X-ray structure determination of the glycine cleavage system protein H of Mycobacterium tuberculosis using an inverse Compton synchrotron X-ray source

Structural genomics discovery projects require ready access to both X-ray diffraction and NMR spectroscopy which support the collection of experimental data needed to solve large numbers of novel protein structures. The most productive X-ray crystal structure determination laboratories make extensive use of tunable synchrotron X-ray light to solve novel structures by anomalous diffraction methods. This requires that frozen cryo-protected crystals be shipped to large multi acre synchrotron facilities for data collection. In this paper we report on the development and use of the first laboratory-scale synchrotron light source capable of performing many of the state-of-the-art synchrotron applications in X-ray science. This Compact Light Source is a first-in-class device that uses inverse Compton scattering to generate X-rays of sufficient flux, tunable wavelength and beam size to allow high-resolution X-ray diffraction data collection from protein crystals. We report on benchmarking tests of X-ray diffraction data collection with hen egg white lysozyme, and the successful high-resolution X-ray structure determination of the Glycine cleavage system protein H from Mycobacterium tuberculosis using diffraction data collected with the Compact Light Source X-ray beam.     

New techniques in macromolecular cryocrystallography: macromolecular crystal annealing and cryogenic helium

Cryocrystallography is used today for almost all X-ray diffraction data collection at synchrotron beam lines, with rotating-anode generators, and micro X-ray sources. Despite the widespread use of flash-cooling to place macromolecular crystals in the cryogenic state, its use can ruin crystals, trips to the synchrotron, and sometimes even an entire project. Annealing of macromolecular crystals takes little time, requires no specialized equipment, and can save crystallographic projects that might otherwise end in failure. Annealing should be tried whenever initial flash-cooling causes an unacceptable increase in mosaicity, results in ice rings, fails to provide adequate diffraction quality, or causes a crystal to be positioned awkwardly. Overall, annealing improves the quality of data and overall success rate at synchrotron beam lines. Its use should be considered whenever problems arise with a flash-cooled crystal. Helium is a more efficient cryogen than nitrogen and will deliver lower temperatures. Experiments suggest that when crystals are cooled with He rather than N2, crystals maintain order and high-resolution data are less affected by increased radiation load. Individually or in combination, these two techniques can enhance the success of crystallographic data collection, and their use should be considered essential for high-throughput programs.     

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.     

Trichromatic concept optimizes MAD experiments in synchrotron X-ray crystallography

The trichromatic concept is a new synchrotron beamline design that optimizes MAD experiments by reducing systematic experimental errors with three-colored and coaxial synchrotron X-ray beams produced by a tandem vertical undulator and trichromator. The concept enables rapid and flexible switching of three defined wavelengths, and extends the flexibility of experimental design for MAD data collection. Thus, we can collect MAD data taking into account time series effects such as radiation damage. The data based on the trichromatic concept gave a better quality electron density map than data collected by conventional methods. It was also revealed that multicolor diffraction using dichromatic or trichromatic X-ray beams is effective in rapid MAD data collection.     

Doses for experiments with microbeams and microcrystals: Monte Carlo simulations in RADDOSE‐3D

Increasingly, microbeams and microcrystals are being used for macromolecular crystallography (MX) experiments at synchrotrons. However, radiation damage remains a major concern since it is a fundamental limiting factor affecting the success of macromolecular structure determination. The rate of radiation damage at cryotemperatures is known to be proportional to the absorbed dose, so to optimize experimental outcomes, accurate dose calculations are required which take into account the physics of the interactions of the crystal constituents. The program RADDOSE‐3D estimates the dose absorbed by samples during MX data collection at synchrotron sources, allowing direct comparison of radiation damage between experiments carried out with different samples and beam parameters. This has aided the study of MX radiation damage and enabled prediction of approximately when it will manifest in diffraction patterns so it can potentially be avoided. However, the probability of photoelectron escape from the sample and entry from the surrounding material has not previously been included in RADDOSE‐3D, leading to potentially inaccurate does estimates for experiments using microbeams or microcrystals. We present an extension to RADDOSE‐3D which performs Monte Carlo simulations of a rotating crystal during MX data collection, taking into account the redistribution of photoelectrons produced both in the sample and the material surrounding the crystal. As well as providing more accurate dose estimates, the Monte Carlo simulations highlight the importance of the size and composition of the surrounding material on the dose and thus the rate of radiation damage to the sample. Minimizing irradiation of the surrounding material or removing it almost completely will be key to extending the lifetime of microcrystals and enhancing the potential benefits of using higher incident X‐ray energies.     

