Fine ϕ-slicing substantially improves scaling statistics and anomalous signal for diffraction data collection with hybrid pixel detectors.
The data-collection parameters used in a macromolecular diffraction experiment have a strong impact on data quality. A careful choice of parameters leads to better data and can make the difference between success and failure in phasing attempts, and will also result in a more accurate atomic model. The selection of parameters has to account for the application of the data in various phasing methods or high-resolution refinement. Furthermore, experimental factors such as crystal characteristics, available experiment time and the properties of the X-ray source and detector have to be considered. For many years, CCD detectors have been the prevalent type of detectors used in macromolecular crystallography. Recently, hybrid pixel X-ray detectors that operate in single-photon-counting mode have become available. These detectors have fundamentally different characteristics compared with CCD detectors and different data-collection strategies should be applied. Fine ϕ-slicing is a strategy that is particularly well suited to hybrid pixel detectors because of the fast readout time and the absence of readout noise. A large number of data sets were systematically collected from crystals of four different proteins in order to investigate the benefit of fine ϕ-slicing on data quality with a noise-free detector. The results show that fine ϕ-slicing can substantially improve scaling statistics and anomalous signal provided that the rotation angle is comparable to half the crystal mosaicity.
diffraction data collection; data-collection strategies; detectors; hybrid pixel detector; single-photon counting
Observations of the dose-rate effect in continuous X-ray diffraction data acquisition at room temperature are presented.
The first study of room-temperature macromolecular crystallography data acquisition with a silicon pixel detector is presented, where the data are collected in continuous sample rotation mode, with millisecond read-out time and no read-out noise. Several successive datasets were collected sequentially from single test crystals of thaumatin and insulin. The dose rate ranged between ∼1320 Gy s−1 and ∼8420 Gy s−1 with corresponding frame rates between 1.565 Hz and 12.5 Hz. The data were analysed for global radiation damage. A previously unreported negative dose-rate effect is observed in the indicators of global radiation damage, which showed an approximately 75% decrease in D
1/2 at sixfold higher dose rate. The integrated intensity decreases in an exponential manner. Sample heating that could give rise to the enhanced radiation sensitivity at higher dose rate is investigated by collecting data between crystal temperatures of 298 K and 353 K. UV-Vis spectroscopy is used to demonstrate that disulfide radicals and trapped electrons do not accumulate at high dose rates in continuous data collection.
room-temperature data collection; PILATUS 6M; dose rate; radiation damage
A system for the automatic reduction of single- and multi-position macromolecular crystallography data is presented.
The development of automated high-intensity macromolecular crystallography (MX) beamlines at synchrotron facilities has resulted in a remarkable increase in sample throughput. Developments in X-ray detector technology now mean that complete X-ray diffraction datasets can be collected in less than one minute. Such high-speed collection, and the volumes of data that it produces, often make it difficult for even the most experienced users to cope with the deluge. However, the careful reduction of data during experimental sessions is often necessary for the success of a particular project or as an aid in decision making for subsequent experiments. Automated data reduction pipelines provide a fast and reliable alternative to user-initiated processing at the beamline. In order to provide such a pipeline for the MX user community of the European Synchrotron Radiation Facility (ESRF), a system for the rapid automatic processing of MX diffraction data from single and multiple positions on a single or multiple crystals has been developed. Standard integration and data analysis programs have been incorporated into the ESRF data collection, storage and computing environment, with the final results stored and displayed in an intuitive manner in the ISPyB (information system for protein crystallography beamlines) database, from which they are also available for download. In some cases, experimental phase information can be automatically determined from the processed data. Here, the system is described in detail.
automation; data processing; macromolecular crystallography; computer programs
Structural genomics discovery projects require ready access to both X-ray and NMR instrumentation 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 frequent 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 government-run synchrotron facilities for data collection. In an effort to eliminate the need to ship crystals for data collection, we have developed 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.
A repetitive measurement of the same diffraction image allows to judge the performance of a data collection facility.
The accuracy of X-ray diffraction data depends on the properties of the crystalline sample and on the performance of the data-collection facility (synchrotron beamline elements, goniostat, detector etc.). However, it is difficult to evaluate the level of performance of the experimental setup from the quality of data sets collected in rotation mode, as various crystal properties such as mosaicity, non-uniformity and radiation damage affect the measured intensities. A multiple-image experiment, in which several analogous diffraction frames are recorded consecutively at the same crystal orientation, allows minimization of the influence of the sample properties. A series of 100 diffraction images of a thaumatin crystal were measured on the SBC beamline 19BM at the APS (Argonne National Laboratory). The obtained data were analyzed in the context of the performance of the data-collection facility. An objective way to estimate the uncertainties of individual reflections was achieved by analyzing the behavior of reflection intensities in the series of analogous diffraction images. The multiple-image experiment is found to be a simple and adequate method to decompose the random errors from the systematic errors in the data, which helps in judging the performance of a data-collection facility. In particular, displaying the intensity as a function of the frame number allows evaluation of the stability of the beam, the beamline elements and the detector with minimal influence of the crystal properties. Such an experiment permits evaluation of the highest possible data quality potentially achievable at the particular beamline.
