evolution and viral membrane biogenesis.« less

The Race To X-ray Microbeam and Nanobeam Science

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ice, Gene E; Budai, John D; Pang, Judy

2011-01-01

X-ray microbeams are an emerging characterization tool with transformational implications for broad areas of science ranging from materials structure and dynamics, geophysics and environmental science to biophysics and protein crystallography. In this review, we discuss the race toward sub-10 nm- x-ray beams with the ability to penetrate tens to hundreds of microns into most materials and with the ability to determine local (crystal) structure. Examples of science enabled by current micro/nanobeam technologies are presented and we provide a perspective on future directions. Applications highlighted are chosen to illustrate the important features of various submicron beam strategies and to highlight themore » directions of current and future research. While it is clear that x-ray microprobes will impact science broadly, the practical limit for hard x-ray beam size, the limit to trace element sensitivity, and the ultimate limitations associated with near-atomic structure determinations are the subject of ongoing research.« less

Cleavage crystallography of liquid metal embrittled aluminum alloys

NASA Technical Reports Server (NTRS)

Reynolds, A. P.; Stoner, G. E.

1991-01-01

The crystallography of liquid metal-induced transgranular cleavage in six aluminum alloys having a variety of microstructures has been determined via Laue X-ray back reflection. The cleavage crystallography was independent of alloy microstructure, and the cleavage plane was 100-plane oriented in all cases. It was further determined that the cleavage crystallography was not influenced by alloy texture. Examination of the fracture surface indicated that there was not a unique direction of crack propagation. In addition, the existence of 100-plane cleavage on alloy 2024 fracture surfaces was inferred by comparison of secondary cleavage crack intersection geometry on the 2024 surfaces with the geometry of secondary cleavage crack intersections on the test alloys.

Resonant inelastic X-ray scattering on synthetic nickel compounds and Ni-Fe hydrogenase protein

NASA Astrophysics Data System (ADS)

Sanganas, Oliver; Löscher, Simone; Pfirrmann, Stefan; Marinos, Nicolas; Glatzel, Pieter; Weng, Tsu-Chien; Limberg, Christian; Driess, Matthias; Dau, Holger; Haumann, Michael

2009-11-01

Ni-Fe hydrogenases are proteins catalyzing the oxidative cleavage of dihydrogen (H2) and proton reduction to H2 at high turnover rates. Their active site is a heterobimetallic center comprising one Ni and one Fe atom. To understand the function of the site, well resolved structural and electronic information is required. Such information is expected to become accessible by high resolution X-ray absorption and emission techniques, which are rapidly developing at third generation synchrotron radiation sources. We studied a number of synthetic Ni compounds, which mimic relevant features of the Ni site in hydrogenases, and the Ni site in the soluble, NAD-reducing hydrogenase (SH) from the bacterium Ralstonia eutropha by resonant inelastic X-ray scattering (RIXS) using a Rowland-type spectrometer at the ESRF. The SH is particularly interesting because its H2-cleavage reaction is highly resistant against inhibition by O2. Kα-fluorescence detected RIXS planes in the 1s→3d region of the X-ray absorption spectrum were recorded on the protein which allow to extract L3-edge type spectra Spectral features of the protein are compared to those of the model compounds.

Coherent convergent-beam time-resolved X-ray diffraction

PubMed Central

Spence, John C. H.; Zatsepin, Nadia A.; Li, Chufeng

2014-01-01

The use of coherent X-ray lasers for structural biology allows the use of nanometre diameter X-ray beams with large beam divergence. Their application to the structure analysis of protein nanocrystals and single particles raises new challenges and opportunities. We discuss the form of these coherent convergent-beam (CCB) hard X-ray diffraction patterns and their potential use for time-resolved crystallography, normally achieved by Laue (polychromatic) diffraction, for which the monochromatic laser radiation of a free-electron X-ray laser is unsuitable. We discuss the possibility of obtaining single-shot, angle-integrated rocking curves from CCB patterns, and the dependence of the resulting patterns on the focused beam coordinate when the beam diameter is larger or smaller than a nanocrystal, or smaller than one unit cell. We show how structure factor phase information is provided at overlapping interfering orders and how a common phase origin between different shots may be obtained. Their use in refinement of the phase-sensitive intensity between overlapping orders is suggested. PMID:24914153

Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers

PubMed Central

Hattne, Johan; Echols, Nathaniel; Tran, Rosalie; Kern, Jan; Gildea, Richard J.; Brewster, Aaron S.; Alonso-Mori, Roberto; Glöckner, Carina; Hellmich, Julia; Laksmono, Hartawan; Sierra, Raymond G.; Lassalle-Kaiser, Benedikt; Lampe, Alyssa; Han, Guangye; Gul, Sheraz; DiFiore, Dörte; Milathianaki, Despina; Fry, Alan R.; Miahnahri, Alan; White, William E.; Schafer, Donald W.; Seibert, M. Marvin; Koglin, Jason E.; Sokaras, Dimosthenis; Weng, Tsu-Chien; Sellberg, Jonas; Latimer, Matthew J.; Glatzel, Pieter; Zwart, Petrus H.; Grosse-Kunstleve, Ralf W.; Bogan, Michael J.; Messerschmidt, Marc; Williams, Garth J.; Boutet, Sébastien; Messinger, Johannes; Zouni, Athina; Yano, Junko; Bergmann, Uwe; Yachandra, Vittal K.; Adams, Paul D.; Sauter, Nicholas K.

2014-01-01

X-ray free-electron laser (XFEL) sources enable the use of crystallography to solve three-dimensional macromolecular structures under native conditions and free from radiation damage. Results to date, however, have been limited by the challenge of deriving accurate Bragg intensities from a heterogeneous population of microcrystals, while at the same time modeling the X-ray spectrum and detector geometry. Here we present a computational approach designed to extract statistically significant high-resolution signals from fewer diffraction measurements. PMID:24633409

An Excel Spreadsheet for a One-Dimensional Fourier Map in X-ray Crystallography

ERIC Educational Resources Information Center

Clegg, William

2004-01-01

The teaching of crystal structure determination with single-crystal X-ray diffraction at undergraduate level faces numerous challenges. Single-crystal X-ray diffraction is used in a vast range of chemical research projects and forms the basis for a high proportion of structural results that are presented to high-school, undergraduate, and graduate…

Serial femtosecond X-ray diffraction of enveloped virus microcrystals

DOE PAGES

Lawrence, Robert M.; Conrad, Chelsie E.; Zatsepin, Nadia A.; ...

2015-08-20

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 a pertinent step toward determining room temperature structures from virus microcrystals that may enable time-resolved studies of enveloped viruses.

Protein Crystallography in Vaccine Research and Development.

PubMed

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

2015-06-09

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

Protein Crystallography in Vaccine Research and Development

PubMed Central

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

2015-01-01

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

High-pressure crystallography of periodic and aperiodic crystals

PubMed Central

Hejny, Clivia; Minkov, Vasily S.

2015-01-01

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

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

PubMed

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

2016-01-01

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

An Exercise in X-Ray Diffraction Using the Polymorphic Transition of Nickel Chromite.

ERIC Educational Resources Information Center

Chipman, David W.

1980-01-01

Describes a laboratory experiment appropriate for a course in either x-ray crystallography or mineralogy. The experiment permits the direct observation of a polymorphic transition in nickel chromite without the use of a special heating stage or heating camera. (Author/GS)

Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers.

PubMed

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.

Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fuller, Franklin D.; Gul, Sheraz; Chatterjee, Ruchira

X-ray crystallography at X-ray free-electron laser (XFEL) sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy (XES), both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing new insights into the interplay between the protein structure/dynamics and chemistry at an active site. However, implementing such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly impacts the data quality. We present here a new, robust way of delivering controlled sample amountsmore » 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.« less

Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers

DOE PAGES

Fuller, Franklin D.; Gul, Sheraz; Chatterjee, Ruchira; ...

2017-02-27

X-ray crystallography at X-ray free-electron laser (XFEL) sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy (XES), both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing new insights into the interplay between the protein structure/dynamics and chemistry at an active site. However, implementing such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly impacts the data quality. We present here a new, robust way of delivering controlled sample amountsmore » 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.« less

Mitigation of X-ray damage in macromolecular crystallography by submicrometre line focusing.

PubMed

Finfrock, Y Zou; Stern, Edward A; Alkire, R W; Kas, Joshua J; Evans-Lutterodt, Kenneth; Stein, Aaron; Duke, Norma; Lazarski, Krzysztof; Joachimiak, Andrzej

2013-08-01

Reported here are measurements of the penetration depth and spatial distribution of photoelectron (PE) damage excited by 18.6 keV X-ray photons in a lysozyme crystal with a vertical submicrometre line-focus beam of 0.7 µm full-width half-maximum (FWHM). The experimental results determined that the penetration depth of PEs is 5 ± 0.5 µm with a monotonically decreasing spatial distribution shape, resulting in mitigation of diffraction signal damage. This does not agree with previous theoretical predication that the mitigation of damage requires a peak of damage outside the focus. A new improved calculation provides some qualitative agreement with the experimental results, but significant errors still remain. The mitigation of radiation damage by line focusing was measured experimentally by comparing the damage in the X-ray-irradiated regions of the submicrometre focus with the large-beam case under conditions of equal exposure and equal volumes of the protein crystal, and a mitigation factor of 4.4 ± 0.4 was determined. The mitigation of radiation damage is caused by spatial separation of the dominant PE radiation-damage component from the crystal region of the line-focus beam that contributes the diffraction signal. The diffraction signal is generated by coherent scattering of incident X-rays (which introduces no damage), while the overwhelming proportion of damage is caused by PE emission as X-ray photons are absorbed.

Fast fluorescence techniques for crystallography beamlines

PubMed Central

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

2011-01-01

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

Single-crystal Raman spectroscopy and X-ray crystallography at beamline X26-C of the NSLS

PubMed Central

Stoner-Ma, Deborah; Skinner, John M.; Schneider, Dieter K.; Cowan, Matt; Sweet, Robert M.; Orville, Allen M.

2011-01-01

Three-dimensional structures derived from X-ray diffraction of protein crystals provide a wealth of information. Features and interactions important for the function of macromolecules can be deduced and catalytic mechanisms postulated. Still, many questions can remain, for example regarding metal oxidation states and the interpretation of ‘mystery density’, i.e. ambiguous or unknown features within the electron density maps, especially at ∼2 Å resolutions typical of most macromolecular structures. Beamline X26-C at the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory (BNL), provides researchers with the opportunity to not only determine the atomic structure of their samples but also to explore the electronic and vibrational characteristics of the sample before, during and after X-ray diffraction data collection. When samples are maintained under cryo-conditions, an opportunity to promote and follow photochemical reactions in situ as a function of X-ray exposure is also provided. Plans are in place to further expand the capabilities at beamline X26-C and to develop beamlines at NSLS-II, currently under construction at BNL, which will provide users access to a wide array of complementary spectroscopic methods in addition to high-quality X-ray diffraction data. PMID:21169688

New developments in crystallography: exploring its technology, methods and scope in the molecular biosciences.

PubMed

Helliwell, John R

2017-08-31

Since the Protein Data Bank (PDB) was founded in 1971, there are now over 120,000 depositions, the majority of which are from X-ray crystallography and 90% of those made use of synchrotron beamlines. At the Cambridge Structure Database (CSD), founded in 1965, there are more than 800,000 'small molecule' crystal structure depositions and a very large number of those are relevant in the biosciences as ligands or cofactors. The technology for crystal structure analysis is still developing rapidly both at synchrotrons and in home labs. Determination of the details of the hydrogen atoms in biological macromolecules is well served using neutrons as probe. Large multi-macromolecular complexes cause major challenges to crystallization; electrons as probes offer unique advantages here. Methods developments naturally accompany technology change, mainly incremental but some, such as the tuneability, intensity and collimation of synchrotron radiation, have effected radical changes in capability of biological crystallography. In the past few years, the X-ray laser has taken X-ray crystallography measurement times into the femtosecond range. In terms of applications many new discoveries have been made in the molecular biosciences. The scope of crystallographic techniques is indeed very wide. As examples, new insights into chemical catalysis of enzymes and relating ligand bound structures to thermodynamics have been gained but predictive power is seen as not yet achieved. Metal complexes are also an emerging theme for biomedicine applications. Our studies of coloration of live and cooked lobsters proved to be an unexpected favourite with the public and schoolchildren. More generally, public understanding of the biosciences and crystallography's role within the field have been greatly enhanced by the United Nations International Year of Crystallography coordinated by the International Union of Crystallography. This topical review describes each of these areas along with

Structural insights into binding of inhibitors to soluble epoxide hydrolase gained by fragment screening and X-ray crystallography.

PubMed

Amano, Yasushi; Yamaguchi, Tomohiko; Tanabe, Eiki

2014-04-15

Soluble epoxide hydrolase (sEH) is a component of the arachidonic acid cascade and is a candidate target for therapies for hypertension or inflammation. Although many sEH inhibitors are available, their scaffolds are not structurally diverse, and knowledge of their specific interactions with sEH is limited. To obtain detailed structural information about protein-ligand interactions, we conducted fragment screening of sEH, analyzed the fragments using high-throughput X-ray crystallography, and determined 126 fragment-bound structures at high resolution. Aminothiazole and benzimidazole derivatives were identified as novel scaffolds that bind to the catalytic triad of sEH with good ligand efficiency. We further identified fragment hits that bound to subpockets of sEH called the short and long branches. The water molecule conserved in the structure plays an important role in binding to the long branch, whereas Asp496 and the main chain of Phe497 form hydrogen bonds with fragment hits in the short branch. Fragment hits and their crystal structures provide structural insights into ligand binding to sEH that will facilitate the discovery of novel and potent inhibitors of sEH. Copyright © 2014 Elsevier Ltd. All rights reserved.

Parabolic single-crystal diamond compound refractive lenses for coherent x-ray imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Terentyev, Sergey; Blank, Vladimir D.; Polyakov, Sergey; Zholudev, Sergey; Snigirev, Anatoly A.; Polikarpov, Maxim; Kolodziej, Tomasz; Qian, Jun; Zhou, Hua; Shvyd'ko, Yuri V.

2016-09-01

We demonstrate parabolic single-crystal diamond compound refractive lenses [1] designed for coherent x-ray imaging resilient to extreme thermal and radiation loading expected from next generation light sources. To ensure the preservation of coherence and resilience, the lenses are manufactured from the highest-quality single-crystalline synthetic diamond material grown by a high-pressure high-temperature technique. Picosecond laser milling is applied to machine lenses to parabolic shapes with a 1-micron precision and surface roughness. A compound refractive lens comprised of six lenses with a radius of curvature R=200 microns at the vertex of the parabola and a geometrical aperture A=900 microns focuses 10 keV x-ray photons from an undulator source at the Advanced Photon Source facility to a focal spot size of 10x40 microns^2 with a gain factor of 100. [1] S. Terentyev, V. Blank, S. Polyakov, S. Zholudev, A. Snigirev, M. Polikarpov, T. Kolodziej, J. Qian, H. Zhou, and Yu. Shvyd'ko Applied Physics Letters 107, 111108 (2015); doi: 10.1063/1.4931357

High-throughput methods for electron crystallography.

PubMed

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

2013-01-01

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

Focusing X-ray free-electron laser pulses using Kirkpatrick-Baez mirrors at the NCI hutch of the PAL-XFEL.

PubMed

Kim, Jangwoo; Kim, Hyo Yun; Park, Jaehyun; Kim, Sangsoo; Kim, Sunam; Rah, Seungyu; Lim, Jun; Nam, Ki Hyun

2018-01-01

The Pohang Accelerator Laboratory X-ray Free-Electron Laser (PAL-XFEL) is a recently commissioned X-ray free-electron laser (XFEL) facility that provides intense ultrashort X-ray pulses based on the self-amplified spontaneous emission process. The nano-crystallography and coherent imaging (NCI) hutch with forward-scattering geometry is located at the hard X-ray beamline of the PAL-XFEL and provides opportunities to perform serial femtosecond crystallography and coherent X-ray diffraction imaging. To produce intense high-density XFEL pulses at the interaction positions between the X-rays and various samples, a microfocusing Kirkpatrick-Baez (KB) mirror system that includes an ultra-precision manipulator has been developed. In this paper, the design of a KB mirror system that focuses the hard XFEL beam onto a fixed sample point of the NCI hutch, which is positioned along the hard XFEL beamline, is described. The focusing system produces a two-dimensional focusing beam at approximately 2 µm scale across the 2-11 keV photon energy range. XFEL pulses of 9.7 keV energy were successfully focused onto an area of size 1.94 µm × 2.08 µm FWHM.

Using Two-Dimensional Colloidal Crystals to Understand Crystallography

ERIC Educational Resources Information Center

Bosse, Stephanie A.; Loening, Nikolaus M.

2008-01-01

X-ray crystallography is an essential technique for modern chemistry and biochemistry, but it is infrequently encountered by undergraduate students owing to lack of access to equipment, the time-scale for generating diffraction-quality molecular crystals, and the level of mathematics involved in analyzing the resulting diffraction patterns.…

SIBYLS - A SAXS and protein crystallography beamline at the ALS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trame, Christine; MacDowell, Alastair A.; Celestre, Richard S.

2003-08-22

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

SIBYLS - a SAXS and Protein Crystallography Beamline at the ALS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trame, C.; MacDowell, A.A.; Celestre, R.S.

2004-05-12

The new Structurally Integrated BiologY for Life Sciences (SIBYLS) beamline at the Advanced Light Source will be dedicated to Macromolecular Crystallography (PX) and Small Angle X-ray Scattering (SAXS). SAXS will provide structural information of macromolecules in solutions and will complement high resolution PX studies on the same systems but in a crystalline state. The x-ray source is one of the 5 Tesla superbend dipoles recently installed at the ALS that allows for a hard x-ray program to be developed on the relatively low energy Advanced Light Source (ALS) ring (1.9 GeV). The beamline is equipped with fast interchangeable monochromator elements,more » consisting of either a pair of single Si(111) crystals for crystallography, or a pair of multilayers for the SAXS mode data collection (E/{delta}E{approx}1/110). Flux rates with Si(111) crystals for PX are measured as 2x1011 hv/sec through a 100{mu}m pinhole at 12.4KeV. For SAXS the flux is up to 3x1013photons/sec at 10KeV with all apertures open when using the multilayer monochromator elements. The performance characteristics of this unique beamline will be described.« less

Single Crystal X-ray Study of 6-Phenyl-4-( p-tolyl)pyridin-2(1 H)-one

NASA Astrophysics Data System (ADS)

Khajuria, Rajni; Sharma, Suresh; Kapoor, Kamal K.; Gupta, Vivek K.

2017-12-01

The title compound 6-phenyl-4-( p-tolyl)pyridin-2(1 H)-one was synthesized via one-pot, three component reaction of ( E)-1-phenyl-3-( p-tolyl)-2-propen-1-one, ethyl 2-nitroacetate and ammonium acetate in refluxing ethanol, as a shiny green crystalline solid in 83% yield. Its structure was characterized by spectral studies and unambiguously corroborated by X-ray diffraction crystallography. The crystals of title compound are monoclinic, sp. gr. P21/ n, a = 11.8346(7) Å, b = 13.4413(9) Å, c = 17.7626(10) Å, β = 99.479(5)°, and Z = 8. All the rings in molecule of the title compound are planar. Hydrogen interactions play significant role in stabilizing the crystal structure and the supramolecular aggregate of molecules is facilitated by strong N-H···O and C-H···O type of hydrogen interactions.

Emerging opportunities in structural biology with X-ray free-electron lasers

PubMed Central

Schlichting, Ilme; Miao, Jianwei

2012-01-01

X-ray free-electron lasers (X-FELs) produce X-ray pulses with extremely brilliant peak intensity and ultrashort pulse duration. It has been proposed that radiation damage can be “outrun” by using an ultra intense and short X-FEL pulse that passes a biological sample before the onset of significant radiation damage. The concept of “diffraction-before-destruction” has been demonstrated recently at the Linac Coherent Light Source, the first operational hard X-ray FEL, for protein nanocrystals and giant virus particles. The continuous diffraction patterns from single particles allow solving the classical “phase problem” by the oversampling method with iterative algorithms. If enough data are collected from many identical copies of a (biological) particle, its three-dimensional structure can be reconstructed. We review the current status and future prospects of serial femtosecond crystallography (SFX) and single-particle coherent diffraction imaging (CDI) with X-FELs. PMID:22922042

Neutron Crystallography for the Study of Hydrogen Bonds in Macromolecules.

PubMed

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

2017-04-07

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

Neutron crystallography for the study of hydrogen bonds in macromolecules

DOE PAGES

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

2017-04-07

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

Neutron crystallography for the study of hydrogen bonds in macromolecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

Towards phasing using high X-ray intensity

DOE PAGES

Galli, Lorenzo; Son, Sang -Kil; Barends, Thomas R. M.; ...

2015-09-30

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 schememore » 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.« less

NMR Crystallography of Enzyme Active Sites: Probing Chemically-Detailed, Three-Dimensional Structure in Tryptophan Synthase

PubMed Central

Dunn, Michael F.

2013-01-01

Conspectus NMR crystallography – the synergistic combination of X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry – offers unprecedented insight into three-dimensional, chemically-detailed structure. From its initial role in refining diffraction data of organic and inorganic solids, NMR crystallography is now being developed for application to active sites in biomolecules, where it reveals chemically-rich detail concerning the interactions between enzyme site residues and the reacting substrate that is not achievable when X-ray, NMR, or computational methodologies are applied in isolation. For example, typical X-ray crystal structures (1.5 to 2.5 Å resolution) of enzyme-bound intermediates identify possible hydrogen-bonding interactions between site residues and substrate, but do not directly identify the protonation state of either. Solid-state NMR can provide chemical shifts for selected atoms of enzyme-substrate complexes, but without a larger structural framework in which to interpret them, only empirical correlations with local chemical structure are possible. Ab initio calculations and molecular mechanics can build models for enzymatic processes, but rely on chemical details that must be specified. Together, however, X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry can provide consistent and testable models for structure and function of enzyme active sites: X-ray crystallography provides a coarse framework upon which models of the active site can be developed using computational chemistry; these models can be distinguished by comparison of their calculated NMR chemical shifts with the results of solid-state NMR spectroscopy experiments. Conceptually, each technique is a puzzle piece offering a generous view of the big picture. Only when correctly pieced together, however, can they reveal the big picture at highest resolution. In this Account, we detail our first steps in the development of NMR

Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishigami, Izumi; Zatsepin, Nadia A.; Hikita, Masahide

Here, cytochrome c oxidase (C cO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine C cO (bC cO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-raymore » crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe–C–O angle of ~142°. In contrast, in the synchrotron structure, the Fe–CO bond is cleaved; CO relocates to a new site near Cu B, which, in turn, moves closer to the heme a 3 iron by ~0.38 Å. Structural comparison reveals that ligand binding to the heme a 3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a 3, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.« less

Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature

DOE PAGES

Ishigami, Izumi; Zatsepin, Nadia A.; Hikita, Masahide; ...

2017-07-11

Here, cytochrome c oxidase (C cO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine C cO (bC cO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-raymore » crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe–C–O angle of ~142°. In contrast, in the synchrotron structure, the Fe–CO bond is cleaved; CO relocates to a new site near Cu B, which, in turn, moves closer to the heme a 3 iron by ~0.38 Å. Structural comparison reveals that ligand binding to the heme a 3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a 3, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.« less

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-03-17

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

EIGER detector: application in macromolecular crystallography.