Secure web book to store structural genomics research data

Recently established collaborative structural genomics programs aim at significantly accelerating the crystal structure analysis of proteins. These large-scale projects require efficient data management systems to ensure seamless collaboration between different groups of scientists working towards the same goal. Within the Berlin-based Protein Structure Factory, the synchrotron X-ray data collection and the subsequent crystal structure analysis tasks are located at BESSY, a third-generation synchrotron source. To organize file-based communication and data transfer at the BESSY site of the Protein Structure Factory, we have developed the web-based BCLIMS, the BESSY Crystallography Laboratory Information Management System. BCLIMS is a relational data management system which is powered by MySQL as the database engine and Apache HTTP as the web server. The database interface routines are written in Python programing language. The software is freely available to academic users. Here we describe the storage, retrieval and manipulation of laboratory information, mainly pertaining to the synchrotron X-ray diffraction experiments and the subsequent protein structure analysis, using BCLIMS.     

X-ray diffraction photographs of chicken gizzard G-actin.DNase I complex crystals taken with synchrotron radiation

X-ray diffraction photographs of a chicken gizzard G-actin.DNase I complex crystal have been recorded using the synchrotron radiation beam emitted by the Synchrotron Radiation Source at Daresbury and the Photon Factory at Tsukuba. The resolution limit was extended to 2.4 A and the exposure time was reduced approximately by a factor of 10, when data recorded at the Photon Factory, were compared with those recorded with a conventional rotating-anode source. Using a newly designed Weissenberg camera equipped with a multi-layer line screen, the diffraction data in a 36 degrees oscillation range were recorded on a single film up to 3.5 A resolution.     

Time-resolved methods in biophysics. 6. Time-resolved Laue crystallography as a tool to investigate photo-activated protein dynamics.

When polychromatic X-rays are shined onto crystalline material, they generate a Laue diffraction pattern. At third generation synchrotron radiation sources, a single X-ray pulse of approximately 100 ps duration is enough to produce interpretable Laue data from biomolecular crystals. Thus, by initiating biological turnover in a crystalline protein, structural changes along the reaction pathway may be filmed by ultra-fast Laue diffraction. Using laser-light as a trigger, transient species in photosensitive macromolecules can be captured at near atomic resolution with sub-nanosecond time-resolution. Such pump-probe Laue experiments have now reached an outstanding level of sophistication and have found a domain of excellence in the investigation of light-sensitive proteins undergoing cyclic photo-reactions and producing stiff crystals. The main theoretical concepts of Laue diffraction and the challenges associated with time-resolved experiments on biological crystals are recalled. The recent advances in the design of experiments are presented in terms of instrumental choices, data collection strategy and data processing, and some of the inherent difficulties of the method are highlighted. The discussion is based on the example of myoglobin, a protein that has traversed the whole history of pump-probe Laue diffraction, and for which a massive amount of data have provided considerable insight into the understanding of protein dynamics.     

Disorder is characteristic of nerve myelin.

An X-ray diffraction pattern from the myelin in frog sciatic nerve has been obtained using the intense synchrotron radiation from the storage ring, SPEAR. Data with good statistical accuracy are obtained in a few minutes by using a scintillation counter or position-sensitive detector. The same indications for stacking disorder are seen as in previous conventional exposures which required one to two days. Thus, the stacking disorder is characteristic of myelin in a freshly dissected nerve. The present data, obtained with a more nearly monochromatic X-ray beam than in the previous study, remove one of two ambiguities which bear on the possible phasing of the higher order Bragg reflections.     

How to avoid premature decay of your macromolecular crystal: a quick soak for long life

Radiation damage to biological samples is currently one of the major limiting factors in macromolecular X-ray crystallography, since it severely and irreversibly affects the quality of the data that can be obtained from a diffraction experiment. However, radiation damage can effectively be reduced by utilizing the electron and radical scavenging potential of certain small-molecule compounds. We propose an approach to protect macromolecular crystals prior to data collection by quick soaking with scavengers. This, in favorable cases, can more than double crystal lifetime in the X-ray beam. The approach has the potential to yield diffraction data of superior quality and hence to increase the amount of high-quality diffraction data and of structural information attainable from a single crystal.     

X-ray diffraction from a left ventricular wall of rat heart

We studied x-ray diffraction from the left ventricular wall of an excised, perfused whole heart of a rat using x rays from the third-generation synchrotron radiation facility, SPring-8. With the beam at right angles to the long axis of the left ventricle, well-oriented, strong equatorial reflections were observed from the epicardium surface. The reflections became vertically split arcs when the beam passed through myocardium deeper in the wall, and rings were observed when the beam passed into the inner myocardium of the wall. These diffraction patterns were explained by employing a layered-spiral model of the arrangement of muscle fibers in the heart. In a quiescent heart with an expanded left ventricle, the muscle fibers at the epicardium surface were found to have a (1,0) lattice spacing smaller than in the rest of the wall. The intensity ratio of the (1,0) and (1,1) equatorial reflections decreased on contraction with a similar time course in all parts of the wall. The results show that it is possible to assign the origin of reflections in a diffraction diagram from a whole heart. This study offers a basis for interpretation of x-ray diffraction from a beating heart under physiologically and pathologically different conditions.     