diffraction data precision; signal-to-noise ratio; measurement uncertainty; beamline performance
It is shown that the anisotropy of anomalous scattering (AAS) is a significant and ubiquitous effect in data sets collected at an absorption edge and that its exploitation can substantially enhance the phasing power of single- or multi-wavelength anomalous diffraction. The improvements in the phases are typically of the same order of magnitude as those obtained in a conventional approach by adding a second-wavelength data set to a SAD experiment.
The X-ray polarization anisotropy of anomalous scattering in crystals of brominated nucleic acids and selenated proteins is shown to have significant effects on the diffraction data collected at an absorption edge. For conventionally collected single- or multi-wavelength anomalous diffraction data, the main manifestation of the anisotropy of anomalous scattering is the breakage of the equivalence between symmetry-related reflections, inducing intensity differences between them that can be exploited to yield extra phase information in the structure-solution process. A new formalism for describing the anisotropy of anomalous scattering which allows these effects to be incorporated into the general scheme of experimental phasing methods using an extended Harker construction is introduced. This requires a paradigm shift in the data-processing strategy, since the usual separation of the data-merging and phasing steps is abandoned. The data are kept unmerged down to the Harker construction, where the symmetry-breaking is explicitly modelled and refined and becomes a source of supplementary phase information. These ideas have been implemented in the phasing program SHARP. Refinements using actual data show that exploitation of the anisotropy of anomalous scattering can deliver substantial extra phasing power compared with conventional approaches using the same raw data. Examples are given that show improvements in the phases which are typically of the same order of magnitude as those obtained in a conventional approach by adding a second-wavelength data set to a SAD experiment. It is argued that such gains, which come essentially for free, i.e. without the collection of new data, are highly significant, since radiation damage can frequently preclude the collection of a second-wavelength data set. Finally, further developments in synchrotron instrumentation and in the design of data-collection strategies that could help to maximize these gains are outlined.
anisotropy of anomalous scattering; phasing; SAD; MAD; polarized resonant diffraction
The crystal structure of perdeuterated diisopropyl fluorophosphatase is reported and compared with the hydrogenated structure. Diffraction guidelines for neutron crystallography experiments are summarized.
The signal-to-noise ratio is one of the limiting factors in neutron macromolecular crystallography. Protein perdeuteration, which replaces all H atoms with deuterium, is a method of improving the signal-to-noise ratio of neutron crystallography experiments by reducing the incoherent scattering of the hydrogen isotope. Detailed analyses of perdeuterated and hydrogenated structures are necessary in order to evaluate the utility of perdeuterated crystals for neutron diffraction studies. The room-temperature X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase) is reported at 2.1 Å resolution. Comparison with an independently refined hydrogenated room-temperature structure of DFPase revealed no major systematic differences, although the crystals of perdeuterated DFPase did not diffract neutrons. The lack of diffraction is examined with respect to data-collection and crystallographic parameters. The diffraction characteristics of successful neutron structure determinations are presented as a guideline for future neutron diffraction studies of macromolecules. X-ray diffraction to beyond 2.0 Å resolution appears to be a strong predictor of successful neutron structures.
diisopropyl fluorophosphatase; perdeuteration
Anomalous diffraction signals can be very weak and sensitive to radiation damage. Here, in application to a poorly diffracting (d
min of 3.5 Å) and relatively large structure (1456 ordered residues), it is shown that data merged from multiple crystals can support SAD structure determination when no single data set is adequate.
Multiwavelength anomalous diffraction (MAD) and single-wavelength anomalous diffraction (SAD) are the two most commonly used methods for de novo determination of macromolecular structures. Both methods rely on the accurate extraction of anomalous signals; however, because of factors such as poor intrinsic order, radiation damage, inadequate anomalous scatterers, poor diffraction quality and other noise-causing factors, the anomalous signal from a single crystal is not always good enough for structure solution. In this study, procedures for extracting more accurate anomalous signals by merging data from multiple crystals are devised and tested. SAD phasing tests were made with a relatively large (1456 ordered residues) poorly diffracting (d
min = 3.5 Å) selenomethionyl protein (20 Se). It is quantified that the anomalous signal, success in substructure determination and accuracy of phases and electron-density maps all improve with an increase in the number of crystals used in merging. Structure solutions are possible when no single crystal can support structural analysis. It is proposed that such multi-crystal strategies may be broadly useful when only weak anomalous signals are available.
anomalous scattering; MAD; multiple crystals; phase determination; SAD
Anomalous diffraction signals from typical native macromolecules are very weak, frustrating their use in structure determination. Here, native SAD procedures are described for enhancing the signal to noise in anomalous diffraction by using multiple crystals are described. Five applications demonstrate that truly routine structure determination is possible without the need for heavy atoms.