PubMed

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

2016-09-01

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

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

PubMed

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

2015-09-01

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

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

PubMed Central

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

2015-01-01

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

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

DOE PAGES

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

2016-10-19

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edlund, Petra; Takala, Heikki; Claesson, Elin

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

High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography

PubMed Central

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

2013-01-01

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

Three-dimensional imaging of nanoscale materials by using coherent x-rays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miao, Jianwei

X-ray crystallography is currently the primary methodology used to determine the 3D structure of materials and macromolecules. However, many nanostructures, disordered materials, biomaterials, hybrid materials and biological specimens are noncrystalline and, hence, their structures are not accessible by X-ray crystallography. Probing these structures therefore requires the employment of different approaches. A very promising technique currently under rapid development is X-ray diffraction microscopy (or lensless imaging), in which the coherent X-ray diffraction pattern of a noncrystalline specimen is measured and then directly phased to obtain a high-resolution image. Through the DOE support over the past three years, we have applied X-raymore » diffraction microscopy to quantitative imaging of GaN quantum dot particles, and revealed the internal GaN-Ga2O3 core shell structure in three dimensions. By exploiting the abrupt change in the scattering cross-section near electronic resonances, we carried out the first experimental demonstration of resonant X-ray diffraction microscopy for element specific imaging. We performed nondestructive and quantitative imaging of buried Bi structures inside a Si crystal by directly phasing coherent X-ray diffraction patterns acquired below and above the Bi M5 edge. We have also applied X-ray diffraction microscopy to nondestructive imaging of mineral crystals inside biological composite materials - intramuscular fish bone - at the nanometer scale resolution. We identified mineral crystals in collagen fibrils at different stages of mineralization and proposed a dynamic mechanism to account for the nucleation and growth of mineral crystals in the collagen matrix. In addition, we have also discovered a novel 3D imaging modality, denoted ankylography, which allows for complete 3D structure determination without the necessity of sample titling or scanning. We showed that when the diffraction pattern of a finite object is sampled at a

Selenium single-wavelength anomalous diffraction de novo phasing using an X-ray-free electron laser

DOE PAGES

Hunter, Mark S.; Yoon, Chun Hong; DeMirci, Hasan; ...

2016-11-04

Structural information about biological macromolecules near the atomic scale provides important insight into the functions of these molecules. To date, X-ray crystallography has been the predominant method used for macromolecular structure determination. However, challenges exist when solving structures with X-rays, including the phase problem and radiation damage. X-ray-free electron lasers (X-ray FELs) have enabled collection of diffraction information before the onset of radiation damage, yet the majority of structures solved at X-ray FELs have been phased using external information via molecular replacement. De novo phasing at X-ray FELs has proven challenging due in part to per-pulse variations in intensity andmore » wavelength. Here we report the solution of a selenobiotinyl-streptavidin structure using phases obtained by the anomalous diffraction of selenium measured at a single wavelength (Se-SAD) at the Linac Coherent Light Source. Finally, our results demonstrate Se-SAD, routinely employed at synchrotrons for novel structure determination, is now possible at X-ray FELs.« less

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

PubMed

Roessler, Christian G; Agarwal, Rakhi; Allaire, Marc; Alonso-Mori, Roberto; Andi, Babak; Bachega, José F R; Bommer, Martin; Brewster, Aaron S; Browne, Michael C; Chatterjee, Ruchira; Cho, Eunsun; Cohen, Aina E; Cowan, Matthew; Datwani, Sammy; Davidson, Victor L; Defever, Jim; Eaton, Brent; Ellson, Richard; Feng, Yiping; Ghislain, Lucien P; Glownia, James M; Han, Guangye; Hattne, Johan; Hellmich, Julia; Héroux, Annie; Ibrahim, Mohamed; Kern, Jan; Kuczewski, Anthony; Lemke, Henrik T; Liu, Pinghua; Majlof, Lars; McClintock, William M; Myers, Stuart; Nelsen, Silke; Olechno, Joe; Orville, Allen M; Sauter, Nicholas K; Soares, Alexei S; Soltis, S Michael; Song, Heng; Stearns, Richard G; Tran, Rosalie; Tsai, Yingssu; Uervirojnangkoorn, Monarin; Wilmot, Carrie M; Yachandra, Vittal; Yano, Junko; Yukl, Erik T; Zhu, Diling; Zouni, Athina

2016-04-05

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography

DOE PAGES

Roessler, Christian G.; Agarwal, Rakhi; Allaire, Marc; ...

2016-03-17

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallizationmore » conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. Lastly, we report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). In addition, samples were screened to demonstrate that these methods can be applied to rare samples« less

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roessler, Christian G.; Agarwal, Rakhi; Allaire, Marc

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallizationmore » conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.« less

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roessler, Christian G.; Agarwal, Rakhi; Allaire, Marc

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallizationmore » conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. Lastly, we report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). In addition, samples were screened to demonstrate that these methods can be applied to rare samples« less

NMR crystallography of 2-acylamino-6-[1 H]-pyridones: Solid-state NMR, GIPAW computational, and single crystal X-ray diffraction studies

NASA Astrophysics Data System (ADS)

Ośmiałowski, Borys; Kolehmainen, Erkki; Ikonen, Satu; Ahonen, Kari; Löfman, Miika

2011-12-01

2-Acylamino-6-[1 H]-pyridones [acyl = RCO, where R = methyl ( 1), ethyl ( 2), iso-propyl ( 3), tert-butyl ( 4), and 1-adamantyl ( 5)] have been synthesized and characterized by NMR spectroscopy. From three congeners, 2, 3 and 5, also single crystal X-ray structures have been solved. For these derivatives GIPAW calculations acts as a "bridge" between solid-state NMR data and calculated chemical shifts based on X-ray determined geometry. In crystals all three compounds exist as pyridone tautomers possessing similar six-membered ring structure stabilized by intramolecular C dbnd O⋯HN hydrogen bond. Theoretical GIPAW calculated and experimental 13C and 15N CPMAS NMR shifts are in excellent agreement with each other.

A Newly Designed Microspectrofluorometer for Kinetic Studies on Protein Crystals in Combination with X-Ray Diffraction

PubMed Central

Klink, Björn U.; Goody, Roger S.; Scheidig, Axel J.

2006-01-01

We present a new design for a fluorescence microspectrophotometer for use in kinetic crystallography in combination with x-ray diffraction experiments. The FLUMIX device (Fluorescence spectroscopy to monitor intermediates in x-ray crystallography) is built for 0° fluorescence detection, which has several advantages in comparison to a conventional fluorometer with 90° design. Due to the reduced spatial requirements and the need for only one objective, the system is highly versatile, easy to handle, and can be used for many different applications. In combination with a conventional stereomicroscope, fluorescence measurements or reaction initiation can be performed directly in a hanging drop crystallization setup. The FLUMIX device can be combined with most x-ray sources, normally without the need of a specialized mechanical support. As a biological model system, we have used H-Ras p21 with an artificially introduced photo-labile GTP precursor (caged GTP) and a covalently attached fluorophore (IANBD amide). Using the FLUMIX system, detailed information about the state of photolyzed crystals of the modified H-Ras p21 (p21(mod)) could be obtained. Measurements in combination with a synchrotron beamline showed significant fluorescence changes in p21(mod) crystals even within a few seconds of x-ray exposure at 100 K. PMID:16698776

Compound focusing mirror and X-ray waveguide optics for coherent imaging and nano-diffraction.

PubMed

Salditt, Tim; Osterhoff, Markus; Krenkel, Martin; Wilke, Robin N; Priebe, Marius; Bartels, Matthias; Kalbfleisch, Sebastian; Sprung, Michael

2015-07-01

A compound optical system for coherent focusing and imaging at the nanoscale is reported, realised by high-gain fixed-curvature elliptical mirrors in combination with X-ray waveguide optics or different cleaning apertures. The key optical concepts are illustrated, as implemented at the Göttingen Instrument for Nano-Imaging with X-rays (GINIX), installed at the P10 coherence beamline of the PETRA III storage ring at DESY, Hamburg, and examples for typical applications in biological imaging are given. Characteristic beam configurations with the recently achieved values are also described, meeting the different requirements of the applications, such as spot size, coherence or bandwidth. The emphasis of this work is on the different beam shaping, filtering and characterization methods.

Insights into photosystem II from isomorphous difference Fourier maps of femtosecond X-ray diffraction data and quantum mechanics/molecular mechanics structural models

DOE PAGES

Wang, Jimin; Askerka, Mikhail; Brudvig, Gary W.; ...

2017-01-12

Understanding structure–function relations in photosystem II (PSII) is important for the development of biomimetic photocatalytic systems. X-ray crystallography, computational modeling, and spectroscopy have played central roles in elucidating the structure and function of PSII. Recent breakthroughs in femtosecond X-ray crystallography offer the possibility of collecting diffraction data from the X-ray free electron laser (XFEL) before radiation damage of the sample, thereby overcoming the main challenge of conventional X-ray diffraction methods. However, the interpretation of XFEL data from PSII intermediates is challenging because of the issues regarding data-processing, uncertainty on the precise positions of light oxygen atoms next to heavy metalmore » centers, and different kinetics of the S-state transition in microcrystals compared to solution. Lastly, we summarize recent advances and outstanding challenges in PSII structure–function determination with emphasis on the implementation of quantum mechanics/molecular mechanics techniques combined with isomorphous difference Fourier maps, direct methods, and high-resolution spectroscopy.« less

Insights into Photosystem II from Isomorphous Difference Fourier Maps of Femtosecond X-ray Diffraction Data and Quantum Mechanics/Molecular Mechanics Structural Models.

PubMed

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

2017-02-10

Understanding structure-function relations in photosystem II (PSII) is important for the development of biomimetic photocatalytic systems. X-ray crystallography, computational modeling, and spectroscopy have played central roles in elucidating the structure and function of PSII. Recent breakthroughs in femtosecond X-ray crystallography offer the possibility of collecting diffraction data from the X-ray free electron laser (XFEL) before radiation damage of the sample, thereby overcoming the main challenge of conventional X-ray diffraction methods. However, the interpretation of XFEL data from PSII intermediates is challenging because of the issues regarding data-processing, uncertainty on the precise positions of light oxygen atoms next to heavy metal centers, and different kinetics of the S-state transition in microcrystals compared to solution. Here, we summarize recent advances and outstanding challenges in PSII structure-function determination with emphasis on the implementation of quantum mechanics/molecular mechanics techniques combined with isomorphous difference Fourier maps, direct methods, and high-resolution spectroscopy.

Insights into photosystem II from isomorphous difference Fourier maps of femtosecond X-ray diffraction data and quantum mechanics/molecular mechanics structural models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Jimin; Askerka, Mikhail; Brudvig, Gary W.

Understanding structure–function relations in photosystem II (PSII) is important for the development of biomimetic photocatalytic systems. X-ray crystallography, computational modeling, and spectroscopy have played central roles in elucidating the structure and function of PSII. Recent breakthroughs in femtosecond X-ray crystallography offer the possibility of collecting diffraction data from the X-ray free electron laser (XFEL) before radiation damage of the sample, thereby overcoming the main challenge of conventional X-ray diffraction methods. However, the interpretation of XFEL data from PSII intermediates is challenging because of the issues regarding data-processing, uncertainty on the precise positions of light oxygen atoms next to heavy metalmore » centers, and different kinetics of the S-state transition in microcrystals compared to solution. Lastly, we summarize recent advances and outstanding challenges in PSII structure–function determination with emphasis on the implementation of quantum mechanics/molecular mechanics techniques combined with isomorphous difference Fourier maps, direct methods, and high-resolution spectroscopy.« less

Noninvasive Synchrotron-Based X-ray Raman Scattering Discriminates Carbonaceous Compounds in Ancient and Historical Materials [ In situ synchrotron-based X-Ray Raman scattering discriminates carbonaceous compounds in ancient and historical materials

DOE PAGES

Gueriau, Pierre; Rueff, Jean -Pascal; Bernard, Sylvain; ...

2017-09-13

Carbon compounds are ubiquitous and occur in a diversity of chemical forms in many systems including ancient and historic materials ranging from cultural heritage to paleontology. Determining their speciation cannot only provide unique information on their origin but may also elucidate degradation processes. Synchrotron-based X-ray absorption near-edge structure (XANES) spectroscopy at the carbon K-edge (280–350 eV) is a very powerful method to probe carbon speciation. However, the short penetration depth of soft X-rays imposes stringent constraints on sample type, preparation, and analytical environment. A hard X-ray probe such as X-ray Raman scattering (XRS) can overcome many of these difficulties. Heremore » we report the use of XRS at ~6 keV incident energy to collect carbon K-edge XANES data and probe the speciation of organic carbon in several specimens relevant to cultural heritage and natural history. This methodology enables the measurement to be done in a nondestructive way, in air, and provides information that is not compromised by surface contamination by ensuring that the dominant signal contribution is from the bulk of the probed material. Using the backscattering geometry at large photon momentum transfer maximizes the XRS signal at the given X-ray energy and enhances nondipole contributions compared to conventional XANES, thereby augmenting the speciation sensitivity. The capabilities and limitations of the technique are discussed. As a result, we show that despite its small cross section, for a range of systems the XRS method can provide satisfactory signals at realistic experimental conditions. XRS constitutes a powerful complement to FT-IR, Raman, and conventional XANES spectroscopy, overcoming some of the limitations of these techniques.« less

Incoherent Diffractive Imaging via Intensity Correlations of Hard X Rays

NASA Astrophysics Data System (ADS)

Classen, Anton; Ayyer, Kartik; Chapman, Henry N.; Röhlsberger, Ralf; von Zanthier, Joachim

2017-08-01

Established x-ray diffraction methods allow for high-resolution structure determination of crystals, crystallized protein structures, or even single molecules. While these techniques rely on coherent scattering, incoherent processes like fluorescence emission—often the predominant scattering mechanism—are generally considered detrimental for imaging applications. Here, we show that intensity correlations of incoherently scattered x-ray radiation can be used to image the full 3D arrangement of the scattering atoms with significantly higher resolution compared to conventional coherent diffraction imaging and crystallography, including additional three-dimensional information in Fourier space for a single sample orientation. We present a number of properties of incoherent diffractive imaging that are conceptually superior to those of coherent methods.

Preparation and reactivity of the heterobimetallic ReIr face-shared bioctahedral compounds Cp*Ir(mu-Cl)(3)Re(CO)(3) and Cp*Ir(mu-SC6H4Me-4)(3)Re(CO)(3): X-ray diffraction structures and redox behavior

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xiaoping; Hammons, Casey; Richmond, Michael G.

2009-01-01

Thermolysis of the dinuclear compound [Cp*IrCl2](2) (1) with ClRe(CO)(5) (2) leads to the formation of the confacial bioctahedral compound Cp*Ir(mu-Cl)(3)Re(CO)(3) (3) in high yield. Whereas the substitution of the chloride ligands in 3 is observed on treatment with excess p-methylbenzenethiol to furnish the sulfido-bridged compound Cp*Ir(mu-SC6H4Me-4)(3)Re(CO)(3) (4), 3 undergoes fragmentation upon reaction with tertiary phosphines [PPh3 and P(OMe)(3)] to furnish the mononuclear compounds CP*IrCl2P and fac-ClRe-(CO3)P-2. Both 3 and 4 have been isolated and fully characterized in solution by IR and H-1 NMR spectroscopies, and their solid-state structures have been established by X-ray crystallography. The redox properties of 3 andmore » 4 have been explored by cyclic voltammetry, and the results are discussed relative to extended Huckel MO calculations.« less

Native sulfur/chlorine SAD phasing for serial femtosecond crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakane, Takanori; Song, Changyong; POSTECH, Pohang 790-784

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.

Protein crystal structure from non-oriented, single-axis sparse X-ray data

DOE PAGES

Wierman, Jennifer L.; Lan, Ti-Yen; Tate, Mark W.; ...

2016-01-01

X-ray free-electron lasers (XFELs) have inspired the development of serial femtosecond crystallography (SFX) as a method to solve the structure of proteins. SFX datasets are collected from a sequence of protein microcrystals injected across ultrashort X-ray pulses. The idea behind SFX is that diffraction from the intense, ultrashort X-ray pulses leaves the crystal before the crystal is obliterated by the effects of the X-ray pulse. The success of SFX at XFELs has catalyzed interest in analogous experiments at synchrotron-radiation (SR) sources, where data are collected from many small crystals and the ultrashort pulses are replaced by exposure times that aremore » kept short enough to avoid significant crystal damage. The diffraction signal from each short exposure is so `sparse' in recorded photons that the process of recording the crystal intensity is itself a reconstruction problem. Using theEMCalgorithm, a successful reconstruction is demonstrated here in a sparsity regime where there are no Bragg peaks that conventionally would serve to determine the orientation of the crystal in each exposure. In this proof-of-principle experiment, a hen egg-white lysozyme (HEWL) crystal rotating about a single axis was illuminated by an X-ray beam from an X-ray generator to simulate the diffraction patterns of microcrystals from synchrotron radiation. Millions of these sparse frames, typically containing only ~200 photons per frame, were recorded using a fast-framing detector. It is shown that reconstruction of three-dimensional diffraction intensity is possible using theEMCalgorithm, even with these extremely sparse frames and without knowledge of the rotation angle. Further, the reconstructed intensity can be phased and refined to solve the protein structure using traditional crystallographic software. In conclusion, this suggests that synchrotron-based serial crystallography of micrometre-sized crystals can be practical with the aid of theEMCalgorithm even in cases

Protein crystal structure from non-oriented, single-axis sparse X-ray data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wierman, Jennifer L.; Lan, Ti-Yen; Tate, Mark W.

X-ray free-electron lasers (XFELs) have inspired the development of serial femtosecond crystallography (SFX) as a method to solve the structure of proteins. SFX datasets are collected from a sequence of protein microcrystals injected across ultrashort X-ray pulses. The idea behind SFX is that diffraction from the intense, ultrashort X-ray pulses leaves the crystal before the crystal is obliterated by the effects of the X-ray pulse. The success of SFX at XFELs has catalyzed interest in analogous experiments at synchrotron-radiation (SR) sources, where data are collected from many small crystals and the ultrashort pulses are replaced by exposure times that aremore » kept short enough to avoid significant crystal damage. The diffraction signal from each short exposure is so `sparse' in recorded photons that the process of recording the crystal intensity is itself a reconstruction problem. Using theEMCalgorithm, a successful reconstruction is demonstrated here in a sparsity regime where there are no Bragg peaks that conventionally would serve to determine the orientation of the crystal in each exposure. In this proof-of-principle experiment, a hen egg-white lysozyme (HEWL) crystal rotating about a single axis was illuminated by an X-ray beam from an X-ray generator to simulate the diffraction patterns of microcrystals from synchrotron radiation. Millions of these sparse frames, typically containing only ~200 photons per frame, were recorded using a fast-framing detector. It is shown that reconstruction of three-dimensional diffraction intensity is possible using theEMCalgorithm, even with these extremely sparse frames and without knowledge of the rotation angle. Further, the reconstructed intensity can be phased and refined to solve the protein structure using traditional crystallographic software. In conclusion, this suggests that synchrotron-based serial crystallography of micrometre-sized crystals can be practical with the aid of theEMCalgorithm even in cases

Effective aperture of X-ray compound refractive lenses.

PubMed

Kohn, V G

2017-05-01

A new definition of the effective aperture of the X-ray compound refractive lens (CRL) is proposed. Both linear (one-dimensional) and circular (two-dimensional) CRLs are considered. It is shown that for a strongly absorbing CRL the real aperture does not influence the focusing properties and the effective aperture is determined by absorption. However, there are three ways to determine the effective aperture in terms of transparent CRLs. In the papers by Kohn [(2002). JETP Lett. 76, 600-603; (2003). J. Exp. Theor. Phys. 97, 204-215; (2009). J. Surface Investig. 3, 358-364; (2012). J. Synchrotron Rad. 19, 84-92; Kohn et al. (2003). Opt. Commun. 216, 247-260; (2003). J. Phys. IV Fr, 104, 217-220], the FWHM of the X-ray beam intensity just behind the CRL was used. In the papers by Lengeler et al. [(1999). J. Synchrotron Rad. 6, 1153-1167; (1998). J. Appl. Phys. 84, 5855-5861], the maximum intensity value at the focus was used. Numerically, these two definitions differ by 50%. The new definition is based on the integral intensity of the beam behind the CRL over the real aperture. The integral intensity is the most physical value and is independent of distance. The new definition gives a value that is greater than that of the Kohn definition by 6% and less than that of the Lengeler definition by 41%. A new approximation for the aperture function of a two-dimensional CRL is proposed which allows one to calculate the two-dimensional CRL through the one-dimensional CRL and to obtain an analytical solution for a complex system of many CRLs.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

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

DOE PAGES

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

2015-08-18

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

Future directions of electron crystallography.

PubMed

Fujiyoshi, Yoshinori

2013-01-01

In biological science, there are still many interesting and fundamental yet difficult questions, such as those in neuroscience, remaining to be answered. Structural and functional studies of membrane proteins, which are key molecules of signal transduction in neural and other cells, are essential for understanding the molecular mechanisms of many fundamental biological processes. Technological and instrumental advancements of electron microscopy have facilitated comprehension of structural studies of biological components, such as membrane proteins. While X-ray crystallography has been the main method of structure analysis of proteins including membrane proteins, electron crystallography is now an established technique to analyze structures of membrane proteins in the lipid bilayer, which is close to their natural biological environment. By utilizing cryo-electron microscopes with helium-cooled specimen stages, structures of membrane proteins were analyzed at a resolution better than 3 Å. Such high-resolution structural analysis of membrane proteins by electron crystallography opens up the new research field of structural physiology. Considering the fact that the structures of integral membrane proteins in their native membrane environment without artifacts from crystal contacts are critical in understanding their physiological functions, electron crystallography will continue to be an important technology for structural analysis. In this chapter, I will present several examples to highlight important advantages and to suggest future directions of this technique.

Serial Femtosecond Crystallography of G Protein-Coupled Receptors

PubMed Central

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

2014-01-01

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

X-ray K-edge absorption spectra of Fe minerals and model compounds: II. EXAFS

NASA Astrophysics Data System (ADS)

Waychunas, Glenn A.; Brown, Gordon E.; Apted, Michael J.

1986-01-01

K-edge extended X-ray absorption fine structure (EXAFS) spectra of Fe in varying environments in a suite of well-characterized silicate and oxide minerals were collected using synchrotron radiation and analyzed using single scattering approximation theory to yield nearest neighbor Fe-O distances and coordination numbers. The partial inverse character of synthetic hercynite spinal was verified in this way. Comparison of the results from all samples with structural data from X-ray diffraction crystal structure refinements indicates that EXAFS-derived first neighbor distances are generally accurate to ±0.02 Å using only theoretically generated phase information, and may be improved over this if similar model compounds are used to determine EXAFS phase functions. Coordination numbers are accurate to ±20 percent and can be similarly improved using model compound EXAFS amplitude information. However, in particular cases the EXAFS-derived distances may be shortened, and the coordination number reduced, by the effects of static and thermal disorder or by partial overlap of the longer Fe-O first neighbor distances with second neighbor distances in the EXAFS structure function. In the former case the total information available in the EXAFS is limited by the disorder, while in the latter case more accurate results can in principle be obtained by multiple neighbor EXAFS analysis. The EXAFS and XANES spectra of Fe in Nain, Labrador osumulite and Lakeview, Oregon plagioclase are also analyzed as an example of the application of X-ray absorption spectroscopy to metal ion site occupation determination in minerals.

Phase Equilibria and Crystallography of Ceramic Oxides

PubMed Central

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

2001-01-01

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

Serial femtosecond crystallography: A revolution in structural biology.

PubMed

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

2016-07-15

Macromolecular crystallography at synchrotron sources has proven to be the most influential method within structural biology, producing thousands of structures since its inception. While its utility has been instrumental in progressing our knowledge of structures of molecules, it suffers from limitations such as the need for large, well-diffracting crystals, and radiation damage that can hamper native structural determination. The recent advent of X-ray free electron lasers (XFELs) and their implementation in the emerging field of serial femtosecond crystallography (SFX) has given rise to a remarkable expansion upon existing crystallographic constraints, allowing structural biologists access to previously restricted scientific territory. SFX relies on exceptionally brilliant, micro-focused X-ray pulses, which are femtoseconds in duration, to probe nano/micrometer sized crystals in a serial fashion. This results in data sets comprised of individual snapshots, each capturing Bragg diffraction of single crystals in random orientations prior to their subsequent destruction. Thus structural elucidation while avoiding radiation damage, even at room temperature, can now be achieved. This emerging field has cultivated new methods for nanocrystallogenesis, sample delivery, and data processing. Opportunities and challenges within SFX are reviewed herein. Published by Elsevier Inc.

