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At least 19 recordsLinked to original sources
PubMed2003

Using a quasi-parallel X-ray beam of ultrashort wavelength for high-pressure virus crystallography: implications for standard macromolecular crystallography.

Data acquisition from crystals of an icosahedral virus, cowpea mosaic virus (CPMV), was carried out to 2.8 A resolution under an elevated hydrostatic pressure of 330 MPa. This was the first example of a complex macromolecular assembly to be studied by high-pressure crystallography. The data were obtained from the ESRF ID30 beamline using a quasi-plane wave of ultrashort wavelength with a diamond anvil cell and an imaging-plate detector. The results of the high-pressure data analysis are given and are compared with those obtained under standard conditions, showing that the experimental procedures implemented are very efficient in terms of diffraction information collected per unit volume of crystal. These results suggest that the use of a quasi-parallel synchrotron radiation beam of ultrashort wavelength should also be considered for conventional macromolecular crystallography data collection.

Comovirus↗
PubMed2004

Mechanistic crystallography. Mechanism of inactivation of gamma-aminobutyric acid aminotransferase by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid as elucidated by crystallography.

(1R,3S,4S)-3-Amino-4-fluorocyclopentane-1-carboxylic acid (7) was previously shown to be a mechanism-based inactivator of gamma-aminobutyric acid aminotransferase (GABA-AT) [Qiu, J. and Silverman, R. B. (2000) J. Med. Chem. 43, 706-720]. Two mechanisms were considered as reasonable possibilities, a Michael addition mechanism and an enamine mechanism. On the basis of a variety of chemical studies, including tedious radiolabeling experiments, it was concluded that inactivation by 7 proceeds by a Michael addition mechanism. Here, a crystal structure of 7 bound to pig liver GABA-AT is reported, which clearly demonstrates that the adduct formed is derived from an enamine mechanism. This represents another example of how crystallography is an important tool for elucidation of inactivation mechanisms.

4-Aminobutyrate Transaminase↗
PubMed1995

Holographic methods in X-ray crystallography. IV. A fast algorithm and its application to macromolecular crystallography.

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

Algorithms↗
PubMed2000

Automated crystal mounting and data collection for protein crystallography.

To increase the efficiency of diffraction data collection for protein crystallographic studies, an automated system designed to store frozen protein crystals, mount them sequentially, align them to the X-ray beam, collect complete data sets, and return the crystals to storage has been developed. Advances in X-ray data collection technology including more brilliant X-ray sources, improved focusing optics, and faster-readout detectors have reduced diffraction data acquisition times from days to hours at a typical protein crystallography laboratory [1,2]. In addition, the number of high-brilliance synchrotron X-ray beam lines dedicated to macromolecular crystallography has increased significantly, and data collection times at these facilities can be routinely less than an hour per crystal. Because the number of protein crystals that may be collected in a 24 hr period has substantially increased, unattended X-ray data acquisition, including automated crystal mounting and alignment, is a desirable goal for protein crystallography. The ability to complete X-ray data collection more efficiently should impact a number of fields, including the emerging structural genomics field [3], structure-directed drug design, and the newly developed screening by X-ray crystallography [4], as well as small molecule applications.

Crystallization↗
PubMed2003

High-throughput crystallography to enhance drug discovery.

Protein crystallography has traditionally been regarded as a resource-intensive, time-consuming technique that, with some notable exceptions, has not made a significant impact on drug discovery. However, inspired by successes in the genome-sequencing initiatives, recent years have seen major changes in X-ray crystallography methodologies and the concept of high-throughput crystallography has emerged. Advances have been made in all phases of the process, including improved molecular biology, protein expression, crystallization and structure determination. This transformation has allowed X-ray crystallography to impact more broadly in the drug-discovery process, extending its utility from structure-based lead optimisation to novel fragment-based lead generation approaches.

Animals↗
PubMed2005

Comparisons of NMR spectral quality and success in crystallization demonstrate that NMR and X-ray crystallography are complementary methods for small protein structure determination.