Structure determinations for biological macromolecules that have no known structural antecedents typically involve the incorporation of heavier atoms than those found natively in biological molecules. Currently, selenomethionyl proteins analyzed using single- or multi-wavelength anomalous diffraction (SAD or MAD) data predominate for such de novo analyses. Naturally occurring metal ions such as zinc or iron often suffice in MAD or SAD experiments, and sulfur SAD has been an option since it was first demonstrated using crambin 30 years ago; however, SAD analyses of structures containing only light atoms (Z
max ≤ 20) have not been common. Here, robust procedures for enhancing the signal to noise in measurements of anomalous diffraction by combining data collected from several crystals at a lower than usual X-ray energy are described. This multi-crystal native SAD method was applied in five structure determinations, using between five and 13 crystals to determine substructures of between four and 52 anomalous scatterers (Z ≤ 20) and then the full structures ranging from 127 to 1200 ordered residues per asymmetric unit at resolutions from 2.3 to 2.8 Å. Tests were devised to assure that all of the crystals used were statistically equivalent. Elemental identities for Ca, Cl, S, P and Mg were proven by f′′ scattering-factor refinements. The procedures are robust, indicating that truly routine structure determination of typical native macromolecules is realised. Synchrotron beamlines that are optimized for low-energy X-ray diffraction measurements will facilitate such direct structural analysis.
anomalous scattering; multiple crystals; phase determination; sulfur SAD
A cross-validation-based method for bias reduction in ‘classical’ iterative density modification of experimental X-ray crystallography maps provides significantly more accurate phase-quality estimates and leads to improved automated model building.
Density modification often suffers from an overestimation of phase quality, as seen by escalated figures of merit. A new cross-validation-based method to address this estimation bias by applying a bias-correction parameter ‘β’ to maximum-likelihood phase-combination functions is proposed. In tests on over 100 single-wavelength anomalous diffraction data sets, the method is shown to produce much more reliable figures of merit and improved electron-density maps. Furthermore, significantly better results are obtained in automated model building iterated with phased refinement using the more accurate phase probability parameters from density modification.
reliable figure-of-merit estimates; density modification; maximum likelihood; bias reduction
Hardware and software solutions for MX data-collection strategies using the EMBL/ESRF miniaturized multi-axis goniometer head are presented.
Most macromolecular crystallography (MX) diffraction experiments at synchrotrons use a single-axis goniometer. This markedly contrasts with small-molecule crystallography, in which the majority of the diffraction data are collected using multi-axis goniometers. A novel miniaturized κ-goniometer head, the MK3, has been developed to allow macromolecular crystals to be aligned. It is available on the majority of the structural biology beamlines at the ESRF, as well as elsewhere. In addition, the Strategy for the Alignment of Crystals (STAC) software package has been developed to facilitate the use of the MK3 and other similar devices. Use of the MK3 and STAC is streamlined by their incorporation into online analysis tools such as EDNA. The current use of STAC and MK3 on the MX beamlines at the ESRF is discussed. It is shown that the alignment of macromolecular crystals can result in improved diffraction data quality compared with data obtained from randomly aligned crystals.
kappa goniometer; crystal alignment; data-collection strategies
A structural characterization of multi-component cellular assemblies is essential to explain the mechanisms governing biological function. Macromolecular architectures may be revealed by integrating information collected from various biophysical sources - for instance, by intepreting low-resolution electron cryomicroscopy reconstructions in relation to the crystal structures of the constituent fragments. A simultaneous registration of multiple components is beneficial when building atomic models as it introduces additional spatial constraints to facilitate the native placement inside the map. The high-dimensional nature of such a search problem prevents the exhaustive exploration of all possible solutions. Here we introduce a novel method based on genetic algorithms, for the effcient exploration of the multi-body registration search space. The classic scheme of a genetic algorithm was enhanced with new genetic operations, tabu search and parallel computing strategies and validated on a benchmark of synthetic and experimental cryo-EM datasets. Even at a low level of detail, for example 35–40Å, the technique successfully registered multiple component biomolecules, measuring accuracies within one order of magnitude of the nominal resolutions of the maps. The algorithm was implemented using the Sculptor molecular modeling framework, which also provides a user-friendly graphical interface and enables an instantaneous, visual exploration of intermediate solutions.