Characterization of single crystals of the large ribosomal particles from Bacillus stearothermophilus

Single, three-dimensional crystals of the 50 S ribosomal subunit from Bacillus stearothermophilus (strain NCA) have been characterized using a synchrotron X-ray source. The crystals are orthorhombic with unit cell dimensions: a = 350 A, b = 670 A, c = 905 A, and contain at least one 2-fold screw axis. With cooling to -2 degrees C, the large crystals (1.0 mm X 0.2 mm X 0.1 mm) diffract to 15 to 18 A resolution and are stable in the synchrotron beam for several hours. Despite the large cell dimensions, the reflections are readily resolved when the X-ray diffraction patterns are densitometered with a 25 microns faster.     

Terahertz Radiation at ANKA, the New Synchrotron Light Source in Karlsruhe

ANKA is a new synchrotron light source atthe Karlsruhe Research Center in southwestGermany. The acronym stands for Ångstrøm Source Karlsruhe.The ANKA-IR beamline provides a highbrilliance infrared beam through the near,mid and far-infrared range. Thefar-infrared range is of particularinterest, since at frequencies lower thanaround 200 cm^-1 (6 THz) synchrotronlight begins to outperform conventionalthermal sources in terms of total intensityas well as brilliance. The extraction ofthe entire flux is a challenge in the THzrange, since the natural verticaldivergence of synchrotron radiationincreases with wavelength and the openingangle for collection is limited by designconstraints. At ANKA-IR, this problem issolved by the collection of radiationemitted from a bending magnet edge source,which has a much smaller verticaldivergence than conventional synchrotronradiation emitted from the constantmagnetic field region within the dipolemagnet. Edge radiation at ANKA permits theextraction of the entire infrared flux downto around 100 cm^-1 (3 THz) while withconventional synchrotron radiation thiswould only be the case for frequencies downto 2500 cm^-1. ANKA-IR provides usableintensity down to 4 cm^-1 (120 GHz).     

Diffraction before destruction

X-ray free-electron lasers have opened up the possibility of structure determination of protein crystals at room temperature, free of radiation damage. The femtosecond-duration pulses of these sources enable diffraction signals to be collected from samples at doses of 1000 MGy or higher. The sample is vaporized by the intense pulse, but not before the scattering that gives rise to the diffraction pattern takes place. Consequently, only a single flash diffraction pattern can be recorded from a crystal, giving rise to the method of serial crystallography where tens of thousands of patterns are collected from individual crystals that flow across the beam and the patterns are indexed and aggregated into a set of structure factors. The high-dose tolerance and the many-crystal averaging approach allow data to be collected from much smaller crystals than have been examined at synchrotron radiation facilities, even from radiation-sensitive samples. Here, we review the interaction of intense femtosecond X-ray pulses with materials and discuss the implications for structure determination. We identify various dose regimes and conclude that the strongest achievable signals for a given sample are attained at the highest possible dose rates, from highest possible pulse intensities.     

New approaches to high-throughput phasing

Recent progress in macromolecular phasing, in part stimulated by the high-throughput structural biology initiatives, has made this crucial stage of the elucidation of crystal structures easier and more automatic. A quick soak in various salts leads to the rapid incorporation of the anomalously scattering ions, suitable for phasing by MAD (multiwavelength anomalous dispersion), SAD (single-wavelength anomalous dispersion) or MIR (multiple isomorphous replacement) methods. The availability of stable synchrotron beam lines equipped with elaborate hardware control and sophisticated data processing programs makes it possible to collect very accurate diffraction data and to solve structures from the very weak anomalous signal of such atoms as sulfur or phosphorus, inherently present in macromolecules. The current progress in phasing, coupled with the parallel advances in protein crystallization, diffraction data collection and so on, suggests that, in the near future, the process of macromolecular crystal structure elucidation may become fully automatic.     

Oxidized and synchrotron cleaved structures of the disulfide redox center in the N-terminal domain of Salmonella typhimurium AhpF

The flavoprotein component (AhpF) of Salmonella typhimurium alkyl hydroperoxide reductase contains an N-terminal domain (NTD) with two contiguous thioredoxin folds but only one redox-active disulfide (within the sequence -Cys129-His-Asn-Cys132-). This active site is responsible for mediating the transfer of electrons from the thioredoxin reductase-like segment of AhpF to AhpC, the peroxiredoxin component of the two-protein peroxidase system. The previously reported crystal structure of AhpF possessed a reduced NTD active site, although fully oxidized protein was used for crystallization. To further investigate this active site, we crystallized an isolated recombinant NTD (rNTD); using diffraction data sets collected first at our in-house X-ray source and subsequently at a synchrotron, we showed that the active site disulfide bond (Cys129-Cys132) is oxidized in the native crystals but becomes reduced during synchrotron data collection. The NTD disulfide bond is apparently particularly sensitive to radiation cleavage compared with other protein disulfides. The two data sets provide the first view of an oxidized (disulfide) form of NTD and show that the changes in conformation upon reduction of the disulfide are localized and small. Furthermore, we report the apparent pKa of the active site thiol to be approximately 5.1, a relatively low pKa given its redox potential (approximately 265 mV) compared with most members of the thioredoxin family.     