Novel protein–inhibitor interactions in site 3 of Ca2+-bound S100B as discovered by X-ray crystallography

PubMed Central

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

2016-01-01

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

Correlations in Scattered X-Ray Laser Pulses Reveal Nanoscale Structural Features of Viruses

NASA Astrophysics Data System (ADS)

Kurta, Ruslan P.; Donatelli, Jeffrey J.; Yoon, Chun Hong; Berntsen, Peter; Bielecki, Johan; Daurer, Benedikt J.; DeMirci, Hasan; Fromme, Petra; Hantke, Max Felix; Maia, Filipe R. N. C.; Munke, Anna; Nettelblad, Carl; Pande, Kanupriya; Reddy, Hemanth K. N.; Sellberg, Jonas A.; Sierra, Raymond G.; Svenda, Martin; van der Schot, Gijs; Vartanyants, Ivan A.; Williams, Garth J.; Xavier, P. Lourdu; Aquila, Andrew; Zwart, Peter H.; Mancuso, Adrian P.

2017-10-01

We use extremely bright and ultrashort pulses from an x-ray free-electron laser (XFEL) to measure correlations in x rays scattered from individual bioparticles. This allows us to go beyond the traditional crystallography and single-particle imaging approaches for structure investigations. We employ angular correlations to recover the three-dimensional (3D) structure of nanoscale viruses from x-ray diffraction data measured at the Linac Coherent Light Source. Correlations provide us with a comprehensive structural fingerprint of a 3D virus, which we use both for model-based and ab initio structure recovery. The analyses reveal a clear indication that the structure of the viruses deviates from the expected perfect icosahedral symmetry. Our results anticipate exciting opportunities for XFEL studies of the structure and dynamics of nanoscale objects by means of angular correlations.

Synthesis, X-ray characterisation and reactions of a trigonal planar palladium(0) carbonyl complex, (tbpx)PdCO.

PubMed

Bellabarba, Ronan M; Tooze, Robert P; Slawin, Alexandra M Z

2003-08-07

The novel complex (tbpx)PdCO (1), the first example of a structurally characterised sixteen electron, trigonal planar palladium(0) carbonyl complex, was prepared, characterised by NMR spectroscopy and X-ray crystallography, and some unusual aspects of its reactivity were studied.

Biophysical and X-ray crystallographic analysis of Mps1 kinase inhibitor complexes.

PubMed

Chu, Matthew L H; Lang, Zhaolei; Chavas, Leonard M G; Neres, João; Fedorova, Olga S; Tabernero, Lydia; Cherry, Mike; Williams, David H; Douglas, Kenneth T; Eyers, Patrick A

2010-03-02

The dual-specificity protein kinase monopolar spindle 1 (Mps1) is a central component of the mitotic spindle assembly checkpoint (SAC), a sensing mechanism that prevents anaphase until all chromosomes are bioriented on the metaphase plate. Partial depletion of Mps1 protein levels sensitizes transformed, but not untransformed, human cells to therapeutic doses of the anticancer agent Taxol, making it an attractive novel therapeutic cancer target. We have previously determined the X-ray structure of the catalytic domain of human Mps1 in complex with the anthrapyrazolone kinase inhibitor SP600125. In order to validate distinct inhibitors that target this enzyme and improve our understanding of nucleotide binding site architecture, we now report a biophysical and structural evaluation of the Mps1 catalytic domain in the presence of ATP and the aspecific model kinase inhibitor staurosporine. Collective in silico, enzymatic, and fluorescent screens also identified several new lead quinazoline Mps1 inhibitors, including a low-affinity compound termed Compound 4 (Cpd 4), whose interaction with the Mps1 kinase domain was further characterized by X-ray crystallography. A novel biophysical analysis demonstrated that the intrinsic fluorescence of SP600125 changed markedly upon Mps1 binding, allowing spectrophotometric displacement analysis and determination of dissociation constants for ATP-competitive Mps1 inhibitors. By illuminating the structure of the Mps1 ATP-binding site our results provide novel biophysical insights into Mps1-ligand interactions that will be useful for the development of specific Mps1 inhibitors, including those employing a therapeutically validated quinazoline template.

Mix-and-diffuse serial synchrotron crystallography

DOE PAGES

Beyerlein, Kenneth R.; Dierksmeyer, Dennis; Mariani, Valerio; ...

2017-10-09

Unravelling the interaction of biological macromolecules with ligands and substrates at high spatial and temporal resolution remains a major challenge in structural biology. The development of serial crystallography methods at X-ray free-electron lasers and subsequently at synchrotron light sources allows new approaches to tackle this challenge. Here, a new polyimide tape drive designed for mix-and-diffuse serial crystallography experiments is reported. The structure of lysozyme bound by the competitive inhibitor chitotriose was determined using this device in combination with microfluidic mixers. The electron densities obtained from mixing times of 2 and 50 s show clear binding of chitotriose to the enzymemore » at a high level of detail. Here, the success of this approach shows the potential for high-throughput drug screening and even structural enzymology on short timescales at bright synchrotron light sources.« less

Mix-and-diffuse serial synchrotron crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beyerlein, Kenneth R.; Dierksmeyer, Dennis; Mariani, Valerio

Unravelling the interaction of biological macromolecules with ligands and substrates at high spatial and temporal resolution remains a major challenge in structural biology. The development of serial crystallography methods at X-ray free-electron lasers and subsequently at synchrotron light sources allows new approaches to tackle this challenge. Here, a new polyimide tape drive designed for mix-and-diffuse serial crystallography experiments is reported. The structure of lysozyme bound by the competitive inhibitor chitotriose was determined using this device in combination with microfluidic mixers. The electron densities obtained from mixing times of 2 and 50 s show clear binding of chitotriose to the enzymemore » at a high level of detail. Here, the success of this approach shows the potential for high-throughput drug screening and even structural enzymology on short timescales at bright synchrotron light sources.« less

Ruthenium-Nitrosyl Complexes with Glycine, l-Alanine, l-Valine, l-Proline, d-Proline, l-Serine, l-Threonine, and l-Tyrosine: Synthesis, X-ray Diffraction Structures, Spectroscopic and Electrochemical Properties, and Antiproliferative Activity

PubMed Central

2014-01-01

The reactions of [Ru(NO)Cl5]2– with glycine (Gly), l-alanine (l-Ala), l-valine (l-Val), l-proline (l-Pro), d-proline (d-Pro), l-serine (l-Ser), l-threonine (l-Thr), and l-tyrosine (l-Tyr) in n-butanol or n-propanol afforded eight new complexes (1–8) of the general formula [RuCl3(AA–H)(NO)]−, where AA = Gly, l-Ala, l-Val, l-Pro, d-Pro, l-Ser, l-Thr, and l-Tyr, respectively. The compounds were characterized by elemental analysis, electrospray ionization mass spectrometry (ESI-MS), 1H NMR, UV–visible and ATR IR spectroscopy, cyclic voltammetry, and X-ray crystallography. X-ray crystallography studies have revealed that in all cases the same isomer type (from three theoretically possible) was isolated, namely mer(Cl),trans(NO,O)-[RuCl3(AA–H)(NO)], as was also recently reported for osmium analogues with Gly, l-Pro, and d-Pro (see Z. Anorg. Allg. Chem.2013, 639, 1590–1597). Compounds 1, 4, 5, and 8 were investigated by ESI-MS with regard to their stability in aqueous solution and reactivity toward sodium ascorbate. In addition, cell culture experiments in three human cancer cell lines, namely, A549 (nonsmall cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon carcinoma), were performed, and the results are discussed in conjunction with the lipophilicity of compounds. PMID:24555845

Preparation and guest-uptake protocol for a porous complex useful for 'crystal-free' crystallography.

PubMed

Inokuma, Yasuhide; Yoshioka, Shota; Ariyoshi, Junko; Arai, Tatsuhiko; Fujita, Makoto

2014-02-01

We recently reported a new method for single-crystal X-ray diffraction (SCD) analysis that does not require the crystallization of the target compound. In this 'crystal-free' crystallography, a tiny crystal of a porous complex is soaked in the solution of the target guest. The guest molecules are absorbed and oriented in the crystal pores and can be analyzed by X-ray diffraction. We describe here a detailed synthetic protocol for the preparation of uniform single crystals of the porous host complex and for the subsequent guest uptake. The protocol describes our most versatile porous complex, which is prepared from commercially available ZnI2 and 2,4,6-tri(4-pyridyl)-1,3,5-triazine. The host complex has large pores with a cross-section of 8 × 5 Å(2). Single crystals of the complex are grown from layered solutions of the two components. The pores of the as-synthesized complex are filled with nitrobenzene, which is replaced with the inert solvent cyclohexane. This solvent exchange is essential for the rapid and effective inclusion of target compounds. The most crucial and delicate step is the selection of high-quality single crystals from the mixture of crystals of various shapes and sizes. We suggest using the facial indices of the single crystals as a criterion for crystal selection. Single-crystal samples for X-ray analysis can be prepared by immersing the selected crystals in a cyclohexane/dichloromethane solution of target compound. After a very slow evaporation of the solvent, typically over 2 d, the final crystal can be picked and directly subjected to SCD analysis. The protocol can be completed within ∼16 d.

Serial femtosecond crystallography at the SACLA: breakthrough to dynamic structural biology.

PubMed

Mizohata, Eiichi; Nakane, Takanori; Fukuda, Yohta; Nango, Eriko; Iwata, So

2018-04-01

X-ray crystallography visualizes the world at the atomic level. It has been used as the most powerful technique for observing the three-dimensional structures of biological macromolecules and has pioneered structural biology. To determine a crystal structure with high resolution, it was traditionally required to prepare large crystals (> 200 μm). Later, synchrotron radiation facilities, such as SPring-8, that produce powerful X-rays were built. They enabled users to obtain good quality X-ray diffraction images even with smaller crystals (ca. 200-50 μm). In recent years, one of the most important technological innovations in structural biology has been the development of X-ray free electron lasers (XFELs). The SPring-8 Angstrom Compact free electron LAser (SACLA) in Japan generates the XFEL beam by accelerating electrons to relativistic speeds and directing them through in-vacuum, short-period undulators. Since user operation started in 2012, we have been involved in the development of serial femtosecond crystallography (SFX) measurement systems using XFEL at the SACLA. The SACLA generates X-rays a billion times brighter than SPring-8. The extremely bright XFEL pulses enable data collection with microcrystals (ca. 50-1 μm). Although many molecular analysis techniques exist, SFX is the only technique that can visualize radiation-damage-free structures of biological macromolecules at room temperature in atomic resolution and fast time resolution. Here, we review the achievements of the SACLA-SFX Project in the past 5 years. In particular, we focus on: (1) the measurement system for SFX; (2) experimental phasing by SFX; (3) enzyme chemistry based on damage-free room-temperature structures; and (4) molecular movie taken by time-resolved SFX.

Measuring and modeling diffuse scattering in protein X-ray crystallography

PubMed Central

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

2016-01-01

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

Measuring and modeling diffuse scattering in protein X-ray crystallography

DOE PAGES

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

2016-03-28

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

Fab Chaperone-Assisted RNA Crystallography (Fab CARC).

PubMed

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

2016-01-01

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

X-ray excited Auger transitions of Pu compounds

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nelson, Art J., E-mail: nelson63@llnl.gov; Grant, William K.; Stanford, Jeff A.

2015-05-15

X-ray excited Pu core–valence–valence and core–core–valence Auger line-shapes were used in combination with the Pu 4f photoelectron peaks to characterize differences in the oxidation state and local electronic structure for Pu compounds. The evolution of the Pu 4f core-level chemical shift as a function of sputtering depth profiling and hydrogen exposure at ambient temperature was quantified. The combination of the core–valence–valence Auger peak energies with the associated chemical shift of the Pu 4f photoelectron line defines the Auger parameter and results in a reliable method for definitively determining oxidation states independent of binding energy calibration. Results show that PuO{sub 2},more » Pu{sub 2}O{sub 3}, PuH{sub 2.7}, and Pu have definitive Auger line-shapes. These data were used to produce a chemical state (Wagner) plot for select plutonium oxides. This Wagner plot allowed us to distinguish between the trivalent hydride and the trivalent oxide, which cannot be differentiated by the Pu 4f binding energy alone.« less

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

NASA Astrophysics Data System (ADS)

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.

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

PubMed

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

2016-04-12

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.

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

PubMed Central

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

2016-01-01

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. PMID:27070998

From Macrocrystals to Microcrystals: A Strategy for Membrane Protein Serial Crystallography.

PubMed

Dods, Robert; Båth, Petra; Arnlund, David; Beyerlein, Kenneth R; Nelson, Garrett; Liang, Mengling; Harimoorthy, Rajiv; Berntsen, Peter; Malmerberg, Erik; Johansson, Linda; Andersson, Rebecka; Bosman, Robert; Carbajo, Sergio; Claesson, Elin; Conrad, Chelsie E; Dahl, Peter; Hammarin, Greger; Hunter, Mark S; Li, Chufeng; Lisova, Stella; Milathianaki, Despina; Robinson, Joseph; Safari, Cecilia; Sharma, Amit; Williams, Garth; Wickstrand, Cecilia; Yefanov, Oleksandr; Davidsson, Jan; DePonte, Daniel P; Barty, Anton; Brändén, Gisela; Neutze, Richard

2017-09-05

Serial protein crystallography was developed at X-ray free-electron lasers (XFELs) and is now also being applied at storage ring facilities. Robust strategies for the growth and optimization of microcrystals are needed to advance the field. Here we illustrate a generic strategy for recovering high-density homogeneous samples of microcrystals starting from conditions known to yield large (macro) crystals of the photosynthetic reaction center of Blastochloris viridis (RC vir ). We first crushed these crystals prior to multiple rounds of microseeding. Each cycle of microseeding facilitated improvements in the RC vir serial femtosecond crystallography (SFX) structure from 3.3-Å to 2.4-Å resolution. This approach may allow known crystallization conditions for other proteins to be adapted to exploit novel scientific opportunities created by serial crystallography. Copyright © 2017 Elsevier Ltd. All rights reserved.

Compound formation and superconductivity in Au-Si: X-ray absorption measurements on ion-beam-mixed Au-Si films

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeon, Y.; Jisrawi, N.; Liang, G.

Multilayered Au-Si thin films have been deposited with the net compositions ''Au/sub 1-//sub x/Si/sub x/,'' x = 0.29, 0.5, and 0.8. After ion-beam mixing these films exhibited superconductivity in the 0.3--1.2 K range despite the nonsuperconducting character of both Au and Si. Near-edge x-ray absorption spectroscopy (XAS) measurements on the Au L/sub 3/ edge in these films indicate that metastable Au-Si compound formation occurs in these ion-mixed materials. Specifically, the XAS measurements indicate changes in Au 5d-orbital occupancy and changes in the local Au structural environment which are both consistent with local compound formation.

Correlations in Scattered X-Ray Laser Pulses Reveal Nanoscale Structural Features of Viruses

DOE PAGES

Kurta, Ruslan P.; Donatelli, Jeffrey J.; Yoon, Chun Hong; ...

2017-10-12

We use extremely bright and ultrashort pulses from an x-ray free-electron laser (XFEL) to measure correlations in x rays scattered from individual bioparticles. This allows us to go beyond the traditional crystallography and single-particle imaging approaches for structure investigations. We employ angular correlations to recover the three-dimensional (3D) structure of nanoscale viruses from x-ray diffraction data measured at the Linac Coherent Light Source. Correlations provide us with a comprehensive structural fingerprint of a 3D virus, which we use both for model-based and ab initio structure recovery. The analyses reveal a clear indication that the structure of the viruses deviates frommore » the expected perfect icosahedral symmetry. Lastly, our results anticipate exciting opportunities for XFEL studies of the structure and dynamics of nanoscale objects by means of angular correlations.« less

Luteolin as reactive oxygen generator by X-ray and UV irradiation

NASA Astrophysics Data System (ADS)

Toyama, Michiru; Mori, Takashi; Takahashi, Junko; Iwahashi, Hitoshi

2018-05-01

Non-toxic X-ray-responsive substances can be used in the radiosensitization of cancer, like porphyrin mediated radiotherapy. However, most X-ray-responsive substances are toxic. To find novel non-toxic X-ray-responsive substances, we studied the X-ray and UV reactivity of 40 non-toxic compounds extracted from plants. Dihydroethidium was used as an indicator to detect reactive oxygen species (ROS) generated by the compounds under X-ray or UV irradiation. We found that 13 of the investigated compounds generated ROS under X-ray irradiation and 17 generated ROS under UV irradiation. Only 4 substances generated ROS under both X-ray and UV. In particular, luteolin exhibited the highest activity among the investigated compounds; therefore, the ROS generated by luteolin were thoroughly characterized. To identify the ROS, we employed a combination of ROS detection reagents and their quenchers. O2·- generation by luteolin was monitored using dihydroethidium and superoxide dismutase (as an O2·- quencher). OH· and 1O2 generation was determined using aminophenyl fluorescein with ethanol (OH· quencher) and Singlet Oxygen Sensor Green® with NaN3 (1O2 quencher), respectively. Generation of O2·- under X-ray and UV irradiation was observed; however, no OH· or 1O2 was detected. The production of ROS from luteolin is surprising, because luteolin is a well-known antioxidant.

The accurate assessment of small-angle X-ray scattering data

DOE PAGES

Grant, Thomas D.; Luft, Joseph R.; Carter, Lester G.; ...

2015-01-23

Small-angle X-ray scattering (SAXS) has grown in popularity in recent times with the advent of bright synchrotron X-ray sources, powerful computational resources and algorithms enabling the calculation of increasingly complex models. However, the lack of standardized data-quality metrics presents difficulties for the growing user community in accurately assessing the quality of experimental SAXS data. Here, a series of metrics to quantitatively describe SAXS data in an objective manner using statistical evaluations are defined. These metrics are applied to identify the effects of radiation damage, concentration dependence and interparticle interactions on SAXS data from a set of 27 previously described targetsmore » for which high-resolution structures have been determined via X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. Studies show that these metrics are sufficient to characterize SAXS data quality on a small sample set with statistical rigor and sensitivity similar to or better than manual analysis. The development of data-quality analysis strategies such as these initial efforts is needed to enable the accurate and unbiased assessment of SAXS data quality.« less

Review: Serial Femtosecond Crystallography: A Revolution in Structural Biology

PubMed Central

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

2016-01-01

Macromolecular crystallography at synchrotron sources has proven to be the most influential method within structural biology, producing thousands of structures since its inception. While its utility has been instrumental in progressing our knowledge of structures of molecules, it suffers from limitations such as the need for large, well-diffracting crystals, and radiation damage that can hamper native structural determination. The recent advent of X-ray free electron lasers (XFELs) and their implementation in the emerging field of serial femtosecond crystallography (SFX) has given rise to a remarkable expansion upon existing crystallographic constraints, allowing structural biologists access to previously restricted scientific territory. SFX relies on exceptionally brilliant, micro-focused X-ray pulses, which are femtoseconds in duration, to probe nano/micrometer sized crystals in a serial fashion. This results in data sets comprised of individual snapshots, each capturing Bragg diffraction of single crystals in random orientations prior to their subsequent destruction. Thus structural elucidation while avoiding radiation damage, even at room temperature, can now be achieved. This emerging field has cultivated new methods for nanocrystallogenesis, sample delivery, and data processing. Opportunities and challenges within SFX are reviewed herein. PMID:27143509

CCD sensors in synchrotron X-ray detectors

NASA Astrophysics Data System (ADS)

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

1988-04-01

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 × 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 a rate of ˜ 1 frame/s or a complete 3-dimensional data set from a single crystal in ˜ 2 min. In electron energy-loss spectroscopy (EELS), the CCD was used in a parallel detection mode which is similar to the mode array detectors are used in dispersive EXAFS. With a beam current corresponding to 3 × 10 9 electron/s on the detector, a series of 64 spectra were recorded on the CCD in a continuous sequence without interruption due to readout. The frame-to-frame pixel signal fluctuations had σ = 0.4% from which DQE = 0.4 was obtained, where the detector conversion efficiency was 2.6 CCD electrons/X-ray photon. These multiple frame series also showed the time-resolved modulation of the electron microscope optics by stray magnetic fields.

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

PubMed

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.

Serial Millisecond Crystallography of Membrane Proteins.

PubMed

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

2016-01-01

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

Does crystallography need a new name?

DOE PAGES

Argryriou, Dimitri

2017-07-01

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

Does crystallography need a new name?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Argryriou, Dimitri

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

Microbeam X-ray analysis in Poland - past and future

NASA Astrophysics Data System (ADS)

Kusinski, J.

2010-02-01

The article provides an overview of the development of electron beam X-ray microanalysis (EPMA) in Poland. Since the introduction by Prof. Bojarski of EMPA over 45 years ago, tremendous advances in methodologies and in instrumentation have been made in order to improve the precision of quantitative compositional analysis, spatial resolution and analytical sensitivity. This was possible due to the activity of Applied Crystallography Committee at the Polish Academy of Sciences, as well as the groups of researches working in the Institute for Ferrous Metallurgy (Gliwice), the Technical University of Warsaw, the Silesian Technical University (Katowice), the AGH-University of Sciences and Technology (Krakow), and the Institute of Materials Science and Metallurgy Polish Academy of Sciences (Krakow). Based on the research examples realized by these teams, conferences, seminars and congresses organized, as well as books and academic textbooks issued, the evolution of electron beam X-ray microanalysis in Poland is demonstrated.

X-ray Fluorescence Holography: Principles, Apparatus, and Applications

NASA Astrophysics Data System (ADS)

Hayashi, Kouichi; Korecki, Pawel

2018-06-01

X-ray fluorescence holography (XFH) is an atomic structure determination technique that combines the capabilities of X-ray diffraction and X-ray fluorescence spectroscopy. It provides a unique means of gaining fully three-dimensional information about the local atomic structure and lattice site positions of selected elements inside compound samples. In this work, we discuss experimental and theoretical aspects that are essential for the efficient recording and analysis of X-ray fluorescence holograms and review the most recent advances in XFH. We describe experiments performed with brilliant synchrotron radiation as well as with tabletop setups that employ conventional X-ray tubes.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, X. Edward; Gao, Xiang; Barty, Anton

Here, 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 solvedmore » 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.« less

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

DOE PAGES

Zhou, X. Edward; Gao, Xiang; Barty, Anton; ...

2016-04-12

Here, 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 solvedmore » 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.« less

A combined experimental and theoretical study of dinitrosyl iron complexes containing chelating bis(diphenyl)phosphinoX (X = benzene, propane and ethylene): X-ray crystal structures and properties influenced by the presence or absence of π-bonds in chelating ligands

PubMed Central

Holloway, Lauren R.; Clough, Andrew J.; Li, Jessica Y.; Tao, Emily L.; Tao, Fu-Ming; Li, Lijuan

2014-01-01

Recent discoveries involving the roles of nitric oxide in humans have stimulated intense interest in transition metal nitrosyl complexes. A series of dinitrosyl iron complexes with the formula [(DPPX)Fe(NO)2], {DPPX = 1,2-bis(diphenylphosphino)benzene (1), 1,3-bis(diphenylphosphino)propane (2), and cis-1,2-bis(diphenylphosphino)ethylene (3)} has been prepared and characterized through a combination of FT-IR, NMR, UV-vis, X-ray crystallography, and electrochemical techniques. Infrared spectroscopy showed NO shifts to the region of 1723 and 1674 cm−1 for complexes 1 and 3, and 1708 and 1660 cm−1 for 2, indicating that ligand 2 acts as a stronger σ–donor. The X-ray crystallographic data showed that 1 and 3 possess the rare repulso conformation while 2 has the attracto conformation. CV studies on compounds 1, 2 and 3 display two quasi-reversible oxidations with the E°1/2 values at 0.101 and 0.186 V, 0.121 and 0.184 V, and 0.019 and 0.342 V, respectively. The larger ΔE value for compound 2 compared with that of 1 and 3 is attributed to the lack of π-bonds between the two phosphorus atoms. Theoretical calculations using density functional theory were carried out on the synthesized compounds and model compounds and the results are consistent with the experimental data. The calculated HOMO-LUMO gaps for compounds 1, 2 and 3 are 3.736, 4.060, and 3.669 eV, respectively, which supports the stronger back-donation for compound 2 than that of compounds 1 and 3. PMID:24860235

BioCARS: a synchrotron resource for time-resolved X-ray science

DOE Office of Scientific and Technical Information (OSTI.GOV)

Graber, T.; Anderson, S.; Brewer, H.