X-ray crystallography and NMR spectroscopy provide the only sources of experimental data from which protein structures can be analyzed at high or even atomic resolution. The degree to which these methods complement each other as sources of structural knowledge is a matter of debate; it is often proposed that small proteins yielding high quality, readily analyzed NMR spectra are a subset of those that readily yield strongly diffracting crystals. We have examined the correlation between NMR spectral quality and success in structure determination by X-ray crystallography for 159 prokaryotic and eukaryotic proteins, prescreened to avoid proteins providing polydisperse and/or aggregated samples. This study demonstrates that, across this protein sample set, the quality of a protein's [15N-1H]-heteronuclear correlation (HSQC) spectrum recorded under conditions generally suitable for 3D structure determination by NMR, a key predictor of the ability to determine a structure by NMR, is not correlated with successful crystallization and structure determination by X-ray crystallography. These results, together with similar results of an independent study presented in the accompanying paper (Yee, et al., J. Am. Chem. Soc., accompanying paper), demonstrate that X-ray crystallography and NMR often provide complementary sources of structural data and that both methods are required in order to optimize success for as many targets as possible in large-scale structural proteomics efforts.

Animals↗
PubMed2003

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.

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.

Crystallography, X-Ray↗
PubMed1998

Electron Crystallography of Two-Dimensional Crystals of Membrane Proteins.

Electron microscopy has become a powerful technique, along with X-ray crystallography and nuclear magnetic resonance spectroscopy, to study the three-dimensional structure of biological molecules. It has evolved into a number of methods dealing with a wide range of biological samples, with electron crystallography of two-dimensional crystals being so far the only method allowing data collection at near-atomic resolution. In this paper, we review the methodology of electron crystallography and its application to membrane proteins, starting with the pioneering work on bacteriorhodopsin, which led to the first visualization of the secondary structure of a membrane protein in 1975. Since then, improvements in instrumentation, sample preparation, and data analysis have led to atomic models for bacteriorhodopsin and light-harvesting complex II from higher plants. The structures of many more membrane proteins have been studied by electron crystallography and in this review examples are included where a resolution of better than 10 Å has been achieved. Indeed, in some of the given examples an atomic model can be expected in the near future. Finally, a brief outlook is given on current and future developments of electron crystallographic methods. Copyright 1998 Academic Press.

Journal Article↗
PubMed2001

New opportunities in biological and chemical crystallography.

Banerjee [Proc. R. Soc. (1933), 141, 188-193] offered a new way of approaching the crystallographic phase problem which not only broke new ground beyond the 'trial and error' structure solution method of that time but also heralded the extremely powerful direct methods of crystallography of the modern era from the 1970s onwards in chemical crystallography. Some 200000 crystal structures are known today. More complex crystal structures such as proteins required new experimental and theoretical methods to solve the phase problem. These are still evolving, and new methods and results involving synchrotron radiation at softer X-ray wavelengths (2 A) are reported. In addition, an overview is given of the new opportunities that are possible for biological and chemical crystallography, especially via harnessing synchrotron radiation and neutron beams.

Concanavalin A↗
PubMed2006

Neutron Laue macromolecular crystallography.

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

Crystallography↗
PubMed2006

Milestones in electron crystallography.

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

Cryoelectron Microscopy↗
PubMed1997

Electron crystallography of macromolecular periodic arrays on phospholipid monolayers.