Crystallization and crystallographic data for new forms of thymidylate synthase from Lactobacillus casei

Several new crystal forms of thymidylate synthase (5,10-methlenetetrahydrofolate:dUMP C-methyltransferase; EC 2.1.1.45) were obtained by controlled pH change. In the crystals the dimeric molecule has a 2-fold symmetry axis coinciding with crystallographic symmetry. The crystals scatter to at least 2.7 A resolution in the synchrotron X-ray beam and appear to be suitable for high-resolution X-ray diffraction analysis. The crystals were successfully derivatized and preliminary results are reported for the covalent inhibitory ternary complex of thymidylate synthase, 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate.     

Atomic structure of a CK2alpha human kinase by microfocus diffraction of extra-small microcrystals grown with nanobiofilm template

Extra-small microcrystals of a human kinase CK2alpha were obtained for the first time by the optimization of a recent protein crystallization method based on highly packed protein nanofilm template. Protein crystal induction and growth appear indeed optimal at high surface pressure of the film template yielding high protein orientation and packing. The resulting extra-small CK2alpha microcrystals (of about 20 microm in diameter) was subsequently used for synchrotron radiation diffraction data collection, which proves possible by means of the Microfocus Beamline at the ESRF Synchrotron in Grenoble. The quality of the resulting crystal diffraction patterns and of its resulting atomic structure at 2.4 A resolution proves the unique validity of the above two combined frontier technologies in defining a new approach to structural proteomics capable to solve the atomic structure of proteins so far never been crystallized and of pharmaceutical relevance. Physical explanation in terms of template dipole moments and possibility of generalization of this method to the wide class of proteins not yet crystallized are finally discussed. The structure of our CK2alpha mutant is in the Protein Data Bank (PDB ID Code 1NA7, deposited on 27 November 2002).     

Inducing phase changes in crystals of macromolecules: status and perspectives for controlled crystal dehydration

The increase in the number of large multi-component complexes and membrane protein crystal structures determined over the last few years can be ascribed to a number of factors such as better protein expression and purification systems, the emergence of high-throughput crystallization techniques and the advent of 3rd generation synchrotron sources. However, many systems tend to produce crystals that can be extremely heterogeneous in their diffraction properties. This prevents, in many cases, the collection of diffraction data of sufficient quality to yield useful biological or phase information. Techniques that can increase the diffraction quality of macromolecular crystals can therefore be essential in the successful conclusion of these challenging projects. No technique is universal but encouraging results have been recently achieved by carrying out the controlled dehydration of crystals of biological macromolecules. A new device that delivers a stream of air with a precisely controlled relative humidity to the complicated sample environment found at modern synchrotron beamlines has been conceived at the EMBL Grenoble and developed by the EMBL and the ESRF as part of the SPINE2 complexes project, a European Commission funded protein structure initiative. The device, the HC1b, has been available for three years at the ESRF macromolecular crystallography beamlines and many systems have benefitted from on-line controlled dehydration. Here we describe a standard dehydration experiment, highlight some successful cases and discuss the different possible uses of the device.     

Crystallization and preliminary X-ray diffraction analysis of foot-and-mouth disease virus

Foot-and-mouth disease virus has been crystallized with the objectives of (1) determining the composition and conformation of the major immunogenic site(s) and (2) comparing its structure with those of the related polio, rhino and Mengo viruses, representing the other three genera of the picornaviruses. Most of the work has been done with virus strain O1BFS 1860, which crystallized as small rhombic dodecahedra of maximum dimension 0.3 mm. Virus recovered from crystals was infectious, and was indistinguishable from native virus both in protein composition and buoyant density. The stability of the crystals in the X-ray beam was comparable with that of other picornavirus crystals and they diffracted to a resolution of better than 2.3 A. Initial analysis of the X-ray diffraction data shows the virus to be positioned on a point of 23 symmetry in a close-packed array so that examples of all the icosahedral symmetry elements, except the 5-fold axes, are expressed crystallographically. The cell dimensions are a = b = c = 345 A, alpha = beta = gamma = 90 degrees, with a space group of I23. The diameter of the virus particle is 300 A. Despite the small size of the crystals, diffraction data have been collected to a reasonable resolution using a synchrotron source. Phasing of the diffraction data will be attempted using the methods of molecular replacement.