2011-08-16

BioCARS, a NIH-supported national user facility for macromolecular time-resolved X-ray crystallography at the Advanced Photon Source (APS), has recently completed commissioning of an upgraded undulator-based beamline optimized for single-shot laser-pump X-ray-probe measurements with time resolution as short as 100 ps. The source consists of two in-line undulators with periods of 23 and 27 mm that together provide high-flux pink-beam capability at 12 keV as well as first-harmonic coverage from 6.8 to 19 keV. A high-heat-load chopper reduces the average power load on downstream components, thereby preserving the surface figure of a Kirkpatrick-Baez mirror system capable of focusing the X-ray beammore » to a spot size of 90 {micro}m horizontal by 20 {micro}m vertical. A high-speed chopper isolates single X-ray pulses at 1 kHz in both hybrid and 24-bunch modes of the APS storage ring. In hybrid mode each isolated X-ray pulse delivers up to {approx}4 x 10{sup 10} photons to the sample, thereby achieving a time-averaged flux approaching that of fourth-generation X-FEL sources. A new high-power picosecond laser system delivers pulses tunable over the wavelength range 450-2000 nm. These pulses are synchronized to the storage-ring RF clock with long-term stability better than 10 ps RMS. Monochromatic experimental capability with Biosafety Level 3 certification has been retained.« less

Systems and methods for detecting x-rays

DOEpatents

Bross, Alan D.; Mellott, Kerry L.; Pla-Dalmau, Anna

2006-05-02

Systems and methods for detecting x-rays are disclosed herein. One or more x-ray-sensitive scintillators can be configured from a plurality of heavy element nano-sized particles and a plastic material, such as polystyrene. As will be explained in greater detail herein, the heavy element nano-sized particles (e.g., PbWO₄) can be compounded into the plastic material with at least one dopant that permits the plastic material to scintillate. X-rays interact with the heavy element nano-sized particles to produce electrons that can deposit energy in the x-ray sensitive scintillator, which in turn can produce light.

Applied Crystallography - Proceedings of the XVth Conference

NASA Astrophysics Data System (ADS)

Morawiec, H.; Ströż, D.

1993-06-01

The Table of Contents for the full book PDF is as follows: * Foreword * The International Centre for Diffraction Data and Its Future Developments * The Rietveld Method - A Historical Perspective * Real Structure in Quantitative Powder Diffraction Phase Analysis * Neutron Focusing Optics in Applied Crystallography * The Crystal Structures of Oxygen Deficient Rare Earth Oxides * Short-Range Order in Layer-Structured Ba1-xSrxBi2Nb2O9 Ferroelectrics * Radial Distribution Function as a Tool of Structural Studies on Noncrystalline Materials * Determination of Radial Distribution Function (RDF) of Electrodeposited Cu-Cd Alloys After Annealing * Spheres Packing as a Factor Describing the Local Environment and Structure Stability * X-Ray Stress Measurement of Samples Combined with Diffraction Line Analysis * Phase Stability and Martensitic Transformation in Cu-Zn and Cu-Zn-Al Single Crystals * Order, Defects, Precipitates and the Martensitic Transformation in β Cu-Zn-Al * Effect of γ Precipitates on the Martensitic Transformation in Cu-Zn-Al Alloys * Phase Transitions and Shape Memory Effect in a Thermomechanically Treated NiTi Alloy * Structure of Martensite and Bainite in CuAlMn Alloys * Glass-Ceramics * Mechanism of Texture Formation at the Rolling of Low Stacking Fault Energy Metals and Alloys * Shear Texture of Zinc and the Conditions of Its Occuring * The Development of Texture of ZnAlMg Sheets Depending on Deformation Geometry * Texture Stability of the D.S. NiAlMoCrTi Alloy After Heat Treatment * X-Ray Diffraction Method for Controlling of Texture Evolution in Layers * Texture and Lattice Imperfections Study of Some Low Alloyed Copper Alloys * Selected Examples of the Calculation of the Orientation Distribution Function for Low Crystal and Sample Symmetries * Automatical X-Ray Quantitative Phase Analysis * Application of a PC Computer for Crystallographic Calculations * Electron Diffraction Analysis using a Personal Computer * CA.R.INE Crystallography Version 2

Opportunities and challenges for time-resolved studies of protein structural dynamics at X-ray free-electron lasers.

PubMed

Neutze, Richard

2014-07-17

X-ray free-electron lasers (XFELs) are revolutionary X-ray sources. Their time structure, providing X-ray pulses of a few tens of femtoseconds in duration; and their extreme peak brilliance, delivering approximately 10(12) X-ray photons per pulse and facilitating sub-micrometre focusing, distinguish XFEL sources from synchrotron radiation. In this opinion piece, I argue that these properties of XFEL radiation will facilitate new discoveries in life science. I reason that time-resolved serial femtosecond crystallography and time-resolved wide angle X-ray scattering are promising areas of scientific investigation that will be advanced by XFEL capabilities, allowing new scientific questions to be addressed that are not accessible using established methods at storage ring facilities. These questions include visualizing ultrafast protein structural dynamics on the femtosecond to picosecond time-scale, as well as time-resolved diffraction studies of non-cyclic reactions. I argue that these emerging opportunities will stimulate a renaissance of interest in time-resolved structural biochemistry.

xMDFF: molecular dynamics flexible fitting of low-resolution X-ray structures.

PubMed

McGreevy, Ryan; Singharoy, Abhishek; Li, Qufei; Zhang, Jingfen; Xu, Dong; Perozo, Eduardo; Schulten, Klaus

2014-09-01

X-ray crystallography remains the most dominant method for solving atomic structures. However, for relatively large systems, the availability of only medium-to-low-resolution diffraction data often limits the determination of all-atom details. A new molecular dynamics flexible fitting (MDFF)-based approach, xMDFF, for determining structures from such low-resolution crystallographic data is reported. xMDFF employs a real-space refinement scheme that flexibly fits atomic models into an iteratively updating electron-density map. It addresses significant large-scale deformations of the initial model to fit the low-resolution density, as tested with synthetic low-resolution maps of D-ribose-binding protein. xMDFF has been successfully applied to re-refine six low-resolution protein structures of varying sizes that had already been submitted to the Protein Data Bank. Finally, via systematic refinement of a series of data from 3.6 to 7 Å resolution, xMDFF refinements together with electrophysiology experiments were used to validate the first all-atom structure of the voltage-sensing protein Ci-VSP.

Tables of X-ray absorption corrections and dispersion corrections: the new versus the old

NASA Astrophysics Data System (ADS)

Creagh, Dudley

1990-11-01

This paper compares the data on X-ray absorption coefficients calculated by Creagh and Hubbell and tabulated in International Tables for Crystallography, vol. C, ed. A.J.C. Wilson (1990) section 4.2.4 [1] with empirical (Saloman, Hubbell and Scofield, At. Data and Nucl. Data Tables 38 (1988) 1, [6]) and semi-empirical (Hubbell, McMaster, Kerr Del Grande and Mallett, in: International Tables for Crystallography, vol. IV, eds. Ibers and Hamilton (Kynoch, Birmingham, 1974) [2]) tabulations as well as the renormalized relativistic Dirac-Hartree-Fock calculations of Scofield [6]. It also makes comparisons of the real part of the dispersion correction ƒ‧(ω, 0) and tabulated in ref. [1], with theoretical data sets (Cromer and Liberman, J. Chem. Phys. 53 (1970) 1891, and Acta Crystallogr. A37 (1981) 267 [4,5]; Wang, Phys. Rev. A34 (1986) 636 [85]; Kissel, in: Workshop Report on New Dimensions in X-ray Scattering, CONF-870459 (Livermore, 1987) p. 9 [86]) and data collected using a variety of experimental techniques. In both cases the data tabulated in ref. [1] is shown to give improved self-consistency and agreement with experiment.

Single-crystal X-ray diffraction and NMR crystallography of a 1:1 cocrystal of dithianon and pyrimethanil.

PubMed

Pöppler, Ann Christin; Corlett, Emily K; Pearce, Harriet; Seymour, Mark P; Reid, Matthew; Montgomery, Mark G; Brown, Steven P

2017-03-01

A single-crystal X-ray diffraction structure of a 1:1 cocrystal of two fungicides, namely dithianon (DI) and pyrimethanil (PM), is reported [systematic name: 5,10-dioxo-5H,10H-naphtho[2,3-b][1,4]dithiine-2,3-dicarbonitrile-4,6-dimethyl-N-phenylpyrimidin-2-amine (1/1), C 14 H 4 N 2 O 2 S 2 ·C 12 H 13 N 2 ]. Following an NMR crystallography approach, experimental solid-state magic angle spinning (MAS) NMR spectra are presented together with GIPAW (gauge-including projector augmented wave) calculations of NMR chemical shieldings. Specifically, experimental 1 H and 13 C chemical shifts are determined from two-dimensional 1 H- 13 C MAS NMR correlation spectra recorded with short and longer contact times so as to probe one-bond C-H connectivities and longer-range C...H proximities, whereas H...H proximities are identified in a 1 H double-quantum (DQ) MAS NMR spectrum. The performing of separate GIPAW calculations for the full periodic crystal structure and for isolated molecules allows the determination of the change in chemical shift upon going from an isolated molecule to the full crystal structure. For the 1 H NMR chemical shifts, changes of 3.6 and 2.0 ppm correspond to intermolecular N-H...O and C-H...O hydrogen bonding, while changes of -2.7 and -1.5 ppm are due to ring current effects associated with C-H...π interactions. Even though there is a close intermolecular S...O distance of 3.10 Å, it is of note that the molecule-to-crystal chemical shifts for the involved sulfur or oxygen nuclei are small.

Resolution of structural heterogeneity in dynamic crystallography.

PubMed

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

2013-06-01

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

Correct interpretation of diffraction properties of quartz crystals for X-ray optics applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Xian-Rong; Gog, Thomas; Kim, Jungho

Quartz has hundreds of strong Bragg reflections that may offer a great number of choices for making fixed-angle X-ray analyzers and polarizers at virtually any hard X-ray energies with selectable resolution. However, quartz crystals, unlike silicon and germanium, are chiral and may thus appear in two different forms of handedness that are mirror images. Furthermore, because of the threefold rotational symmetry along thecaxis, the {h 1h 2h 3L} and {h 2h 1h 3L} Bragg reflections may have quite different Darwin bandwidth, reflectivity and angular acceptance, although they have the same Bragg angle. The design of X-ray optics from quartz crystalsmore » therefore requires unambiguous determination of the orientation, handedness and polarity of the crystals. The Laue method and single-axis diffraction technique can provide such information, but the variety of conventions used in the literature to describe quartz structures has caused widespread confusion. The current studies give detailed guidelines for design and fabrication of quartz X-ray optics, with special emphasis on the correct interpretation of Laue patterns in terms of the crystallography and diffraction properties of quartz. Meanwhile, the quartz crystals examined were confirmed by X-ray topography to have acceptably low densities of dislocations and other defects, which is the foundation for developing high-resolution quartz-based X-ray optics.« less

Low-dose fixed-target serial synchrotron crystallography.

PubMed

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

2017-04-01

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

Polymer X-ray refractive nano-lenses fabricated by additive technology.

PubMed

Petrov, A K; Bessonov, V O; Abrashitova, K A; Kokareva, N G; Safronov, K R; Barannikov, A A; Ershov, P A; Klimova, N B; Lyatun, I I; Yunkin, V A; Polikarpov, M; Snigireva, I; Fedyanin, A A; Snigirev, A

2017-06-26

The present work demonstrates the potential applicability of additive manufacturing to X-Ray refractive nano-lenses. A compound refractive lens with a radius of 5 µm was produced by the two-photon polymerization induced lithography. It was successfully tested at the X-ray microfocus laboratory source and a focal spot of 5 μm was measured. An amorphous nature of polymer material combined with the potential of additive technologies may result in a significantly enhanced focusing performance compared to the best examples of modern X-ray compound refractive lenses.

Covering complete proteomes with X-ray structures: A current snapshot

DOE PAGES

Mizianty, Marcin J.; Fan, Xiao; Yan, Jing; ...

2014-10-23

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 obtainedmore » 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.« less

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens.

PubMed

MacLachlan, Bruce J; Greenshields-Watson, Alexander; Mason, Georgina H; Schauenburg, Andrea J; Bianchi, Valentina; Rizkallah, Pierre J; Sewell, Andrew K; Fuller, Anna; Cole, David K

2017-02-06

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

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

PubMed

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.

In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

PubMed Central

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

2016-01-01

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

In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

DOE PAGES

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

2016-03-01

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

In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

Solvent minimization induces preferential orientation and crystal clustering in serial micro-crystallography on micro-meshes, in situ plates and on a movable crystal conveyor belt.

PubMed

Soares, Alexei S; Mullen, Jeffrey D; Parekh, Ruchi M; McCarthy, Grace S; Roessler, Christian G; Jackimowicz, Rick; Skinner, John M; Orville, Allen M; Allaire, Marc; Sweet, Robert M

2014-11-01

X-ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial micro-crystallography: MiTeGen MicroMeshes™, Greiner Bio-One Ltd in situ micro-plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X-ray beam. 6° wedges of data were taken from ∼100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face that preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X-ray beam was incident normal to the data-collection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 µm were observed to clump together into clusters of crystals. Clustering required that the X-ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. High-quality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies.

Dynamic X-ray diffraction sampling for protein crystal positioning

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scarborough, Nicole M.; Godaliyadda, G. M. Dilshan P.; Ye, Dong Hye

A sparse supervised learning approach for dynamic sampling (SLADS) is described for dose reduction in diffraction-based protein crystal positioning. Crystal centering is typically a prerequisite for macromolecular diffraction at synchrotron facilities, with X-ray diffraction mapping growing in popularity as a mechanism for localization. In X-ray raster scanning, diffraction is used to identify the crystal positions based on the detection of Bragg-like peaks in the scattering patterns; however, this additional X-ray exposure may result in detectable damage to the crystal prior to data collection. Dynamic sampling, in which preceding measurements inform the next most information-rich location to probe for image reconstruction,more » significantly reduced the X-ray dose experienced by protein crystals during positioning by diffraction raster scanning. The SLADS algorithm implemented herein is designed for single-pixel measurements and can select a new location to measure. In each step of SLADS, the algorithm selects the pixel, which, when measured, maximizes the expected reduction in distortion given previous measurements. Ground-truth diffraction data were obtained for a 5 µm-diameter beam and SLADS reconstructed the image sampling 31% of the total volume and only 9% of the interior of the crystal greatly reducing the X-ray dosage on the crystal. Furthermore, by usingin situtwo-photon-excited fluorescence microscopy measurements as a surrogate for diffraction imaging with a 1 µm-diameter beam, the SLADS algorithm enabled image reconstruction from a 7% sampling of the total volume and 12% sampling of the interior of the crystal. When implemented into the beamline at Argonne National Laboratory, without ground-truth images, an acceptable reconstruction was obtained with 3% of the image sampled and approximately 5% of the crystal. The incorporation of SLADS into X-ray diffraction acquisitions has the potential to significantly minimize the impact of X-ray exposure

Dynamic X-ray diffraction sampling for protein crystal positioning

PubMed Central

Scarborough, Nicole M.; Godaliyadda, G. M. Dilshan P.; Ye, Dong Hye; Kissick, David J.; Zhang, Shijie; Newman, Justin A.; Sheedlo, Michael J.; Chowdhury, Azhad U.; Fischetti, Robert F.; Das, Chittaranjan; Buzzard, Gregery T.; Bouman, Charles A.; Simpson, Garth J.

2017-01-01

A sparse supervised learning approach for dynamic sampling (SLADS) is described for dose reduction in diffraction-based protein crystal positioning. Crystal centering is typically a prerequisite for macromolecular diffraction at synchrotron facilities, with X-ray diffraction mapping growing in popularity as a mechanism for localization. In X-ray raster scanning, diffraction is used to identify the crystal positions based on the detection of Bragg-like peaks in the scattering patterns; however, this additional X-ray exposure may result in detectable damage to the crystal prior to data collection. Dynamic sampling, in which preceding measurements inform the next most information-rich location to probe for image reconstruction, significantly reduced the X-ray dose experienced by protein crystals during positioning by diffraction raster scanning. The SLADS algorithm implemented herein is designed for single-pixel measurements and can select a new location to measure. In each step of SLADS, the algorithm selects the pixel, which, when measured, maximizes the expected reduction in distortion given previous measurements. Ground-truth diffraction data were obtained for a 5 µm-diameter beam and SLADS reconstructed the image sampling 31% of the total volume and only 9% of the interior of the crystal greatly reducing the X-ray dosage on the crystal. Using in situ two-photon-excited fluorescence microscopy measurements as a surrogate for diffraction imaging with a 1 µm-diameter beam, the SLADS algorithm enabled image reconstruction from a 7% sampling of the total volume and 12% sampling of the interior of the crystal. When implemented into the beamline at Argonne National Laboratory, without ground-truth images, an acceptable reconstruction was obtained with 3% of the image sampled and approximately 5% of the crystal. The incorporation of SLADS into X-ray diffraction acquisitions has the potential to significantly minimize the impact of X-ray exposure on the crystal by

Dynamic X-ray diffraction sampling for protein crystal positioning.

PubMed

Scarborough, Nicole M; Godaliyadda, G M Dilshan P; Ye, Dong Hye; Kissick, David J; Zhang, Shijie; Newman, Justin A; Sheedlo, Michael J; Chowdhury, Azhad U; Fischetti, Robert F; Das, Chittaranjan; Buzzard, Gregery T; Bouman, Charles A; Simpson, Garth J

2017-01-01

A sparse supervised learning approach for dynamic sampling (SLADS) is described for dose reduction in diffraction-based protein crystal positioning. Crystal centering is typically a prerequisite for macromolecular diffraction at synchrotron facilities, with X-ray diffraction mapping growing in popularity as a mechanism for localization. In X-ray raster scanning, diffraction is used to identify the crystal positions based on the detection of Bragg-like peaks in the scattering patterns; however, this additional X-ray exposure may result in detectable damage to the crystal prior to data collection. Dynamic sampling, in which preceding measurements inform the next most information-rich location to probe for image reconstruction, significantly reduced the X-ray dose experienced by protein crystals during positioning by diffraction raster scanning. The SLADS algorithm implemented herein is designed for single-pixel measurements and can select a new location to measure. In each step of SLADS, the algorithm selects the pixel, which, when measured, maximizes the expected reduction in distortion given previous measurements. Ground-truth diffraction data were obtained for a 5 µm-diameter beam and SLADS reconstructed the image sampling 31% of the total volume and only 9% of the interior of the crystal greatly reducing the X-ray dosage on the crystal. Using in situ two-photon-excited fluorescence microscopy measurements as a surrogate for diffraction imaging with a 1 µm-diameter beam, the SLADS algorithm enabled image reconstruction from a 7% sampling of the total volume and 12% sampling of the interior of the crystal. When implemented into the beamline at Argonne National Laboratory, without ground-truth images, an acceptable reconstruction was obtained with 3% of the image sampled and approximately 5% of the crystal. The incorporation of SLADS into X-ray diffraction acquisitions has the potential to significantly minimize the impact of X-ray exposure on the crystal by

Dynamic X-ray diffraction sampling for protein crystal positioning

DOE PAGES

Scarborough, Nicole M.; Godaliyadda, G. M. Dilshan P.; Ye, Dong Hye; ...

2017-01-01

A sparse supervised learning approach for dynamic sampling (SLADS) is described for dose reduction in diffraction-based protein crystal positioning. Crystal centering is typically a prerequisite for macromolecular diffraction at synchrotron facilities, with X-ray diffraction mapping growing in popularity as a mechanism for localization. In X-ray raster scanning, diffraction is used to identify the crystal positions based on the detection of Bragg-like peaks in the scattering patterns; however, this additional X-ray exposure may result in detectable damage to the crystal prior to data collection. Dynamic sampling, in which preceding measurements inform the next most information-rich location to probe for image reconstruction,more » significantly reduced the X-ray dose experienced by protein crystals during positioning by diffraction raster scanning. The SLADS algorithm implemented herein is designed for single-pixel measurements and can select a new location to measure. In each step of SLADS, the algorithm selects the pixel, which, when measured, maximizes the expected reduction in distortion given previous measurements. Ground-truth diffraction data were obtained for a 5 µm-diameter beam and SLADS reconstructed the image sampling 31% of the total volume and only 9% of the interior of the crystal greatly reducing the X-ray dosage on the crystal. Furthermore, by usingin situtwo-photon-excited fluorescence microscopy measurements as a surrogate for diffraction imaging with a 1 µm-diameter beam, the SLADS algorithm enabled image reconstruction from a 7% sampling of the total volume and 12% sampling of the interior of the crystal. When implemented into the beamline at Argonne National Laboratory, without ground-truth images, an acceptable reconstruction was obtained with 3% of the image sampled and approximately 5% of the crystal. The incorporation of SLADS into X-ray diffraction acquisitions has the potential to significantly minimize the impact of X-ray exposure

Combination of X-ray crystallography, SAXS and DEER to obtain the structure of the FnIII-3, 4 domains of integrin α6β4

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alonso-García, Noelia; García-Rubio, Inés; Academia General Militar, Carretera de Huesca s/n, 50090 Zaragoza

The structure of the FnIII-3, 4 region of integrin β4 was solved using a hybrid approach that combines crystallographic structures, SAXS, DEER and molecular modelling. The structure helps in understanding how integrin β4 might bind to other hemidesmosomal proteins and mediate signalling. Integrin α6β4 is a major component of hemidesmosomes that mediate the stable anchorage of epithelial cells to the underlying basement membrane. Integrin α6β4 has also been implicated in cell proliferation and migration and in carcinoma progression. The third and fourth fibronectin type III domains (FnIII-3, 4) of integrin β4 mediate binding to the hemidesmosomal proteins BPAG1e and BPAG2,more » and participate in signalling. Here, it is demonstrated that X-ray crystallography, small-angle X-ray scattering and double electron–electron resonance (DEER) complement each other to solve the structure of the FnIII-3, 4 region. The crystal structures of the individual FnIII-3 and FnIII-4 domains were solved and the relative arrangement of the FnIII domains was elucidated by combining DEER with site-directed spin labelling. Multiple structures of the interdomain linker were modelled by Monte Carlo methods complying with DEER constraints, and the final structures were selected against experimental scattering data. FnIII-3, 4 has a compact and cambered flat structure with an evolutionary conserved surface that is likely to correspond to a protein-interaction site. Finally, this hybrid method is of general application for the study of other macromolecules and complexes.« less

Monitoring X-Ray Emission from X-Ray Bursters

NASA Technical Reports Server (NTRS)

Halpern, Jules P.; Kaaret, Philip

1999-01-01

The scientific goal of this project was to monitor a selected sample of x-ray bursters using data from the All-Sky Monitor (ASM) on the Rossi X-Ray Timing Explorer together with data from the Burst and Transient Source Experiment (BATSE) on the Compton Gamma-Ray Observatory to study the long-term temporal evolution of these sources in the x-ray and hard x-ray bands. The project was closely related to "Long-Term Hard X-Ray Monitoring of X-Ray Bursters", NASA project NAG5-3891, and and "Hard x-ray emission of x-ray bursters", NASA project NAG5-4633, and shares publications in common with both of these. The project involved preparation of software for use in monitoring and then the actual monitoring itself. These efforts have lead to results directly from the ASM data and also from Target of Opportunity Observations (TOO) made with the Rossi X-Ray Timing Explorer based on detection of transient hard x-ray outbursts with the ASM and BATSE.