Electron crystallography has the potential of yielding structural information equivalent to x-ray diffraction. The major difficulty has been preparing specimens with the required structural order and size for diffraction and imaging in the electron microscope. 2D crystallization on phospholipid monolayers is capable of fulfilling both of these requirements. Crystals can form as a result of specific interactions with a protein's ligand or an analog, suitably linked to a lipid tail; or on a surface of complementary head-group charge. With such choices, the availability of a suitable lipid is limited only by synthetic chemistry. Ultimately, it is the quality and regularity of the protein-protein interactions that determine the crystalline order, as it is with any protein crystal. In the case of streptavidin, the monolayer crystal diffracts beyond 2.5 A. A 3 A projection map reconstructed from electron diffraction amplitudes and phases from images shows density which can be interpreted as beta-sheets and clusters of side chains. It remains to be shown that the monolayer crystals are flat and diffract as well at high tilt angle as untilted. Technological issues such as charging must be resolved. With parallel advances in data collection and processing, electron crystallography of monolayer macromolecular crystals will eventually take its place beside x-ray crystallography and NMR as a routine and efficient structural technique.

Crystallography↗
PubMed2005

X-ray damage to the Mn4Ca complex in single crystals of photosystem II: a case study for metalloprotein crystallography.

X-ray absorption spectroscopy was used to measure the damage caused by exposure to x-rays to the Mn(4)Ca active site in single crystals of photosystem II as a function of dose and energy of x-rays, temperature, and time. These studies reveal that the conditions used for structure determination by x-ray crystallography cause serious damage specifically to the metal-site structure. The x-ray absorption spectra show that the structure changes from one that is characteristic of a high-valent Mn(4)(III(2),IV(2)) oxo-bridged Mn(4)Ca cluster to that of Mn(II) in aqueous solution. This damage to the metal site occurs at a dose that is more than one order of magnitude lower than the dose that results in loss of diffractivity and is commonly considered safe for protein crystallography. These results establish quantitative x-ray dose parameters that are applicable to redox-active metalloproteins. This case study shows that a careful evaluation of the structural intactness of the active site(s) by spectroscopic techniques can validate structures derived from crystallography and that it can be a valuable complementary method before structure-function correlations of metalloproteins can be made on the basis of high-resolution x-ray crystal structures.

Calcium↗
PubMed1989

Direct methods in protein crystallography.

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

Crystallography↗
PubMed2005

Current state and prospects of macromolecular crystallography.

The current situation and possible future development of macromolecular crystallography are reviewed. The rapid progress and maturation of this field in recent years have to a large extent been made possible by the inspiration and generous support of several active structural genomics initiatives. Two tendencies can be currently observed: one which treats protein crystallography as a highly automatic tool for investigating various biological problems without the need to engage in the intricacies of the technique and a second approach where this method is applied to crystals of difficult, large and complex biological systems, requiring a deeper knowledge of various aspects of crystallography. In the near future it is expected that these two trends will coexist, developing in a parallel fashion.

Crystallography, X-Ray↗
PubMed1998

Crystallography & NMR system: A new software suite for macromolecular structure determination.

A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs, the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible. The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular structure determination by X-ray crystallography and solution NMR.

Computer Simulation↗
PubMed2001

Overview of the tunable beamlines for protein crystallography at the EMBL Hamburg Outstation; an analysis of current and future usage and developments.

The EMBL Hamburg Outstation currently operates two tunable protein crystallography beamlines suitable for single and multiple anomalous diffraction (SAD/MAD) experiments. The first beamline, designated X31, is located on a bending magnet of the DORIS III storage ring whereas the second beamline, BW7A, is positioned at a multipole wiggler at the same storage ring. X31 is equipped with an energy stabilization device to ensure constant wavelength during longer data-collection periods. The in-house built crystallographic end-station is now equipped with a Mar345 imaging-plate scanner as a detector. The wiggler beamline BW7A features a novel sagitally focusing monochromator. The end-station used here has also been developed and built in-house. The beamline is currently operated with a Mar 165 CCD detector. In this paper the hardware and software developments of the last years will be summarized and the outlook for substantial upgrades will be given. The future plans include the design and construction of a third tunable beamline, designated X12, for protein crystallography. The development of automated beamlines for protein crystallography is of particular importance with respect to structural genomics initiatives. The analysis of the projects of the last years shows the wide range of anomalous scatterer used on the tunable beamlines thus demonstrating the need of a wide range of accessible energies and fast and reliable energy changes.

Crystallography↗