Scalable, large area compound array refractive lens for hard X-rays

NASA Astrophysics Data System (ADS)

Reich, Stefan; dos Santos Rolo, Tomy; Letzel, Alexander; Baumbach, Tilo; Plech, Anton

2018-04-01

We demonstrate the fabrication of a 2D Compound Array Refractive Lens (CARL) for multi-contrast X-ray imaging. The CARL consists of six stacked polyimide foils with each displaying a 2D array of lenses with a 65 μm pitch aiming for a sensitivity on sub-micrometer structures with a (few-)micrometer resolution in sensing through phase and scattering contrast at multiple keV. The parabolic lenses are formed by indents in the foils by a paraboloid needle. The ability for fast single-exposure multi-contrast imaging is demonstrated by filming the kinetics of pulsed laser ablation in liquid. The three contrast channels, absorption, differential phase, and scattering, are imaged with a time resolution of 25 μs. By changing the sample-detector distance, it is possible to distinguish between nanoparticles and microbubbles.

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

PubMed Central

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

2014-01-01

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

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

DOE PAGES

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

2014-10-31

The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. With smaller crystals, high-density grids were usedmore » to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β 2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.« less

NSLS-II biomedical beamlines for micro-crystallography, FMX, and for highly automated crystallography, AMX: New opportunities for advanced data collection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fuchs, Martin R., E-mail: mfuchs@bnl.gov; Bhogadi, Dileep K.; Jakoncic, Jean

We present the final design of the x-ray optics and experimental stations of two macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source-II. The microfocusing FMX beamline will deliver a flux of ∼5×10{sup 12} ph/s at 1 Å into a 1 – 20 µm spot, its flux density surpassing current MX beamlines by up to two orders of magnitude. It covers an energy range from 5 – 30 keV. The highly automated AMX beamline is optimized for high throughput, with beam sizes from 4 – 100 µm, an energy range of 5 – 18 keV and a flux atmore » 1 Å of ∼10{sup 13} ph/s. A focus in designing the beamlines lay on achieving high beam stability, for example by implementing a horizontal bounce double crystal monochromator at FMX. A combination of compound refractive lenses and bimorph mirror optics at FMX supports rapid beam size changes. Central components of the in-house developed experimental stations are horizontal axis goniometers with a target sphere of confusion of 100 nm, piezo-slits for dynamic beam size changes during diffraction experiments, dedicated secondary goniometers for data collection from specimen in crystallization plates, and next generation pixel array detectors. FMX and AMX will support a broad range of biomedical structure determination methods from serial crystallography on micron-sized crystals, to structure determination of complexes in large unit cells, to rapid sample screening and room temperature data collection of crystals in trays.« less

Novel bimetallic thiocyanate-bridged Cu(II)-Hg(II) compounds-synthesis, X-Ray studies and magnetic properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Machura, B., E-mail: basia@ich.us.edu.pl; Switlicka, A.; Zwolinski, P.

2013-01-15

Seven novel heterobimetallic Cu/Hg polymers based on thiocyanate bridges have been synthesised and characterised by means of IR, EPR, magnetic measurements and single crystal X-Ray. Three of them, [Cu(pzH){sub 4}Hg(SCN){sub 4}]{sub n} (1) [Cu(indH){sub 4}Hg(SCN){sub 4}]{sub n} (2) and [Cu(ampy){sub 2}Hg(SCN){sub 4}]{sub n} (3), have one-dimensional coordination structure. Two compounds [Cu(pzH){sub 2}Hg(SCN){sub 4}]{sub n} (4) and [Cu(abzimH)Hg(SCN){sub 4}]{sub n} (5) form two-dimensional nets, whereas the complexes [Cu(pyCN){sub 2}Hg(SCN){sub 4}]{sub n} (6) and [Cu(pyCH(OH)(OMe)){sub 2}Hg(SCN){sub 4}]{sub n} (7) are three-dimensional coordination polymers. The chains of 1 are connected by the intermolecular N-H Bullet Bullet Bullet N hydrogen bonds to the threemore » dimensional net. In 2 the N-H Bullet Bullet Bullet S hydrogen bonds link the polymeric chains to the two dimensional layer extending along crystallographic (0 0 1) plane. The polymeric chains of compound 3 are joined by the intermolecular N-H Bullet Bullet Bullet N and N-H Bullet Bullet Bullet S hydrogen bonds to the three dimensional net. The polymeric layers of 4 are connected by the intermolecular N-H Bullet Bullet Bullet N hydrogen bonds to the three dimensional net. - Graphical abstract: Novel bimetallic thiocyanate-bridged Cu(II)-Hg(II) compound-synthesis,X-Ray studies and magnetic properties. Highlights: Black-Right-Pointing-Pointer Novel heterobimetallic Cu/Hg coordination polymers were synthesised. Black-Right-Pointing-Pointer The multidimensional structures have been proved by single X-ray analysIs. Black-Right-Pointing-Pointer A variation in the crystalline architectures was observed depending on auxiliary ligands. Black-Right-Pointing-Pointer Magnetic measurements indicate weak exchange interaction between Cu(II) in the crystal lattices below 10 K.« less

SPring-8 BL41XU, a high-flux macromolecular crystallography beamline

PubMed Central

Hasegawa, Kazuya; Shimizu, Nobutaka; Okumura, Hideo; Mizuno, Nobuhiro; Baba, Seiki; Hirata, Kunio; Takeuchi, Tomoyuki; Yamazaki, Hiroshi; Senba, Yasunori; Ohashi, Haruhiko; Yamamoto, Masaki; Kumasaka, Takashi

2013-01-01

SPring-8 BL41XU is a high-flux macromolecular crystallography beamline using an in-vacuum undulator as a light source. The X-rays are monochromated by a liquid-nitrogen-cooling Si double-crystal monochromator, and focused by Kirkpatrick–Baez mirror optics. The focused beam size at the sample is 80 µm (H) × 22 µm (V) with a photon flux of 1.1 × 1013 photons s−1. A pinhole aperture is used to collimate the beam in the range 10–50 µm. This high-flux beam with variable size provides opportunities not only for micro-crystallography but also for data collection effectively making use of crystal volume. The beamline also provides high-energy X-rays covering 20.6–35.4 keV which allows ultra-high-resolution data to be obtained and anomalous diffraction using the K-edge of Xe and I. Upgrade of BL41XU for more rapid and accurate data collection is proceeding. Here, details of BL41XU are given and an outline of the upgrade project is documented. PMID:24121338

X-ray diffraction study of the caged magnetic compound DyFe 2 Zn 20 at low temperatures

NASA Astrophysics Data System (ADS)

Ohashi, M.; Ohashi, K.; Sawabu, M.; Miyagawa, M.; Maeta, K.; Isikawa, Y.

2018-05-01

We have carried out high-angle X-ray powder diffraction measurements of the caged magnetic compound DyFe2Zn20 at low temperature between 14 and 300 K. Even though a strong magnetic anisotropy exists in the magnetization and magnetic susceptibility due to strong exchange interaction between Fe and Dy, almost all X-ray powder diffraction peaks correspond to Bragg reflections of the cubic structural models not only at room temperature paramagnetic state but also at low temperature magnetic ordering state. The Debye temperature is obtained to be 227 K from the results of the volumetric thermal expansion coefficient, which is approximately coincident with that of CeRu2Zn20 (245 K) and that of pure Zn metal (235 K).

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

PubMed

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.

Resolution of structural heterogeneity in dynamic crystallography

PubMed Central

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

2013-01-01

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

BioCARS: a synchrotron resource for time-resolved X-ray science

PubMed Central

Graber, T.; Anderson, S.; Brewer, H.; Chen, Y.-S.; Cho, H. S.; Dashdorj, N.; Henning, R. W.; Kosheleva, I.; Macha, G.; Meron, M.; Pahl, R.; Ren, Z.; Ruan, S.; Schotte, F.; Šrajer, V.; Viccaro, P. J.; Westferro, F.; Anfinrud, P.; Moffat, K.

2011-01-01

BioCARS, a NIH-supported national user facility for macromolecular time-resolved X-ray crystallography at the Advanced Photon Source (APS), has recently completed commissioning of an upgraded undulator-based beamline optimized for single-shot laser-pump X-ray-probe measurements with time resolution as short as 100 ps. The source consists of two in-line undulators with periods of 23 and 27 mm that together provide high-flux pink-beam capability at 12 keV as well as first-harmonic coverage from 6.8 to 19 keV. A high-heat-load chopper reduces the average power load on downstream components, thereby preserving the surface figure of a Kirkpatrick–Baez mirror system capable of focusing the X-ray beam to a spot size of 90 µm horizontal by 20 µm vertical. A high-speed chopper isolates single X-ray pulses at 1 kHz in both hybrid and 24-bunch modes of the APS storage ring. In hybrid mode each isolated X-ray pulse delivers up to ∼4 × 1010 photons to the sample, thereby achieving a time-averaged flux approaching that of fourth-generation X-FEL sources. A new high-power picosecond laser system delivers pulses tunable over the wavelength range 450–2000 nm. These pulses are synchronized to the storage-ring RF clock with long-term stability better than 10 ps RMS. Monochromatic experimental capability with Biosafety Level 3 certification has been retained. PMID:21685684

X-ray filter for x-ray powder diffraction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sinsheimer, John Jay; Conley, Raymond P.; Bouet, Nathalie C. D.

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 wallsmore » 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.« less

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

PubMed Central

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

Large-volume protein crystal growth for neutron macromolecular crystallography.

PubMed

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

2015-04-01

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

Exploring the palladium- and platinum-bis(pyridine) complex motif by NMR spectroscopy, X-ray crystallography, (tandem) mass spectrometry, and isothermal titration calorimetry: do substituent effects follow chemical intuition?

PubMed

Weilandt, Torsten; Löw, Nora L; Schnakenburg, Gregor; Daniels, Jörg; Nieger, Martin; Schalley, Christoph A; Lützen, Arne

2012-12-21

A series of ten palladium-bis(pyridine) complexes, as well as their corresponding platinum complexes, have been synthesized. The pyridine ligands in each series carried different σ-donor and/or π-acceptor/donor substituents at the para-position of their pyridine rings. These complexes were analysed by NMR spectroscopy, X-ray crystallography, (tandem) MS, and isothermal titration calorimetry (ITC) to validate whether these methods allowed us to obtain a concise and systematic picture of the relative and absolute thermodynamic stabilities of the complexes, as determined by the electronic effects of the substituents. Interestingly, the NMR spectroscopic data hardly correlated with the expected substituent effects but the heteronuclear platinum-phosphorus coupling constants did. Crystallographic data were found to be blurred by packing effects. Instead, tandem MS and ITC data were in line with each other and followed the expected trends. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Solvent minimization induces preferential orientation and crystal clustering in serial micro-crystallography on micro-meshes, in situ plates and on a movable crystal conveyor belt

PubMed Central

Soares, Alexei S.; Mullen, Jeffrey D.; Parekh, Ruchi M.; McCarthy, Grace S.; Roessler, Christian G.; Jackimowicz, Rick; Skinner, John M.; Orville, Allen M.; Allaire, Marc; Sweet, Robert M.

2014-01-01

X-ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial micro-crystallography: MiTeGen MicroMeshes™, Greiner Bio-One Ltd in situ micro-plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X-ray beam. 6° wedges of data were taken from ∼100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face that preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X-ray beam was incident normal to the data-collection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 µm were observed to clump together into clusters of crystals. Clustering required that the X-ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. High-quality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies. PMID:25343789

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

PubMed

Sakabe, N; Sakabe, K; Sasaki, K

2004-01-01

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

Solvent minimization induces preferential orientation and crystal clustering in serial micro-crystallography on micro-meshes, in situ plates and on a movable crystal conveyor belt

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soares, Alexei S.; Mullen, Jeffrey D.; Parekh, Ruchi M.

X-ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial micro-crystallography: MiTeGen MicroMeshes™, Greiner Bio-One Ltdin situmicro-plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X-ray beam. 6° wedges of data were taken from ~100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face thatmore » preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X-ray beam was incident normal to the data-collection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 µm were observed to clump together into clusters of crystals. Clustering required that the X-ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. Lastly, high-quality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies.« less

Solvent minimization induces preferential orientation and crystal clustering in serial micro-crystallography on micro-meshes, in situ plates and on a movable crystal conveyor belt

DOE PAGES

Soares, Alexei S.; Mullen, Jeffrey D.; Parekh, Ruchi M.; ...

2014-10-09

X-ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial micro-crystallography: MiTeGen MicroMeshes™, Greiner Bio-One Ltdin situmicro-plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X-ray beam. 6° wedges of data were taken from ~100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face thatmore » preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X-ray beam was incident normal to the data-collection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 µm were observed to clump together into clusters of crystals. Clustering required that the X-ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. Lastly, high-quality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies.« less

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

PubMed

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

2013-11-01

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

Lipidic cubic phase serial millisecond crystallography using synchrotron radiation

PubMed Central

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

2015-01-01

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

Dielectric and Impedance Characteristics of Nickel-Modified BiFeO3-BaTiO3 Electronic Compound

NASA Astrophysics Data System (ADS)

Das, S. N.; Pardhan, S. K.; Bhuyan, S.; Sahoo, S.; Choudhary, R. N. P.; Goswami, M. N.

2018-01-01

The temperature- and field-dependent capacitive, resistive and conducting characteristics of nickel-modified binary electronic systems of bismuth ferrite (BiFeO3) and barium titanate (BaTiO3) have been investigated using dielectric and impedance spectroscopy techniques. The orthorhombic crystal structures of the solid solution (Bi1-2xNixBax)(Fe1-2xTi0.2x)O3 (with x = 0.10, 0.15, 0.20 and 0.25) have been identified from powder x-ray crystallography. The micrographs exhibit the development of dense samples with reduced grain size for higher percentage of Ni in the BiFeO3-BaTiO3. The stoichiometric content of each sample has been realized using the energy dispersive x-ray technique. The relationship between micro-structural study and frequency-temperature-dependent electrical properties of the compound has revealed a negative temperature coefficient of resistance behavior. A non-Debye-type relaxation process is observed from the Niquist plot. The studied compound presents important dielectric properties for the formulation of electronic devices.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wall, Michael E.

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.

Implications of the focal beam profile in serial femtosecond crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

High-speed fixed-target serial virus crystallography

DOE PAGES

Roedig, Philip; Ginn, Helen M.; Pakendorf, Tim; ...

2017-06-19

Here, we report a method for serial X-ray crystallography at X-ray free-electron lasers (XFELs), which allows for full use of the current 120-Hz repetition rate of the Linear Coherent Light Source (LCLS). Using a micropatterned silicon chip in combination with the high-speed Roadrunner goniometer for sample delivery, we were able to determine the crystal structures of the picornavirus bovine enterovirus 2 (BEV2) and the cytoplasmic polyhedrosis virus type 18 polyhedrin, with total data collection times of less than 14 and 10 min, respectively. Our method requires only micrograms of sample and should therefore broaden the applicability of serial femtosecond crystallographymore » to challenging projects for which only limited sample amounts are available. By synchronizing the sample exchange to the XFEL repetition rate, our method allows for most efficient use of the limited beam time available at XFELs and should enable a substantial increase in sample throughput at these facilities.« less

High-speed fixed-target serial virus crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roedig, Philip; Ginn, Helen M.; Pakendorf, Tim

Here, we report a method for serial X-ray crystallography at X-ray free-electron lasers (XFELs), which allows for full use of the current 120-Hz repetition rate of the Linear Coherent Light Source (LCLS). Using a micropatterned silicon chip in combination with the high-speed Roadrunner goniometer for sample delivery, we were able to determine the crystal structures of the picornavirus bovine enterovirus 2 (BEV2) and the cytoplasmic polyhedrosis virus type 18 polyhedrin, with total data collection times of less than 14 and 10 min, respectively. Our method requires only micrograms of sample and should therefore broaden the applicability of serial femtosecond crystallographymore » to challenging projects for which only limited sample amounts are available. By synchronizing the sample exchange to the XFEL repetition rate, our method allows for most efficient use of the limited beam time available at XFELs and should enable a substantial increase in sample throughput at these facilities.« less

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

PubMed Central

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

2015-01-01

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

Advanced High Brilliance X-Ray Source

NASA Technical Reports Server (NTRS)

Gibson, Walter M.

1998-01-01

The possibility to dramatically increase the efficiency of laboratory based protein structure measurements through the use of polycapillary X-ray optics was investigated. This project initiated April 1, 1993 and concluded December 31, 1996 (including a no cost extension from June 31, 1996). This is a final report of the project. The basis for the project is the ability to collect X-rays from divergent electron bombardment laboratory X-ray sources and redirect them into quasiparallel or convergent (focused) beams. For example, a 0.1 radian (approx. 6 deg) portion of a divergent beam collected by a polycapillary collimator and transformed into a quasiparallel beam of 3 millradian (0.2 deg) could give a gain of 6(exp 2)/0.2(exp 2) x T for the intensity of a diffracted beam from a crystal with a 0.2 deg diffraction width. T is the transmission efficiency of the polycapillary diffraction optic, and for T=0.5, the gain would be 36/0.04 x O.5=45. In practice, the effective collection angle will depend on the source spot size, the input focal length of the optic (usually limited by the source spot-to-window distance on the x-ray tube) and the size of the crystal relative to the output diameter of the optic. The transmission efficiency, T, depends on the characteristics (fractional open area, surface roughness, shape and channel diameter) of the polycapillary optic and is typically in the range 0.2-0.4. These effects could substantially reduce the expected efficiency gain. During the course of this study, the possibility to use a weakly focused beam (0.5 deg convergence) was suggested which could give an additional 10-20 X efficiency gain for small samples . Weakly focused beams from double focusing mirrors are frequently used for macromolecular crystallography studies. Furthermore the crystals are typically oscillated by as much as 2 deg during each X-ray exposure in order to increase the reciprocal space (number of crystal planes) sampled and use of a slightly convergent

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

PubMed

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.

Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies.

PubMed

Sutton, Kristin A; Black, Paul J; Mercer, Kermit R; Garman, Elspeth F; Owen, Robin L; Snell, Edward H; Bernhard, William A

2013-12-01

Electron paramagnetic resonance (EPR) and online UV-visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage. Online UV-visible spectroscopy showed that upon X-irradiation, disulfide radicalization appeared to saturate at an absorbed dose of approximately 0.5-0.8 MGy, in contrast to the saturating dose of ∼0.2 MGy observed using EPR at much lower dose rates. The observations suggest that a multi-track model involving product formation owing to the interaction of two separate tracks is a valid model for radiation damage in protein crystals. The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied. The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized. Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure.

Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies

PubMed Central

Sutton, Kristin A.; Black, Paul J.; Mercer, Kermit R.; Garman, Elspeth F.; Owen, Robin L.; Snell, Edward H.; Bernhard, William A.

2013-01-01

Electron paramagnetic resonance (EPR) and online UV–visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage. Online UV–visible spectroscopy showed that upon X-irradiation, disulfide radicalization appeared to saturate at an absorbed dose of approximately 0.5–0.8 MGy, in contrast to the saturating dose of ∼0.2 MGy observed using EPR at much lower dose rates. The observations suggest that a multi-track model involving product formation owing to the interaction of two separate tracks is a valid model for radiation damage in protein crystals. The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied. The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized. Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure. PMID:24311579

XAFS and Protein Crystallography Beamline BL38B1 at SPring-8

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanida, Hajime; Miura, Keiko; Takeshita, Kunikazu

2004-05-12

The SPring-8 bending magnet beamline BL38B1 is designed for R and D of optics, detectors, experiments for XAFS and protein X-ray crystallography (PX). This beamline has a multi-purpose hutch for two experimental stations of XAFS and PX, and removable optical benches used for R and D of detectors and instruments. The design and the performance of the beamline are presented.

High transmission Ni compound refractive lens for high energy X-rays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brancewicz, M., E-mail: brancew@spring8.or.jp; Itou, M.; Sakurai, Y.

We present a new planar Ni compound refractive lens for high energy X-rays (116 keV). The lens is composed of identical plano-concave elements with longitudinal parabolic grooves manufactured by a punch technique. In order to increase the lens transmission, the thickness of the single lens at the parabolic groove vertex was reduced to less than 5 μm and the radius of curvature was reduced to about 20 μm. The small radius of curvature allowed us to reduce the number of single elements needed to get the focal length of 3 m to 54 single lenses. The gain parameter has beenmore » significantly improved compared to the previous lenses due to higher transmission, but the focused beam size and its gain are not as good as expected, mostly due to the aberrations caused by the lens shape imperfections.« less

Time-lapse crystallography snapshots of a double-strand break repair polymerase in action.

PubMed

Jamsen, Joonas A; Beard, William A; Pedersen, Lars C; Shock, David D; Moon, Andrea F; Krahn, Juno M; Bebenek, Katarzyna; Kunkel, Thomas A; Wilson, Samuel H

2017-08-15

DNA polymerase (pol) μ is a DNA-dependent polymerase that incorporates nucleotides during gap-filling synthesis in the non-homologous end-joining pathway of double-strand break repair. Here we report time-lapse X-ray crystallography snapshots of catalytic events during gap-filling DNA synthesis by pol μ. Unique catalytic intermediates and active site conformational changes that underlie catalysis are uncovered, and a transient third (product) metal ion is observed in the product state. The product manganese coordinates phosphate oxygens of the inserted nucleotide and PP i . The product metal is not observed during DNA synthesis in the presence of magnesium. Kinetic analyses indicate that manganese increases the rate constant for deoxynucleoside 5'-triphosphate insertion compared to magnesium. The likely product stabilization role of the manganese product metal in pol μ is discussed. These observations provide insight on structural attributes of this X-family double-strand break repair polymerase that impact its biological function in genome maintenance.DNA polymerase (pol) μ functions in DNA double-strand break repair. Here the authors use time-lapse X-ray crystallography to capture the states of pol µ during the conversion from pre-catalytic to product complex and observe a third transiently bound metal ion in the product state.

Designing a diverse high-quality library for crystallography-based FBDD screening.

PubMed

Tounge, Brett A; Parker, Michael H

2011-01-01

A well-chosen set of fragments is able to cover a large chemical space using a small number of compounds. The actual size and makeup of the fragment set is dependent on the screening method since each technique has its own practical limits in terms of the number of compounds that can be screened and requirements for compound solubility. In this chapter, an overview of the general requirements for a fragment library is presented for different screening platforms. In the case of the FBDD work at Johnson & Johnson Pharmaceutical Research and Development, L.L.C., our main screening technology is X-ray crystallography. Since every soaked protein crystal needs to be diffracted and a protein structure determined to delineate if a fragment binds, the size of our initial screening library cannot be a rate-limiting factor. For this reason, we have chosen 900 as the appropriate primary fragment library size. To choose the best set, we have developed our own mix of simple property ("Rule of 3") and "bad" substructure filtering. While this gets one a long way in terms of limiting the fragment pool, there are still tens of thousands of compounds to choose from after this initial step. Many of the choices left at this stage are not drug-like, so we have developed an FBDD Score to help select a 900-compound set. The details of this score and the filtering are presented. Copyright © 2011 Elsevier Inc. All rights reserved.

Exposing hidden alternative backbone conformations in X-ray crystallography using qFit

DOE PAGES

Keedy, Daniel A.; Fraser, James S.; van den Bedem, Henry; ...

2015-10-27

Proteins must move between different conformations of their native ensemble to perform their functions. Crystal structures obtained from high-resolution X-ray diffraction data reflect this heterogeneity as a spatial and temporal conformational average. Although movement between natively populated alternative conformations can be critical for characterizing molecular mechanisms, it is challenging to identify these conformations within electron density maps. Alternative side chain conformations are generally well separated into distinct rotameric conformations, but alternative backbone conformations can overlap at several atomic positions. Our model building program qFit uses mixed integer quadratic programming (MIQP) to evaluate an extremely large number of combinations of sidechainmore » conformers and backbone fragments to locally explain the electron density. Here, we describe two major modeling enhancements to qFit: peptide flips and alternative glycine conformations. We find that peptide flips fall into four stereotypical clusters and are enriched in glycine residues at the n+1 position. The potential for insights uncovered by new peptide flips and glycine conformations is exemplified by HIV protease, where different inhibitors are associated with peptide flips in the “flap” regions adjacent to the inhibitor binding site. Our results paint a picture of peptide flips as conformational switches, often enabled by glycine flexibility, that result in dramatic local rearrangements. Our results furthermore demonstrate the power of large-scale computational analysis to provide new insights into conformational heterogeneity. Furthermore, improved modeling of backbone heterogeneity with high-resolution X-ray data will connect dynamics to the structure-function relationship and help drive new design strategies for inhibitors of biomedically important systems.« less

Large-volume protein crystal growth for neutron macromolecular crystallography

DOE PAGES

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

2015-03-30

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

Large-volume protein crystal growth for neutron macromolecular crystallography

PubMed Central

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

2015-01-01

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

Large-volume protein crystal growth for neutron macromolecular crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

Unified Theory for Decoding the Signals from X-Ray Florescence and X-Ray Diffraction of Mixtures.

PubMed

Chung, Frank H

2017-05-01

For research and development or for solving technical problems, we often need to know the chemical composition of an unknown mixture, which is coded and stored in the signals of its X-ray fluorescence (XRF) and X-ray diffraction (XRD). X-ray fluorescence gives chemical elements, whereas XRD gives chemical compounds. The major problem in XRF and XRD analyses is the complex matrix effect. The conventional technique to deal with the matrix effect is to construct empirical calibration lines with standards for each element or compound sought, which is tedious and time-consuming. A unified theory of quantitative XRF analysis is presented here. The idea is to cancel the matrix effect mathematically. It turns out that the decoding equation for quantitative XRF analysis is identical to that for quantitative XRD analysis although the physics of XRD and XRF are fundamentally different. The XRD work has been published and practiced worldwide. The unified theory derives a new intensity-concentration equation of XRF, which is free from the matrix effect and valid for a wide range of concentrations. The linear decoding equation establishes a constant slope for each element sought, hence eliminating the work on calibration lines. The simple linear decoding equation has been verified by 18 experiments.

Structural dissection of human metapneumovirus phosphoprotein using small angle x-ray scattering.

PubMed

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.

X-ray Magnetic Scattering From Surfaces^*

NASA Astrophysics Data System (ADS)

Gibbs, Doon

1997-03-01

In the last several years, there have been continuing efforts to probe long-ranged magnetic order at surfaces by x-ray and neutron diffraction, following many earlier studies by low energy electron diffraction. The main motivation has been to discover how bulk magnetic structures are modified near a surface, where the crystal symmetry is broken. In this talk, we describe x-ray scattering studies of the magnetic structure observed near the (001) surface of the antiferromagnet uranium dioxide.(G. M. Watson, Doon Gibbs, G. H. Lander, B. D. Gaulin, L.E. Berman, Hj. Matzke and W. Ellis, Phys. Rev. Lett. 77), 751 (1996). Within about 50 Åof the surface, the intensity of the magnetic scattering decreases continuously as the bulk Neel temperature is approached from below. This contrasts with the bulk magnetic ordering transition which is discontinuous. Recent measurements of the specular magnetic reflectivity suggest that the width of the magnetic interface diverges as a power-law in reduced temperature reminiscent of surface induced disorder. Related experiments concerned with magnetic crystallography of Co_3-Pt(111) surfaces(S. Ferrer, P. Fajardo, F. de Bergevin, J. Alvarez, X. Torrelles, H. A. van der Vegt and V. H. Etgens, Phys. Rev. Lett. 77), 747 (1996). and interfacial magnetic roughness of Co/Cu multilayers(J. F. MacKay, C. Teichert, D.E. Savage and M.G. Lagally, Phys. Rev. Lett. 77), 3925 (1996). will also be discussed. ^* Work at Brookhaven National Laboratory is supported by the U.S. DOE under Contract No. DE-AC02-CH7600016.

Au133(SPh-tBu)52 nanomolecules: X-ray crystallography, optical, electrochemical, and theoretical analysis.

PubMed

Dass, Amala; Theivendran, Shevanuja; Nimmala, Praneeth Reddy; Kumara, Chanaka; Jupally, Vijay Reddy; Fortunelli, Alessandro; Sementa, Luca; Barcaro, Giovanni; Zuo, Xiaobing; Noll, Bruce C

2015-04-15

Crystal structure determination has revolutionized modern science in biology, chemistry, and physics. However, the difficulty in obtaining periodic crystal lattices which are needed for X-ray crystal analysis has hindered the determination of atomic structure in nanomaterials, known as the "nanostructure problem". Here, by using rigid and bulky ligands, we have overcome this limitation and successfully solved the X-ray crystallographic structure of the largest reported thiolated gold nanomolecule, Au133S52. The total composition, Au133(SPh-tBu)52, was verified using high resolution electrospray ionization mass spectrometry (ESI-MS). The experimental and simulated optical spectra show an emergent surface plasmon resonance that is more pronounced than in the slightly larger Au144(SCH2CH2Ph)60. Theoretical analysis indicates that the presence of rigid and bulky ligands is the key to the successful crystal formation.

Single crystal X-ray structure of the artists' pigment zinc yellow

NASA Astrophysics Data System (ADS)

Simonsen, Kim Pilkjær; Christiansen, Marie Bitsch; Vinum, Morten Gotthold; Sanyova, Jana; Bendix, Jesper

2017-08-01

The artists' pigment zinc yellow is in general described as a complex potassium zinc chromate with the empirical formula 4ZnCrO4·K2O·3H2O. Even though the pigment has been in use since the second half of the 19th century also in large-scale industrial applications, the exact structure had hitherto been unknown. In this work, zinc yellow was synthesised by precipitation from an aqueous solution of zinc nitrate and potassium chromate under both neutral and basic conditions, and the products were compared with the pigment used in industrial paints. Analyses by Raman microscopy (MRS), scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and powder X-ray diffraction (PXRD), showed that the synthesised products and the industrial pigment were identical. Single-crystal X-ray crystallography determined the structure of zinc yellow as KZn2(CrO4)2(H2O)(OH) or as KZn2(CrO4)2(H3O2) emphasizing the μ-H3O2- moiety. Notably, the zinc yellow is isostructural to the recently structurally characterized cadmium analog and both belong to the natrochalcite structure type.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sierra, Raymond G.; Gati, Cornelius; Laksmono, Hartawan

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

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

PubMed

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

2016-01-01

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

Study of chemical shift in Kα, Kβ1,3 and Kβ// X-ray emission lines of 37Rb compounds with WDXRF

NASA Astrophysics Data System (ADS)

Kainth, Harpreet Singh; Singh, Ranjit; Singh, Tejbir; Mehta, D.; Shahi, J. S.; Kumar, Sanjeev

2018-05-01

The positive and negative chemical shifts in Kα, Kβ1,3 and Kβ// X-ray emission lines of rubidium compounds were measured with high resolution WDXRF spectrometer. The measured energy shifts in Kα emission lines ranges from -2.95 eV to -3.64 eV, Kβ1,3 emission lines ranges from 1.16 eV to 1.32 eV and Kβ// emission lines ranges from 1.31 eV to 4.36 eV respectively. In the present work, it has been found that chemical shift in Kβ// X-ray emission lines were found to be larger than Kα and Kβ1,3 X-ray emission lines. To find the cause of chemical shift, various factors like effective charge, line intensity ratio, bond length and electro-negativity were calculated and correlated with the chemical shift.

Analysis of RNA structure using small-angle X-ray scattering

PubMed Central

Cantara, William A.; Olson, Erik D.; Musier-Forsyth, Karin

2016-01-01

In addition to their role in correctly attaching specific amino acids to cognate tRNAs, aminoacyl-tRNA synthetases (aaRS) have been found to possess many alternative functions and often bind to and act on other nucleic acids. In contrast to the well-defined 3D structure of tRNA, the structures of many of the other RNAs recognized by aaRSs have not been solved. Despite advances in the use of X-ray crystallography (XRC), nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM) for structural characterization of biomolecules, significant challenges to solving RNA structures still exist. Recently, small-angle X-ray scattering (SAXS) has been increasingly employed to characterize the 3D structures of RNAs and RNA-protein complexes. SAXS is capable of providing low-resolution tertiary structure information under physiological conditions and with less intensive sample preparation and data analysis requirements than XRC, NMR and cryo-EM. In this article, we describe best practices involved in the process of RNA and RNA-protein sample preparation, SAXS data collection, data analysis, and structural model building. PMID:27777026

Sulfur K-edge extended X-ray absorption fine structure spectroscopy of homoleptic thiolato complexes with Zn(II) and Cd(II).

PubMed

Matsunaga, Yuki; Fujisawa, Kiyoshi; Ibi, Naoko; Fujita, Mitsuharu; Ohashi, Tetuya; Amir, Nagina; Miyashita, Yoshitaro; Aika, Ken-Ichi; Izumi, Yasuo; Okamoto, Ken-Ichi

2006-02-01

The sulfur K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy is applied to homoleptic thiolato complexes with Zn(II) and Cd(II), (Et(4)N)[Zn(SAd)(3)] (1), (Et(4)N)(2)[{Zn(ScHex)(2)}(2)(mu-ScHex)(2)] (2), (Et(4)N)(2)[{Cd(ScHex)(2)}(2)(mu-ScHex)(2)] (3), (Et(4)N)(2)[{Cd(ScHex)}(4)(mu-ScHex)(6)] (4), [Zn(mu-SAd)(2)](n) (5), and [Cd(mu-SAd)(2)](n) (6) (HSAd=1-adamantanethiol, HScHex=cyclohexanethiol). The EXAFS results are consistent with the X-ray crystal data of 1-4. The structures of 5 and 6, which have not been determined by X-ray crystallography, are proposed to be polynuclear structures on the basis of the sulfur K-edge EXAFS, far-IR spectra, and elemental analysis. Clear evidences of the S...S interactions (between bridging atoms or neighboring sulfur atoms) and the S...C(far) interactions (in which C(far) atom is next to carbon atom directly bonded to sulfur atom) were observed in the EXAFS data for all complexes and thus lead to the reliable determination of the structures of 5 and 6 in combination with conventional zinc K-edge EXAFS analysis for 5. This new methodology, sulfur K-edge EXAFS, could be applied for the structural determination of in vivo metalloproteins as well as inorganic compounds.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feld, Geoffrey K.; Heymann, Michael; Benner, W. Henry

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 constructedmore » 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.« less

"XANSONS for COD": a new small BOINC project in crystallography

NASA Astrophysics Data System (ADS)

Neverov, Vladislav S.; Khrapov, Nikolay P.

2018-04-01

"XANSONS for COD" (http://xansons4cod.com) is a new BOINC project aimed at creating the open-access database of simulated x-ray and neutron powder diffraction patterns for nanocrystalline phase of materials from the collection of the Crystallography Open Database (COD). The project uses original open-source software XaNSoNS to simulate diffraction patterns on CPU and GPU. This paper describes the scientific problem this project solves, the project's internal structure, its operation principles and organization of the final database.

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

PubMed

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.

A Practical Approach to Protein Crystallography.

PubMed

Ilari, Andrea; Savino, Carmelinda

2017-01-01

Macromolecular crystallography is a powerful tool for structural biology. The resolution of a protein crystal structure is becoming much easier than in the past, thanks to developments in computing, automation of crystallization techniques and high-flux synchrotron sources to collect diffraction datasets. The aim of this chapter is to provide practical procedures to determine a protein crystal structure, illustrating the new techniques, experimental methods, and software that have made protein crystallography a tool accessible to a larger scientific community.It is impossible to give more than a taste of what the X-ray crystallographic technique entails in one brief chapter and there are different ways to solve a protein structure. Since the number of structures available in the Protein Data Bank (PDB) is becoming ever larger (the protein data bank now contains more than 100,000 entries) and therefore the probability to find a good model to solve the structure is ever increasing, we focus our attention on the Molecular Replacement method. Indeed, whenever applicable, this method allows the resolution of macromolecular structures starting from a single data set and a search model downloaded from the PDB, with the aid only of computer work.

Au133(SPh-tBu)52 Nanomolecules: X-ray Crystallography, Optical, Electrochemical, and Theoretical Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dass, Amala; Theivendran, Shevanuja; Nimmala, Praneeth Reddy

2015-04-15

Crystal structure determination has revolutionized modern science in biology, chemistry, and physics. However, the difficulty in obtaining periodic crystal lattices which are needed for X-ray crystal analysis has hindered the determination of atomic structure in nanomaterials, known as the “nanostructure problem”. Here, by using rigid and bulky ligands, we have overcome this limitation and successfully solved the X-ray crystallographic structure of the largest reported thiolated gold nanomolecule, Au133S52. The total composition, Au133(SPh-tBu)52, was verified using high resolution electrospray ionization mass spectrometry (ESI-MS). The experimental and simulated optical spectra show an emergent surface plasmon resonance that is more pronounced than inmore » the slightly larger Au144(SCH2CH2Ph)60. Theoretical analysis indicates that the presence of rigid and bulky ligands is the key to the successful crystal formation.« less

Au 133 (SPh - t Bu) 52 Nanomolecules: X-ray Crystallography, Optical, Electrochemical, and Theoretical Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dass, Amala; Theivendran, Shevanuja; Nimmala, Praneeth Reddy

2015-04-15

Crystal structure determination has revolutionized modern science in biology, chemistry, and physics. However, the difficulty in obtaining periodic crystal lattices which are needed for X-ray crystal analysis has hindered the determination of atomic structure in nanomaterials, known as the "nanostructure problem". Here, by using rigid and bulky ligands, we have overcome this limitation and successfully solved the X-ray crystallographic structure of the largest reported thiolated gold nanomolecule, Au133S52. The total composition, Au-133(SPh-tBu)(52), was verified using high resolution electrospray ionization mass spectrometry (ESI-MS). The experimental and simulated optical spectra show an emergent surface plasmon resonance that is more pronounced than inmore » the slightly larger Au-144(SCH2CH2Ph)(60). Theoretical analysis indicates that the presence of rigid and bulky ligands is the key to the successful crystal formation.« less

Synthesis, X-ray crystallography, spectroscopic (FT-IR, 1H &13C NMR and UV), computational (DFT/B3LYP) and enzymes inhibitory studies of 7-hydroximinocholest-5-en-3-ol acetate

NASA Astrophysics Data System (ADS)

Ahmad, Faheem; Parveen, Mehtab; Alam, Mahboob; Azaz, Shaista; Malla, Ali Mohammed; Alam, Mohammad Jane; Lee, Dong-Ung; Ahmad, Shabbir

2016-07-01

The present study reports the synthesis of 7-Hydroximinocholest-5-en-3-ol acetate (syn. 3β-acetoxycholest-5-en-7-one oxime; in general, steroidal oxime). The identity of steroidal molecule was confirmed by NMR, FT-IR, MS, CHN microanalysis and X-ray crystallography. DFT calculations on the titled molecule have been performed. The molecular structure and spectra interpreted by Gaussian hybrid computational analysis theory (B3LYP) are found to be in good correlation with the experimental data obtained from the various spectrophotometric techniques. The vibrational bands appearing in the FTIR are assigned with great accuracy using harmonic frequencies along with intensities and animated modes. Molecular properties like HOMO-LUMO analysis, chemical reactivity descriptors, MEP mapping, dipole moment and natural atomic charges have been presented at the same level of theory. Moreover, the Hirshfeld analysis was carried out to ascertain the secondary interactions and associated 2D fingerprint plots. The percentages of various interactions are pictorialized by fingerprint plots of Hirshfeld surface. Steroidal oxime exhibited promising inhibitory activity against acetylcholinesterase (AChE) as compared to the reference drug, tacrine. Molecular docking was performed to introduce steroidal molecules into the X-ray crystal structures of acetylcholinesterase at the active site to find out the probable binding mode. The results of molecular docking admitted that steroidal oxime may exhibit enzyme inhibitor activity.

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

PubMed

Ishchenko, Andrii; Cherezov, Vadim; Liu, Wei

2016-09-20

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

High-resolution x-ray absorption spectroscopy studies of metal compounds in neurodegenerative brain tissue

NASA Astrophysics Data System (ADS)

Collingwood, J. F.; Mikhaylova, A.; Davidson, M. R.; Batich, C.; Streit, W. J.; Eskin, T.; Terry, J.; Barrea, R.; Underhill, R. S.; Dobson, J.

2005-01-01

Fluorescence mapping and microfocus X-ray absorption spectroscopy are used to detect, locate and identify iron biominerals and other inorganic metal accumulations in neurodegenerative brain tissue at sub-cellular resolution (<5 microns). Recent progress in developing the technique is reviewed. Synchrotron X-rays are used to map tissue sections for metals of interest, and XANES and XAFS are used to characterise anomalous concentrations of the metals in-situ so that they can be correlated with tissue structures and disease pathology. Iron anomalies associated with biogenic magnetite, ferritin and haemoglobin are located and identified in an avian tissue model with a pixel resolution ~5 microns. Subsequent studies include brain tissue sections from transgenic Huntington's mice, and the first high-resolution mapping and identification of iron biominerals in human Alzheimer's and control autopsy brain tissue. Technical developments include use of microfocus diffraction to obtain structural information about biominerals in-situ, and depositing sample location grids by lithography for the location of anomalies by conventional microscopy. The combined techniques provide a breakthrough in the study of both intra- and extra-cellular iron compounds and related metals in tissue. The information to be gained from this approach has implications for future diagnosis and treatment of neurodegeneration, and for our understanding of the mechanisms involved.

Fine refinement of solid-state molecular structures of Leu- and Met-enkephalins by NMR crystallography.

PubMed

Pawlak, Tomasz; Potrzebowski, Marek J

2014-03-27

This paper presents a methodology that allows the fine refinement of the crystal and molecular structure for compounds for which the data deposited in the crystallographic bases are of poor quality. Such species belong to the group of samples with molecular disorder. In the Cambridge Crystallographic Data Center (CCDC), there are approximately 22,000 deposited structures with an R-factor over 10. The powerful methodology we present employs crystal data for Leu-enkephalin (two crystallographic forms) with R-factor values of 14.0 and 8.9 and for Met-enkephalin (one form) with an R-factor of 10.5. NMR crystallography was employed in testing the X-ray data and the quality of the structure refinement. The GIPAW (gauge invariant projector augmented wave) method was used to optimize the coordinates of the enkephalins and to compute NMR parameters. As we reveal, this complementary approach makes it possible to generate a reasonable set of new coordinates that better correlate to real samples. This methodology is general and can be employed in the study of each compound possessing magnetically active nuclei.

X-Rays

MedlinePlus

X-rays are a type of radiation called electromagnetic waves. X-ray imaging creates pictures of the inside of ... different amounts of radiation. Calcium in bones absorbs x-rays the most, so bones look white. Fat ...

Structural investigation of (2E)-2-(ethoxycarbonyl)-3-[(4-methoxyphenyl)amino]prop-2-enoic acid: X-ray crystal structure, spectroscopy and DFT

NASA Astrophysics Data System (ADS)

Venkatesan, Perumal; Rajakannan, Venkatachalam; Venkataramanan, Natarajan S.; Ilangovan, Andivelu; Sundius, Tom; Thamotharan, Subbiah

2016-09-01

The title compound, (2E)-2-(ethoxycarbonyl)-3-[(4-methoxyphenyl)amino]prop-2-enoic acid is characterized by means of X-ray crystallography, spectroscopic methods and quantum chemical calculations. The title compound crystallizes in centrosymmetric space group P21/c. Moreover, the crystal structure is primarily stabilized through intramolecular Nsbnd H⋯O and Osbnd H⋯O and intermolecular Nsbnd H⋯O and Csbnd H⋯O interactions along with carbonyl⋯carbonyl and Csbnd H⋯C contacts. These intermolecular interactions are analysed and quantified by using Hirshfeld surface analysis, PIXEL energy, NBO, AIM and DFT calculations. The overall lattice energies of the title and parent compounds suggest that the title compound is stabilized by a 4.5 kcal mol-1 higher energy than the parent compound. The additional stabilization force comes from the methoxy substitution on the title molecule, which is evident since the methoxy group is involved in the intermolecular Csbnd H⋯O interaction as an acceptor. The vibrational modes of the interacting groups are investigated using both experimental and theoretical FT-IR and FT-Raman spectra. The experimental and theoretical UV-Vis spectra agree well. The time dependent DFT spectra show that the ligand-to-ligand charge transfer is responsible for the intense absorbance of the compound.

Refractive Optics for Hard X-ray Transmission Microscopy

NASA Astrophysics Data System (ADS)

Simon, M.; Ahrens, G.; Last, A.; Mohr, J.; Nazmov, V.; Reznikova, E.; Voigt, A.

2011-09-01

For hard x-ray transmission microscopy at photon energies higher than 15 keV we design refractive condenser and imaging elements to be used with synchrotron light sources as well as with x-ray tube sources. The condenser lenses are optimized for low x-ray attenuation—resulting in apertures greater than 1 mm—and homogeneous intensity distribution on the detector plane, whereas the imaging enables high-resolution (<100 nm) full-field imaging. To obtain high image quality at reasonable exposure times, custom-tailored matched pairs of condenser and imaging lenses are being developed. The imaging lenses (compound refractive lenses, CRLs) are made of SU-8 negative resist by deep x-ray lithography. SU-8 shows high radiation stability. The fabrication technique enables high-quality lens structures regarding surface roughness and arrangement precision with arbitrary 2D geometry. To provide point foci, crossed pairs of lenses are used. Condenser lenses have been made utilizing deep x-ray lithographic patterning of thick SU-8 layers, too, whereas in this case, the aperture is limited due to process restrictions. Thus, in terms of large apertures, condenser lenses made of structured and rolled polyimide film are more attractive. Both condenser types, x-ray mosaic lenses and rolled x-ray prism lenses (RXPLs), are considered to be implemented into a microscope setup. The x-ray optical elements mentioned above are characterized with synchrotron radiation and x-ray laboratory sources, respectively.

Fast two-dimensional grid and transmission X-ray microscopy scanning methods for visualizing and characterizing protein crystals

PubMed Central

Wojdyla, Justyna Aleksandra; Panepucci, Ezequiel; Martiel, Isabelle; Ebner, Simon; Huang, Chia-Ying; Caffrey, Martin; Bunk, Oliver; Wang, Meitian

2016-01-01

A fast continuous grid scan protocol has been incorporated into the Swiss Light Source (SLS) data acquisition and analysis software suite on the macromolecular crystallography (MX) beamlines. Its combination with fast readout single-photon counting hybrid pixel array detectors (PILATUS and EIGER) allows for diffraction-based identification of crystal diffraction hotspots and the location and centering of membrane protein microcrystals in the lipid cubic phase (LCP) in in meso in situ serial crystallography plates and silicon nitride supports. Diffraction-based continuous grid scans with both still and oscillation images are supported. Examples that include a grid scan of a large (50 nl) LCP bolus and analysis of the resulting diffraction images are presented. Scanning transmission X-ray microscopy (STXM) complements and benefits from fast grid scanning. STXM has been demonstrated at the SLS beamline X06SA for near-zero-dose detection of protein crystals mounted on different types of sample supports at room and cryogenic temperatures. Flash-cooled crystals in nylon loops were successfully identified in differential and integrated phase images. Crystals of just 10 µm thickness were visible in integrated phase images using data collected with the EIGER detector. STXM offers a truly low-dose method for locating crystals on solid supports prior to diffraction data collection at both synchrotron microfocusing and free-electron laser X-ray facilities. PMID:27275141

Cost-effective masks for deep x-ray lithography

NASA Astrophysics Data System (ADS)

Scheunemann, Heinz-Ulrich; Loechel, Bernd; Jian, Linke; Schondelmaier, Daniel; Desta, Yohannes M.; Goettert, Jost

2003-04-01

The production of X-ray masks is one of the key techniques for X-ray lithography and the LIGA process. Different ways for the fabrication of X-ray masks has been established. Very sophisticated, difficult and expensive procedures are required to produce high precision and high quality X-ray masks. In order to minimize the cost of an X-ray mask, the mask blank must be inexpensive and readily available. The steps involved in the fabrication process must also be minimal. In the past, thin membranes made of titanium, silicon carbide, silicon nitride (2-5μm) or thick beryllium substrates (500μm) have been used as mask blanks. Thin titanium and silicon compounds have very high transparency for X-rays; therefore, these materials are predestined for use as mask membrane material. However, the handling and fabrication of thin membranes is very difficult, thus expensive. Beryllium is highly transparent to X-rays, but the processing and use of beryllium is risky due to potential toxicity. During the past few years graphite based X-ray masks have been in use at various research centers, but the sidewall quality of the generated resist patterns is in the range of 200-300 nm Ra. We used polished graphite to improve the sidewall roughness, but polished graphite causes other problems in the fabrication of X-ray masks. This paper describes the advantages associated with the use of polished graphite as mask blank as well as the fabrication process for this low cost X-ray mask. Alternative membrane materials will also be discussed.

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

PubMed Central

Kang, Yanyong; Zhou, X. Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W.; Ke, Jiyuan; Eileen Tan, M. H.; Zhang, Chenghai; Moeller, Arne; West, Graham M.; Pascal, Bruce; Van Eps, Ned; Caro, Lydia N.; Vishnivetskiy, Sergey A.; Lee, Regina J.; Suino-Powell, Kelly M.; Gu, Xin; Pal, Kuntal; Ma, Jinming; Zhi, Xiaoyong; Boutet, Sébastien; Williams, Garth J.; Messerschmidt, Marc; Gati, Cornelius; Zatsepin, Nadia A.; Wang, Dingjie; James, Daniel; Basu, Shibom; Roy-Chowdhury, Shatabdi; Conrad, Chelsie; Coe, Jesse; Liu, Haiguang; Lisova, Stella; Kupitz, Christopher; Grotjohann, Ingo; Fromme, Raimund; Jiang, Yi; Tan, Minjia; Yang, Huaiyu; Li, Jun; Wang, Meitian; Zheng, Zhong; Li, Dianfan; Howe, Nicole; Zhao, Yingming; Standfuss, Jörg; Diederichs, Kay; Dong, Yuhui; Potter, Clinton S; Carragher, Bridget; Caffrey, Martin; Jiang, Hualiang; Chapman, Henry N.; Spence, John C. H.; Fromme, Petra; Weierstall, Uwe; Ernst, Oliver P.; Katritch, Vsevolod; Gurevich, Vsevolod V.; Griffin, Patrick R.; Hubbell, Wayne L.; Stevens, Raymond C.; Cherezov, Vadim; Melcher, Karsten; Xu, H. Eric

2015-01-01

G protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly, in which rhodopsin uses distinct structural elements, including TM7 and Helix 8 to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotation between the N- and C- domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signaling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology. PMID:26200343

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

PubMed

Kang, Yanyong; Zhou, X Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W; Ke, Jiyuan; Tan, M H Eileen; Zhang, Chenghai; Moeller, Arne; West, Graham M; Pascal, Bruce D; Van Eps, Ned; Caro, Lydia N; Vishnivetskiy, Sergey A; Lee, Regina J; Suino-Powell, Kelly M; Gu, Xin; Pal, Kuntal; Ma, Jinming; Zhi, Xiaoyong; Boutet, Sébastien; Williams, Garth J; Messerschmidt, Marc; Gati, Cornelius; Zatsepin, Nadia A; Wang, Dingjie; James, Daniel; Basu, Shibom; Roy-Chowdhury, Shatabdi; Conrad, Chelsie E; Coe, Jesse; Liu, Haiguang; Lisova, Stella; Kupitz, Christopher; Grotjohann, Ingo; Fromme, Raimund; Jiang, Yi; Tan, Minjia; Yang, Huaiyu; Li, Jun; Wang, Meitian; Zheng, Zhong; Li, Dianfan; Howe, Nicole; Zhao, Yingming; Standfuss, Jörg; Diederichs, Kay; Dong, Yuhui; Potter, Clinton S; Carragher, Bridget; Caffrey, Martin; Jiang, Hualiang; Chapman, Henry N; Spence, John C H; Fromme, Petra; Weierstall, Uwe; Ernst, Oliver P; Katritch, Vsevolod; Gurevich, Vsevolod V; Griffin, Patrick R; Hubbell, Wayne L; Stevens, Raymond C; Cherezov, Vadim; Melcher, Karsten; Xu, H Eric

2015-07-30

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

A novel one-dimensional manganese(II) coordination polymer containing both dicyanamide and pyrazinamide ligands: Synthesis, spectroscopic investigations, X-ray studies and evaluation of biological activities

NASA Astrophysics Data System (ADS)

Tabrizi, Leila; Chiniforoshan, Hossein; McArdle, Patrick

2015-03-01

A novel 1D coordination polymer {[Mn(μ1,5-dca)2(PZA)2](PZA)2}n, 1, has been synthesized and characterized by single crystal X-ray crystallography. The coordination mode of dicyanamide (dca) and pyrazinamide (PZA) ligands was inferred by IR spectroscopy. The compound 1 was evaluated for in vitro antimycobacterial and antitumor activities. It demonstrated better in vitro activity against Mycobacterium tuberculosis than pyrazinamide and its MIC value was determined. Complex 1 was also screened for its in vitro antitumor activity towards LM3 and LP07 murine cancer cell lines. In addition, the antibacterial activity of complex 1 has been tested against Gram(+) and Gram(-) bacteria and it has shown promising broad range anti-bacterial activity.

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

PubMed

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sierra, Raymond G.; Gati, Cornelius; Laksmono, Hartawan

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

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

DOE PAGES

Sierra, Raymond G.; Gati, Cornelius; Laksmono, Hartawan; ...

2015-11-30

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

Overview of electron crystallography of membrane proteins: crystallization and screening strategies using negative stain electron microscopy.

PubMed

Nannenga, Brent L; Iadanza, Matthew G; Vollmar, Breanna S; Gonen, Tamir

2013-01-01

Electron cryomicroscopy, or cryoEM, is an emerging technique for studying the three-dimensional structures of proteins and large macromolecular machines. Electron crystallography is a branch of cryoEM in which structures of proteins can be studied at resolutions that rival those achieved by X-ray crystallography. Electron crystallography employs two-dimensional crystals of a membrane protein embedded within a lipid bilayer. The key to a successful electron crystallographic experiment is the crystallization, or reconstitution, of the protein of interest. This unit describes ways in which protein can be expressed, purified, and reconstituted into well-ordered two-dimensional crystals. A protocol is also provided for negative stain electron microscopy as a tool for screening crystallization trials. When large and well-ordered crystals are obtained, the structures of both protein and its surrounding membrane can be determined to atomic resolution.

Two-dimensional single crystal diamond refractive x-ray lens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Antipov, S., E-mail: s.antipov@euclidtechlabs.com; Baryshev, S. V.; Butler, J. E.

2016-07-27

The next generation light sources such as diffraction-limited storage rings and high repetition rate x-ray free-electron lasers will generate x-ray beams with significantly increased brilliance. These future machines will require X-ray optical components that are capable of handling higher instantaneous and average power densities while tailoring the properties of the x-ray beams for a variety of scientific experiments. Single crystal diamond is one of the best bulk materials for this application, because it is radiation hard, has a suitable uniform index of refraction and the best available thermal properties. In this paper we report on fabrication and experimental testing ofmore » a two-dimensional (2D) single crystal diamond compound refractive X-ray lenses (CRL). These lenses were manufactured using femto-second laser cutting and tested at the Advanced Photon Source of Argonne National Laboratory.« less

Unusual mode of protein binding by a cytotoxic π-arene ruthenium(ii) piano-stool compound containing an O,S-chelating ligand.

PubMed

Hildebrandt, Jana; Görls, Helmar; Häfner, Norman; Ferraro, Giarita; Dürst, Matthias; Runnebaum, Ingo B; Weigand, Wolfgang; Merlino, Antonello

2016-08-02

A new pseudo-octahedral π-arene ruthenium(ii) piano-stool compound, containing an O,S-bidentate ligand (compound 1) and showing significant cytotoxic activity in vitro, was synthesized and characterized. In solution stability and interaction with the model protein bovine pancreatic ribonuclease (RNase A) were investigated by using UV-Vis absorption spectroscopy. Its crystal structure and that of the adduct formed upon reaction with RNase A were obtained by X-ray crystallography. The comparison between the structure of purified compound 1 and that of the fragment bound to RNase A reveals an unusual mode of protein binding that includes ligand exchange and alteration of coordination sphere geometry.

Predicting X-ray diffuse scattering from translation–libration–screw structural ensembles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Benschoten, Andrew H.; Afonine, Pavel V.; Terwilliger, Thomas C.

2015-07-28

A method of simulating X-ray diffuse scattering from multi-model PDB files is presented. Despite similar agreement with Bragg data, different translation–libration–screw refinement strategies produce unique diffuse intensity patterns. Identifying the intramolecular motions of proteins and nucleic acids is a major challenge in macromolecular X-ray crystallography. Because Bragg diffraction describes the average positional distribution of crystalline atoms with imperfect precision, the resulting electron density can be compatible with multiple models of motion. Diffuse X-ray scattering can reduce this degeneracy by reporting on correlated atomic displacements. Although recent technological advances are increasing the potential to accurately measure diffuse scattering, computational modeling andmore » validation tools are still needed to quantify the agreement between experimental data and different parameterizations of crystalline disorder. A new tool, phenix.diffuse, addresses this need by employing Guinier’s equation to calculate diffuse scattering from Protein Data Bank (PDB)-formatted structural ensembles. As an example case, phenix.diffuse is applied to translation–libration–screw (TLS) refinement, which models rigid-body displacement for segments of the macromolecule. To enable the calculation of diffuse scattering from TLS-refined structures, phenix.tls-as-xyz builds multi-model PDB files that sample the underlying T, L and S tensors. In the glycerophosphodiesterase GpdQ, alternative TLS-group partitioning and different motional correlations between groups yield markedly dissimilar diffuse scattering maps with distinct implications for molecular mechanism and allostery. These methods demonstrate how, in principle, X-ray diffuse scattering could extend macromolecular structural refinement, validation and analysis.« less

High pressure effects on U L 3 x-ray absorption in partial fluorescence yield mode and single crystal x-ray diffraction in the heavy fermion compound UCd 11

DOE PAGES

Nasreen, Farzana; Antonio, Daniel; VanGennep, Derrick; ...

2016-02-15

© 2016 IOP Publishing Ltd. We report a study of high pressure x-ray absorption (XAS) performed in the partial fluorescence yield mode (PFY) at the U L 3 edge (0-28.2 GPa) and single crystal x-ray diffraction (SXD) (0-20 GPa) on the UCd 11 heavy fermion compound at room temperature. Under compression, the PFY-XAS results show that the white line is shifted by +4.1(3) eV at the highest applied pressure of 28.2 GPa indicating delocalization of the 5f electrons. The increase in full width at half maxima and decrease in relative amplitude of the white line with respect to the edgemore » jump point towards 6d band broadening under high pressure. A bulk modulus of K 0 = 62(1) GPa and its pressure derivative, = 4.9(2) was determined from high pressure SXD results. Both the PFY-XAS and diffraction results do not show any sign of a structural phase transition in the applied pressure range.« less

X-ray scattering data and structural genomics

NASA Astrophysics Data System (ADS)

Doniach, Sebastian

2003-03-01

High throughput structural genomics has the ambitious goal of determining the structure of all, or a very large number of protein folds using the high-resolution techniques of protein crystallography and NMR. However, the program is facing significant bottlenecks in reaching this goal, which include problems of protein expression and crystallization. In this talk, some preliminary results on how the low-resolution technique of small-angle X-ray solution scattering (SAXS) can help ameliorate some of these bottlenecks will be presented. One of the most significant bottlenecks arises from the difficulty of crystallizing integral membrane proteins, where only a handful of structures are available compared to thousands of structures for soluble proteins. By 3-dimensional reconstruction from SAXS data, the size and shape of detergent-solubilized integral membrane proteins can be characterized. This information can then be used to classify membrane proteins which constitute some 25% of all genomes. SAXS may also be used to study the dependence of interparticle interference scattering on solvent conditions so that regions of the protein solution phase diagram which favor crystallization can be elucidated. As a further application, SAXS may be used to provide physical constraints on computational methods for protein structure prediction based on primary sequence information. This in turn can help in identifying structural homologs of a given protein, which can then give clues to its function. D. Walther, F. Cohen and S. Doniach. "Reconstruction of low resolution three-dimensional density maps from one-dimensional small angle x-ray scattering data for biomolecules." J. Appl. Cryst. 33(2):350-363 (2000). Protein structure prediction constrained by solution X-ray scattering data and structural homology identification Zheng WJ, Doniach S JOURNAL OF MOLECULAR BIOLOGY , v. 316(#1) pp. 173-187 FEB 8, 2002

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

PubMed

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.

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

PubMed Central

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

2013-01-01

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

Femtosecond response of polyatomic molecules to ultra-intense hard X-rays.

PubMed

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

Femtosecond response of polyatomic molecules to ultra-intense hard X-rays

DOE PAGES

Rudenko, A.; Inhester, L.; Hanasaki, K.; ...

2017-05-31

We report 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 molecularmore » 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

Femtosecond response of polyatomic molecules to ultra-intense hard X-rays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rudenko, A.; Inhester, L.; Hanasaki, K.

We report 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 molecularmore » 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

Crystallization and preliminary X-ray analysis of a low density lipoprotein from human plasma.

PubMed

Prassl, R; Chapman, J M; Nigon, F; Sara, M; Eschenburg, S; Betzel, C; Saxena, A; Laggner, P

1996-11-15

Single crystals of human plasma low density lipoprotein (LDL), the major transport vehicle for cholesterol in blood, have been produced with a view to analysis of the three-dimensional structure by x-ray crystallography. Crystals with dimensions of approximately 200 x 100 x 50 microm have been reproducibly obtained from highly homogeneous LDL particle subspecies, isolated in the density ranges d = 1.0271-1. 0297 g/ml and d = 1.0297-1.0327 g/ml. Electron microscopic imaging of ultrathin-sectioned preparations of the crystals confirmed the existence of a regular, quasihexagonal arrangement of spherical particles of approximately 18 nm in diameter, thereby resembling the dimensions characteristic of LDL after dehydration and fixation. X-ray diffraction with synchrotron radiation under cryogenic conditions revealed the presence of well resolved diffraction spots, to a resolution of about 29 A. The diffraction patterns are indexed in terms of a triclinic lattice with unit cell dimensions of a = 16. 1 nm, b = 39.0 nm, c = 43.9 nm; alpha = 96.2 degrees, beta = 92.1 degrees, gamma = 102 degrees, and with space group P1.

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

PubMed

Ubarretxena-Belandia, Iban; Stokes, David L

2010-01-01

Membrane proteins are critical to cell physiology, playing roles in signaling, trafficking, transport, adhesion, and recognition. Despite their relative abundance in the proteome and their prevalence as targets of therapeutic drugs, structural information about membrane proteins is in short supply. This chapter describes the use of electron crystallography as a tool for determining membrane protein structures. Electron crystallography offers distinct advantages relative to the alternatives of X-ray crystallography and NMR spectroscopy. Namely, membrane proteins are placed in their native membranous environment, which is likely to favor a native conformation and allow changes in conformation in response to physiological ligands. Nevertheless, there are significant logistical challenges in finding appropriate conditions for inducing membrane proteins to form two-dimensional arrays within the membrane and in using electron cryo-microscopy to collect the data required for structure determination. A number of developments are described for high-throughput screening of crystallization trials and for automated imaging of crystals with the electron microscope. These tools are critical for exploring the necessary range of factors governing the crystallization process. There have also been recent software developments to facilitate the process of structure determination. However, further innovations in the algorithms used for processing images and electron diffraction are necessary to improve throughput and to make electron crystallography truly viable as a method for determining atomic structures of membrane proteins. Copyright © 2010 Elsevier Inc. All rights reserved.

Present and future of membrane protein structure determination by electron crystallography

PubMed Central

Ubarretxena-Belandia, Iban; Stokes, David L.

2011-01-01

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

Quantitative study of the f occupation in CeMIn 5 and other cerium compounds with hard X-rays

DOE PAGES

Sundermann, M.; Strigari, F.; Willers, T.; ...

2016-02-28

We present bulk-sensitive hard X-ray photoelectron spectroscopy (HAXPES) data of the Ce3d core levels and lifetime-reduced L-edge X-ray absorption spectroscopy (XAS) in the partial fluorescence yield (PFY) mode of the CeMIn 5 family with M = Co, Rh, and Ir. The HAXPES data are analyzed quantitatively with a combination of full multiplet and configuration interaction model which allows correcting for the strong plasmons in the CeMIn 5 HAXPES data, and reliable weights w n of the different f n contributions in the ground state are determined. The CeMIn 5 results are compared to HAXPES data of other heavy fermion compoundsmore » and a systematic decrease of the hybridization strength V eff from CePd 3 to CeRh 3B 2 to CeRu 2Si 2 is observed, while it is smallest for the three CeMIn 5 compounds. The f-occupation, however, increases in the same sequence and is close to one for the CeMIn 5 family. The PFY-XAS data confirm an identical f-occupation in the three CeMIn 5 compounds and a phenomenological fit to these PFY-XAS data combined with a configuration interaction model yields consistent results.« less

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

PubMed

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

2012-09-11

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

Biological X-ray absorption spectroscopy (BioXAS): a valuable tool for the study of trace elements in the life sciences.

PubMed

Strange, Richard W; Feiters, Martin C

2008-10-01

Using X-ray absorption spectroscopy (XAS) the binding modes (type and number of ligands, distances and geometry) and oxidation states of metals and other trace elements in crystalline as well as non-crystalline samples can be revealed. The method may be applied to biological systems as a 'stand-alone' technique, but it is particularly powerful when used alongside other X-ray and spectroscopic techniques and computational approaches. In this review, we highlight how biological XAS is being used in concert with crystallography, spectroscopy and computational chemistry to study metalloproteins in crystals, and report recent applications on relatively rare trace elements utilised by living organisms and metals involved in neurodegenerative diseases.

X-ray beamsplitter

DOEpatents

Ceglio, Natale M.; Stearns, Daniel S.; Hawryluk, Andrew M.; Barbee, Jr., Troy W.

1989-01-01

An x-ray beamsplitter which splits an x-ray beam into two coherent parts by reflecting and transmitting some fraction of an incident beam has applications for x-ray interferometry, x-ray holography, x-ray beam manipulation, and x-ray laser cavity output couplers. The beamsplitter is formed of a wavelength selective multilayer thin film supported by a very thin x-ray transparent membrane. The beamsplitter resonantly transmits and reflects x-rays through thin film interference effects. A thin film is formed of 5-50 pairs of alternate Mo/Si layers with a period of 20-250 A. The support membrane is 10-200 nm of silicon nitride or boron nitride. The multilayer/support membrane structure is formed across a window in a substrate by first forming the structure on a solid substrate and then forming a window in the substrate to leave a free-standing structure over the window.

X-Ray Attenuation and Absorption for Materials of Dosimetric Interest

National Institute of Standards and Technology Data Gateway

SRD 126 X-Ray Attenuation and Absorption for Materials of Dosimetric Interest (Web, free access)   Tables and graphs of the photon mass attenuation coefficient and the mass energy-absorption coefficient are presented for all of the elements Z = 1 to 92, and for 48 compounds and mixtures of radiological interest. The tables cover energies of the photon (x-ray, gamma ray, bremsstrahlung) from 1 keV to 20 MeV.

Predicting X-ray diffuse scattering from translation–libration–screw structural ensembles

PubMed Central

Van Benschoten, Andrew H.; Afonine, Pavel V.; Terwilliger, Thomas C.; Wall, Michael E.; Jackson, Colin J.; Sauter, Nicholas K.; Adams, Paul D.; Urzhumtsev, Alexandre; Fraser, James S.

2015-01-01

Identifying the intramolecular motions of proteins and nucleic acids is a major challenge in macromolecular X-ray crystallography. Because Bragg diffraction describes the average positional distribution of crystalline atoms with imperfect precision, the resulting electron density can be compatible with multiple models of motion. Diffuse X-ray scattering can reduce this degeneracy by reporting on correlated atomic displacements. Although recent technological advances are increasing the potential to accurately measure diffuse scattering, computational modeling and validation tools are still needed to quantify the agreement between experimental data and different parameterizations of crystalline disorder. A new tool, phenix.diffuse, addresses this need by employing Guinier’s equation to calculate diffuse scattering from Protein Data Bank (PDB)-formatted structural ensembles. As an example case, phenix.diffuse is applied to translation–libration–screw (TLS) refinement, which models rigid-body displacement for segments of the macromolecule. To enable the calculation of diffuse scattering from TLS-refined structures, phenix.tls_as_xyz builds multi-model PDB files that sample the underlying T, L and S tensors. In the glycerophos­phodiesterase GpdQ, alternative TLS-group partitioning and different motional correlations between groups yield markedly dissimilar diffuse scattering maps with distinct implications for molecular mechanism and allostery. These methods demonstrate how, in principle, X-ray diffuse scattering could extend macromolecular structural refinement, validation and analysis. PMID:26249347

Predicting X-ray diffuse scattering from translation–libration–screw structural ensembles

DOE PAGES

Van Benschoten, Andrew H.; Afonine, Pavel V.; Terwilliger, Thomas C.; ...

2015-07-28

Identifying the intramolecular motions of proteins and nucleic acids is a major challenge in macromolecular X-ray crystallography. Because Bragg diffraction describes the average positional distribution of crystalline atoms with imperfect precision, the resulting electron density can be compatible with multiple models of motion. Diffuse X-ray scattering can reduce this degeneracy by reporting on correlated atomic displacements. Although recent technological advances are increasing the potential to accurately measure diffuse scattering, computational modeling and validation tools are still needed to quantify the agreement between experimental data and different parameterizations of crystalline disorder. A new tool, phenix.diffuse, addresses this need by employing Guinier'smore » equation to calculate diffuse scattering from Protein Data Bank (PDB)-formatted structural ensembles. As an example case, phenix.diffuse is applied to translation–libration–screw (TLS) refinement, which models rigid-body displacement for segments of the macromolecule. To enable the calculation of diffuse scattering from TLS-refined structures, phenix.tls_as_xyz builds multi-model PDB files that sample the underlying T, L and S tensors. In the glycerophosphodiesterase GpdQ, alternative TLS-group partitioning and different motional correlations between groups yield markedly dissimilar diffuse scattering maps with distinct implications for molecular mechanism and allostery. In addition, these methods demonstrate how, in principle, X-ray diffuse scattering could extend macromolecular structural refinement, validation and analysis.« less

Search for Hard X-Ray Emission from the Soft X-Ray Transient Aquila X-1

NASA Astrophysics Data System (ADS)

Harmon, B. A.; Zhang, S. N.; Paciesas, W. S.; Tavani, M.; Kaaret, P.; Ford, E.

1994-12-01

We are investigating the possibility of hard x-ray emission from the recurrent soft x-ray transient and x-ray burst source Aquila X-1 (Aql X-1). Outbursts of this source are relatively frequent with a spacing of ~ 4-10 months (Kitamoto, S. et al. 1993, ApJ, 403, 315). The recent detections of hard tails (\\(>\\)20 keV) in low luminosity x-ray bursters (Barret, D. & Vedrenne, G. 1994, ApJ Supp. S. 92, 505) suggest that neutron star transient systems such as Aql X-1 can produce hard x-ray emission which is detectable by BATSE. We are correlating reported optical and soft x-ray observations since 1991 of Aql X-1 with BATSE observations in order to search for hard x-ray emission episodes, and to study their temporal and spectral evolution. We will present preliminary results of this search in the 20-1000 keV band using the Earth occultation technique applied to the large area detectors. If this work is successful, we hope to alert the astronomical community for the next Aql X-1 outburst expected in 1995. Simultaneous x-ray/hard x-ray and optical observations of Aql X-1 during outburst would be of great importance for the modeling of soft x-ray transients and related systems.

Elemental mapping and microimaging by x-ray capillary optics.

PubMed

Hampai, D; Dabagov, S B; Cappuccio, G; Longoni, A; Frizzi, T; Cibin, G; Guglielmotti, V; Sala, M

2008-12-01

Recently, many experiments have highlighted the advantage of using polycapillary optics for x-ray fluorescence studies. We have developed a special confocal scheme for micro x-ray fluorescence measurements that enables us to obtain not only elemental mapping of the sample but also simultaneously its own x-ray imaging. We have designed the prototype of a compact x-ray spectrometer characterized by a spatial resolution of less than 100 microm for fluorescence and less than 10 microm for imaging. A couple of polycapillary lenses in a confocal configuration together with a silicon drift detector allow elemental studies of extended samples (approximately 3 mm) to be performed, while a CCD camera makes it possible to record an image of the same samples with 6 microm spatial resolution, which is limited only by the pixel size of the camera. By inserting a compound refractive lens between the sample and the CCD camera, we hope to develop an x-ray microscope for more enlarged images of the samples under test.

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

ERIC Educational Resources Information Center

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

2016-01-01

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

X-ray dense cellular inclusions in the cells of the green alga Chlamydomonas reinhardtii as seen by soft-x-ray microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stead, A.D.; Ford, T.W.; Page, A.M.

1997-04-01

Soft x-rays, having a greater ability to penetrate biological material than electrons, have the potential for producing images of intact, living cells. In addition, by using the so-called {open_quotes}water window{close_quotes} area of the soft x-ray spectrum, a degree of natural contrast is introduced into the image due to differential absorption of the wavelengths by compounds with a high carbon content compared to those with a greater oxygen content. The variation in carbon concentration throughout a cell therefore generates an image which is dependent upon the carbon density within the specimen. Using soft x-ray contact microscopy the authors have previously examinedmore » the green alga Chlamydomonas reinhardtii, and the most prominent feature of the cells are the numerous x-ray absorbing spheres, But they were not seen by conventional transmission electron microscopy. Similar structures have also been reported by the Goettingen group using their cryo transmission x-ray microscope at BESSY. Despite the fact that these spheres appear to occupy up to 20% or more of the cell volume when seen by x-ray microscopy, they are not visible by transmission electron microscopy. Given the difficulties and criticisms associated with soft x-ray contact microscopy, the present study was aimed at confirming the existence of these cellular inclusions and learning more of their possible chemical composition.« less

Mg(1 + x)Ir(1 - x) (x = 0, 0.037 and 0.054), a binary intermetallic compound with a new orthorhombic structure type determined from powder and single-crystal X-ray diffraction.

PubMed

Cerný, Radovan; Renaudin, Guillaume; Favre-Nicolin, Vincent; Hlukhyy, Viktor; Pöttgen, Rainer

2004-06-01

The new binary compound Mg(1 + x)Ir(1 - x) (x = 0-0.054) was prepared by melting the elements in the Mg:Ir ratio 2:3 in a sealed tantalum tube under an argon atmosphere in an induction furnace (single crystals) or by annealing cold-pressed pellets of the starting composition Mg:Ir 1:1 in an autoclave under an argon atmosphere (powder sample). The structure was independently solved from high-resolution synchrotron powder and single-crystal X-ray data: Pearson symbol oC304, space group Cmca, lattice parameters from synchrotron powder data a = 18.46948 (6), b = 16.17450 (5), c = 16.82131 (5) A. Mg(1 + x)Ir(1 - x) is a topologically close-packed phase, containing 13 Ir and 12 Mg atoms in the asymmetric unit, and has a narrow homogeneity range. Nearly all the atoms have Frank-Kasper-related coordination polyhedra, with the exception of two Ir atoms, and this compound contains the shortest Ir-Ir distances ever observed. The solution of a rather complex crystal structure from powder diffraction, which was fully confirmed by the single-crystal method, shows the power of powder diffraction in combination with the high-resolution data and the global optimization method.

Analytical transmission cross-coefficients for pink beam X-ray microscopy based on compound refractive lenses.

PubMed

Falch, Ken Vidar; Detlefs, Carsten; Snigirev, Anatoly; Mathiesen, Ragnvald H

2018-01-01

Analytical expressions for the transmission cross-coefficients for x-ray microscopes based on compound refractive lenses are derived based on Gaussian approximations of the source shape and energy spectrum. The effects of partial coherence, defocus, beam convergence, as well as lateral and longitudinal chromatic aberrations are accounted for and discussed. Taking the incoherent limit of the transmission cross-coefficients, a compact analytical expression for the modulation transfer function of the system is obtained, and the resulting point, line and edge spread functions are presented. Finally, analytical expressions for optimal numerical aperture, coherence ratio, and bandwidth are given. Copyright © 2017 Elsevier B.V. All rights reserved.

X-ray beamsplitter

DOEpatents

Ceglio, N.M.; Stearns, D.G.; Hawryluk, A.M.; Barbee, T.W. Jr.

1987-08-07

An x-ray beamsplitter which splits an x-ray beam into two coherent parts by reflecting and transmitting some fraction of an incident beam has applications for x-ray interferometry, x-ray holography, x-ray beam manipulation, and x-ray laser cavity output couplers. The beamsplitter is formed of a wavelength selective multilayer thin film supported by a very thin x-ray transparent membrane. The beamsplitter resonantly transmits and reflects x-rays through thin film interference effects. A thin film is formed of 5--50 pairs of alternate Mo/Si layers with a period of 20--250 A. The support membrane is 10--200 nm of silicon nitride or boron nitride. The multilayer/support membrane structure is formed across a window in a substrate by first forming the structure on a solid substrate and then forming a window in the substrate to leave a free-standing structure over the window. 6 figs.

X-ray attenuation coefficients of high-atomic-number, hexanuclear transition metal cluster compounds: a new paradigm for radiographic contrast agents.

PubMed

Mullan, B F; Madsen, M T; Messerle, L; Kolesnichenko, V; Kruger, J

2000-04-01

The purpose of this study was to examine the radiologic attenuation properties of the parent cluster compounds, particularly attenuation as a function of discrete photon energy, before investigating ligand substitutions, which are necessary to improve cluster biocompatibility and to impart desirable physicochemical properties. The linear attenuation coefficients for solutions of the cluster compounds Ta6Br14, K8Ta6O19, and (H3O)2W6Cl14 were determined at 60, 80, 103, 122, and 140 keV from gamma-ray transmission measurements with americium-241, xenon-133, gadolinium-153, cobalt-57, and technetium-99m radioactive sources. Transmission measurements were obtained for a fixed time interval that ensured a statistically accurate count distribution exceeding 20,000 counts through the sample for each trial. On a strictly mole per liter basis, a 0.075 mol/L aqueous solution of K8Ta6O19 showed 1.08 times the attenuation of 0.063 mol/L aqueous iohexol at 60 keV and 3.30 times the attenuation at 80 keV. Similarly, a 0.05 mol/L methanolic solution of (H3O)2W6Cl4 showed 0.96 times (96%) the attenuation of 0.063 mol/L aqueous iohexol at 60 keV but 3.09 times the attenuation of the iohexol solution at 80 keV. Attenuations of 0.063 mol/L aqueous iohexol and 0.0125 mol/L Ta6Br14 (ie, at approximately one-fifth the iohexol concentration) were comparable at greater than 60 keV. These results confirm the theoretic potential for use of early transition metal cluster compounds as radiographic contrast agents. At higher x-ray energies, cluster compounds demonstrate multiplied x-ray attenuation relative to iodinated contrast agents.

X-ray Full Field Microscopy at 30 keV

NASA Astrophysics Data System (ADS)

Marschall, F.; Last, A.; Simon, M.; Kluge, M.; Nazmov, V.; Vogt, H.; Ogurreck, M.; Greving, I.; Mohr, J.

2014-04-01

In our X-ray full field microscopy experiments, we demonstrated a resolution better than 260 nm over the entire field of view of 80 μm × 80 μm at 30 keV. Our experimental setup at PETRA III, P05, had a length of about 5 m consisting of an illumination optics, an imaging lens and a detector. For imaging, we used a compound refractive lens (CLR) consisting of mr-L negative photo resist, which was fabricated by deep X-ray lithography. As illumination optics, we choose a refractive rolled X-ray prism lens, which was adapted to the numerical aperture of the imaging lens.

Syntheses, spectral characterization, X-ray studies and in vitro cytotoxic activities of triorganotin(IV) derivatives of p-substituted N-methylbenzylaminedithiocarbamates

NASA Astrophysics Data System (ADS)

Khan, Naqeebullah; Farina, Yang; Mun, Lo Kong; Rajab, Nor Fadilah; Awang, Normah

2014-11-01

Two new organotin(IV) complexes of the type R3SnL, where (L = p-bromo-N-methylbenzylaminedithiocarbamate and p-fluoro-N-methylbenzylaminedithiocarbamate, and R = phenyl) have been synthesized in 1:1 molar ratio with good yields and isolated as crystalline solids. The newly synthesized compounds gave fairly sharp melting points indicating that the compounds were pure. A systematic investigation of the derivatives were carried out both in solid and in solution and were suitably characterized by elemental analysis, FT-IR, 1H, 13C, 119Sn NMR spectroscopies. The dithiocarbamate ligands chelated to the tin metal monodentately using only one sulfur atom showing a pair of bands due to ν(Cdbnd S) below 1000 cm-1. This phenomenon was supported by the occurrence of new medium to weak absorptions in the region 411-545, in the spectra of complexes, assigned to ν(Snsbnd S) and ν(Snsbnd C). The crystal structures of the two triorganotin(IV) complexes have been determined by X-ray crystallography. Both the complexes crystallized in the monoclinic, P2(1)/n space group. The spectral investigations and single crystal X-ray diffraction data illustrate that the two dithiocarbamato ligands in the triphenyltin(IV) derivatives 1 and 2 are monodentate and the geometry at tin is best described as a distorted tetrahedron. The in vitro antiproliferative tests of these two derivatives on three human cell lines, leukemic lymphoblastoma Jurkat cells, lymphoblastoma K-562 cells, hepatoblastoma HepG2 cells and one mouse fibroblast cells L929 show dose-dependent decrease of cell proliferation in all cell lines.

Self-consistent method for quantifying indium content from X-ray spectra of thick compound semiconductor specimens in a transmission electron microscope.

PubMed

Walther, T; Wang, X

2016-05-01

Based on Monte Carlo simulations of X-ray generation by fast electrons we calculate curves of effective sensitivity factors for analytical transmission electron microscopy based energy-dispersive X-ray spectroscopy including absorption and fluorescence effects, as a function of Ga K/L ratio for different indium and gallium containing compound semiconductors. For the case of InGaN alloy thin films we show that experimental spectra can thus be quantified without the need to measure specimen thickness or density, yielding self-consistent values for quantification with Ga K and Ga L lines. The effect of uncertainties in the detector efficiency are also shown to be reduced. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Fixed Target combined with Spectral Mapping: Approaching 100% Hit Rates for Serial Crystallography

PubMed Central

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

2018-01-01

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

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

PubMed

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

2016-08-01

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

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

DOEpatents

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

1997-01-01

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

X-ray microscopy of human malaria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Magowan, C.; Brown, J.T.; Mohandas, N.

Associations between intracellular organisms and host cells are complex and particularly difficult to examine. X-ray microscopy provides transmission images of subcellular structures in intact cells at resolutions superior to available methodologies. The spatial resolution is 50-60nm with a 1 micron depth of focus, superior to anything achievable with light microscopy. Image contrast is generated by differences in photoelectric absorption by the atoms in different areas (i.e. subcellular structures) throughout the full thickness of the sample. Absorption due to carbon dominates among all the elements in the sample at 2.4 nm x-ray wavelength. Thus images show features or structures, in amore » way not usually seen by other types of microscopy. The authors used soft x-ray microscopy to investigate structural development of Plasmodium falciparum malaria parasites in normal and genetically abnormal erythrocytes, and in infected erythrocytes treated with compounds that have anti-malarial effects. X-ray microscopy showed newly elaborated structures in the cytosol of unstained, intact erythrocytes, redistribution of mass (carbon) in infected erythrocytes, and aberrant parasite morphology. Better understanding of the process of intracellular parasite maturation and the interactions between the parasite and its host erythrocyte can help define new approaches to the control of this deadly disease.« less

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

PubMed

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

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

PubMed Central

2017-01-01

Conspectus 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 [TeW6O24]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

X-Ray Polarization from High Mass X-Ray Binaries

NASA Technical Reports Server (NTRS)

Kallman, T.; Dorodnitsyn, A.; Blondin, J.

2015-01-01

X-ray astronomy allows study of objects which may be associated with compact objects, i.e. neutron stars or black holes, and also may contain strong magnetic fields. Such objects are categorically non-spherical, and likely non-circular when projected on the sky. Polarization allows study of such geometric effects, and X-ray polarimetry is likely to become feasible for a significant number of sources in the future. A class of potential targets for future X-ray polarization observations is the high mass X-ray binaries (HMXBs), which consist of a compact object in orbit with an early type star. In this paper we show that X-ray polarization from HMXBs has a distinct signature which depends on the source inclination and orbital phase. The presence of the X-ray source displaced from the star creates linear polarization even if the primary wind is spherically symmetric whenever the system is viewed away from conjunction. Direct X-rays dilute this polarization whenever the X-ray source is not eclipsed; at mid-eclipse the net polarization is expected to be small or zero if the wind is circularly symmetric around the line of centers. Resonance line scattering increases the scattering fraction, often by large factors, over the energy band spanned by resonance lines. Real winds are not expected to be spherically symmetric, or circularly symmetric around the line of centers, owing to the combined effects of the compact object gravity and ionization on the wind hydrodynamics. A sample calculation shows that this creates polarization fractions ranging up to tens of percent at mid-eclipse.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaponov, Yu.A.; Igarashi, N.; Hiraki, M.

2004-05-12

An integrated controlling system and a unified database for high throughput protein crystallography experiments have been developed. Main features of protein crystallography experiments (purification, crystallization, crystal harvesting, data collection, data processing) were integrated into the software under development. All information necessary to perform protein crystallography experiments is stored (except raw X-ray data that are stored in a central data server) in a MySQL relational database. The database contains four mutually linked hierarchical trees describing protein crystals, data collection of protein crystal and experimental data processing. A database editor was designed and developed. The editor supports basic database functions to view,more » 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.« less

Be/X-ray Binary Science for Future X-ray Timing Missions

NASA Technical Reports Server (NTRS)

Wilson-Hodge, Colleen A.

2011-01-01

For future missions, the Be/X-ray binary community needs to clearly define our science priorities for the future to advocate for their inclusion in future missions. In this talk, I will describe current designs for two potential future missions and Be X-ray binary science enabled by these designs. The Large Observatory For X-ray Timing (LOFT) is an X-ray timing mission selected in February 2011 for the assessment phase from the 2010 ESA M3 call for proposals. The Advanced X-ray Timing ARray (AXTAR) is a NASA explorer concept X-ray timing mission. This talk is intended to initiate discussions of our science priorities for the future.

Abdomen X-Ray (Radiography)

MedlinePlus

... News Physician Resources Professions Site Index A-Z X-ray (Radiography) - Abdomen Abdominal x-ray uses a ... of an abdominal x-ray? What is abdominal x-ray? An x-ray (radiograph) is a noninvasive ...

A search for X-ray polarization in cosmic X-ray sources. [binary X-ray sources and supernovae remnants

NASA Technical Reports Server (NTRS)

Hughes, J. P.; Long, K. S.; Novick, R.

1983-01-01

Fifteen strong X-ray sources were observed by the X-ray polarimeters on board the OSO-8 satellite from 1975 to 1978. The final results of this search for X-ray polarization in cosmic sources are presented in the form of upper limits for the ten sources which are discussed elsewhere. These limits in all cases are consistent with a thermal origin for the X-ray emission.

X-Ray Emission from the Soft X-Ray Transient Aquila X-1

NASA Technical Reports Server (NTRS)

Tavani, Marco

1998-01-01

Aquila X-1 is the most prolific of soft X-ray transients. It is believed to contain a rapidly spinning neutron star sporadically accreting near the Eddington limit from a low-mass companion star. The interest in studying the repeated X-ray outbursts from Aquila X-1 is twofold: (1) studying the relation between optical, soft and hard X-ray emission during the outburst onset, development and decay; (2) relating the spectral component to thermal and non-thermal processes occurring near the magnetosphere and in the boundary layer of a time-variable accretion disk. Our investigation is based on the BATSE monitoring of Aquila X-1 performed by our group. We observed Aquila X-1 in 1997 and re-analyzed archival information obtained in April 1994 during a period of extraordinary outbursting activity of the source in the hard X-ray range. Our results allow, for the first time for this important source, to obtain simultaneous spectral information from 2 keV to 200 keV. A black body (T = 0.8 keV) plus a broken power-law spectrum describe accurately the 1994 spectrum. Substantial hard X-ray emission is evident in the data, confirming that the accretion phase during sub-Eddington limit episodes is capable of producing energetic hard emission near 5 x 10(exp 35) ergs(exp -1). A preliminary paper summarizes our results, and a more comprehensive account is being written. We performed a theoretical analysis of possible emission mechanisms, and confirmed that a non-thermal emission mechanism triggered in a highly sheared magnetosphere at the accretion disk inner boundary can explain the hard X-ray emission. An anticorrelation between soft and hard X-ray emission is indeed prominently observed as predicted by this model.

Conserved Binding Mode of Human [beta subscript 2] Adrenergic Receptor Inverse Agonists and Antagonist Revealed by X-ray Crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wacker, Daniel; Fenalti, Gustavo; Brown, Monica A.

2010-11-15

G protein-coupled receptors (GPCRs) represent a large fraction of current pharmaceutical targets, and of the GPCRs, the {beta}{sub 2} adrenergic receptor ({beta}{sub 2}AR) is one of the most extensively studied. Previously, the X-ray crystal structure of {beta}{sub 2}AR has been determined in complex with two partial inverse agonists, but the global impact of additional ligands on the structure or local impacts on the binding site are not well-understood. To assess the extent of such ligand-induced conformational differences, we determined the crystal structures of a previously described engineered {beta}{sub 2}AR construct in complex with two inverse agonists: ICI 118,551 (2.8 {angstrom}),more » a recently described compound (2.8 {angstrom}) (Kolb et al, 2009), and the antagonist alprenolol (3.1 {angstrom}). The structures show the same overall fold observed for the previous {beta}{sub 2}AR structures and demonstrate that the ligand binding site can accommodate compounds of different chemical and pharmacological properties with only minor local structural rearrangements. All three compounds contain a hydroxy-amine motif that establishes a conserved hydrogen bond network with the receptor and chemically diverse aromatic moieties that form distinct interactions with {beta}{sub 2}AR. Furthermore, receptor ligand cross-docking experiments revealed that a single {beta}{sub 2}AR complex can be suitable for docking of a range of antagonists and inverse agonists but also indicate that additional ligand-receptor structures may be useful to further improve performance for in-silico docking or lead-optimization in drug design.« less

X Ray Mask Of Gold-Carbon Mixture Absorber On BCN Compound Substrate Fabricated By Plasma Processes

NASA Astrophysics Data System (ADS)

Aiyer, Chandrasekhar R.; Itoh, Satoshi; Yamada, Hitomi; Morita, Shinzo; Hattori, Shuzo

1988-06-01

X-ray mask fabrication based on BCN compound membrane and gold containing polymeric carbon ( Au-C ) absorber by totally dry processes is proposed. The Au-C films were depo-sited by plasma polymerization of propylene or styrene monomers and co-evaporation of gold. These films have 2 to 5 times higher etching rate than that of pure gold for 09 RIE, depending on the Au content. The stress in the films could be reduced to 1.9 E 7 N/m2 by annealing. The BCN films were deposited on silicon wafers by rf (13.56 MHz) plasma CVD with diborane, methane and nitrogen as source gases at typical deposition rate of 30 nm/min. The optical (633nm) and X ray (Pd L~) transparencies were nearly 80% for film thickness of 6 um. Patterning of Au-C was achieved by using tungsten as intermediate layer and PMMA electron beam resist. CF4 RIE was used to etch the tungsten layer which in turn acted as mask for the gold carbide 02 RIE. The process parameters and the characteristics of the Au-C and BCN films are presented.

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

PubMed Central

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

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

PubMed

Jaskolski, Mariusz; Dauter, Zbigniew; Wlodawer, Alexander

2014-09-01

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

"X-Ray Transients in Star-Forming Regions" and "Hard X-Ray Emission from X-Ray Bursters"

NASA Technical Reports Server (NTRS)

Halpern, Jules P.; Kaaret, Philip

1999-01-01

This grant funded work on the analysis of data obtained with the Burst and Transient Experiment (BATSE) on the Compton Gamma-Ray Observatory. The goal of the work was to search for hard x-ray transients in star forming regions using the all-sky hard x-ray monitoring capability of BATSE. Our initial work lead to the discovery of a hard x-ray transient, GRO J1849-03. Follow-up observations of this source made with the Wide Field Camera on BeppoSAX showed that the source should be identified with the previously known x-ray pulsar GS 1843-02 which itself is identified with the x-ray source X1845-024 originally discovered with the SAS-3 satellite. Our identification of the source and measurement of the outburst recurrence time, lead to the identification of the source as a Be/X-ray binary with a spin period of 94.8 s and an orbital period of 241 days. The funding was used primarily for partial salary and travel support for John Tomsick, then a graduate student at Columbia University. John Tomsick, now Dr. Tomsick, received his Ph.D. from Columbia University in July 1999, based partially on results obtained under this investigation. He is now a postdoctoral research scientist at the University of California, San Diego.

Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals

DOE PAGES

Dao, E. Han; Sierra, Raymond G.; Laksmono, Hartawan; ...

2015-04-30

In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS) using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecondmore » X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser.« less

NMR crystallography of α-poly(L-lactide).

PubMed

Pawlak, Tomasz; Jaworska, Magdalena; Potrzebowski, Marek J

2013-03-07

A complementary approach that combines NMR measurements, analysis of X-ray and neutron powder diffraction data and advanced quantum mechanical calculations was employed to study the α-polymorph of L-polylactide. Such a strategy, which is known as NMR crystallography, to the best of our knowledge, is used here for the first time for the fine refinement of the crystal structure of a synthetic polymer. The GIPAW method was used to compute the NMR shielding parameters for the different models, which included the α-PLLA structure obtained by 2-dimensional wide-angle X-ray diffraction (WAXD) at -150 °C (model M1) and at 25 °C (model M2), neutron diffraction (WAND) measurements (model M3) and the fully optimized geometry of the PLLA chains in the unit cell with defined size (model M4). The influence of changes in the chain conformation on the (13)C σ(ii) NMR shielding parameters is shown. The correlation between the σ(ii) and δ(ii) values for the M1-M4 models revealed that the M4 model provided the best fit. Moreover, a comparison of the experimental (13)C NMR spectra with the spectra calculated using the M1-M4 models strongly supports the data for the M4 model. The GIPAW method, via verification using NMR measurements, was shown to be capable of the fine refinement of the crystal structures of polymers when coarse X-ray diffraction data for powdered samples are available.

Lumbosacral spine x-ray

MedlinePlus

X-ray - lumbosacral spine; X-ray - lower spine ... The test is done in a hospital x-ray department or your health care provider's office by an x-ray technician. You will be asked to lie on the x-ray ...

X-ray ptychography

NASA Astrophysics Data System (ADS)

Pfeiffer, Franz

2018-01-01

X-ray ptychographic microscopy combines the advantages of raster scanning X-ray microscopy with the more recently developed techniques of coherent diffraction imaging. It is limited neither by the fabricational challenges associated with X-ray optics nor by the requirements of isolated specimen preparation, and offers in principle wavelength-limited resolution, as well as stable access and solution to the phase problem. In this Review, we discuss the basic principles of X-ray ptychography and summarize the main milestones in the evolution of X-ray ptychographic microscopy and tomography over the past ten years, since its first demonstration with X-rays. We also highlight the potential for applications in the life and materials sciences, and discuss the latest advanced concepts and probable future developments.

Sample mounts for microcrystal crystallography

NASA Technical Reports Server (NTRS)

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

2007-01-01

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