simultaneous registration; multi-body registration; multicomponent; macromolecular assembly; cryo-electron microscopy; cryo-EM; multi-resolution modeling; genetic algorithms; tabu search
Crystal structure analyses for biological macromolecules without known structural relatives entail solving the crystallographic phase problem. Typical de novo phase evaluations depend on incorporating heavier atoms than those found natively; most commonly, multi- or single-wavelength anomalous diffraction (MAD or SAD) experiments exploit selenomethionyl proteins. Here we realize routine structure determination using intrinsic anomalous scattering from native macromolecules. We devised robust procedures for enhancing signal-to-noise in the slight anomalous scattering from generic native structures by combining data measured from multiple crystals at lower-than-usual x-ray energy. Using this multi-crystal SAD method (5–13 equivalent crystals), we determined structures at modest resolution (2.8Å-2.3Å) for native proteins varying in size (127–1148 unique residues) and number of sulfur sites (3–28). With no requirement for heavy-atom incorporation, such experiments provide an attractive alternative to selenomethionyl SAD experiments.
The effect of the X-ray dose on room-temperature time-resolved Laue data is discussed.
Protein X-ray structures are determined with ionizing radiation that damages the protein at high X-ray doses. As a result, diffraction patterns deteriorate with the increased absorbed dose. Several strategies such as sample freezing or scavenging of X-ray-generated free radicals are currently employed to minimize this damage. However, little is known about how the absorbed X-ray dose affects time-resolved Laue data collected at physiological temperatures where the protein is fully functional in the crystal, and how the kinetic analysis of such data depends on the absorbed dose. Here, direct evidence for the impact of radiation damage on the function of a protein is presented using time-resolved macromolecular crystallography. The effect of radiation damage on the kinetic analysis of time-resolved X-ray data is also explored.
radiation damage; X-ray dose; room temperature; time-resolved crystallography; Laue crystallography
The catalytic domain of CHBI was purified from a cellular extract of T. harzianum. Diffraction-quality crystals were obtained and a native X-ray data set was collected using a synchrotron source.
The filamentous fungus Trichoderma harzianum has a considerable cellulolytic activity that is mediated by a complex of enzymes which are essential for the hydrolysis of microcrystalline cellulose. These enzymes were produced by the induction of T. harzianum with microcrystalline cellulose (Avicel) under submerged fermentation in a bioreactor. The catalytic core domain (CCD) of cellobiohydrolase I (CBHI) was purified from the extracellular extracts and submitted to robotic crystallization. Diffraction-quality CBHI CCD crystals were grown and an X-ray diffraction data set was collected under cryogenic conditions using a synchrotron-radiation source.
cellobiohydrolases; Trichoderma harzianum; cellulases
Correction for ice-rings in diffraction images is demonstrated as an alternative to exclusion of affected reflections. Completeness can be increased without significant loss of quality in the integrated data.
Macromolecular structures are routinely determined at cryotemperatures using samples flash-cooled in the presence of cryoprotectants. However, sometimes the best diffraction is obtained under conditions where ice formation is not completely ablated, with the result that characteristic ice rings are superimposed on the macromolecular diffraction. In data processing, the reflections that are most affected by the ice rings are usually excluded. Here, an alternative approach of subtracting the ice diffraction is tested. High completeness can be retained with little adverse effect upon the quality of the integrated data. This offers an alternate strategy when high levels of cryoprotectant lead to loss of crystal quality.
cryotemperatures; data collection; data processing; ice rings; DeIce
The X-CHIP (X-ray Crystallography High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process.
The X-CHIP (X-ray Crystallization High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process. The system has been designed for crystallization condition screening, visual crystal inspection, initial X-ray screening and data collection in a high-throughput fashion. X-ray diffraction data acquisition can be performed directly on-the-chip at room temperature using an in situ approach. The capabilities of the chip eliminate the necessity for manual crystal handling and cryoprotection of crystal samples, while allowing data collection from multiple crystals in the same drop. This technology would be especially beneficial for projects with large volumes of data, such as protein-complex studies and fragment-based screening. The platform employs hydrophilic and hydrophobic concentric ring surfaces on a miniature plate transparent to visible light and X-rays to create a well defined and stable microbatch crystallization environment. The results of crystallization and data-collection experiments demonstrate that high-quality well diffracting crystals can be grown and high-resolution diffraction data sets can be collected using this technology. Furthermore, the quality of a single-wavelength anomalous dispersion data set collected with the X-CHIP at room temperature was sufficient to generate interpretable electron-density maps. This technology is highly resource-efficient owing to the use of nanolitre-scale drop volumes. It does not require any modification for most in-house and synchrotron beamline systems and offers a promising opportunity for full automation of the X-ray structure-determination process.
protein crystallization devices; in situ X-ray analysis; crystallization; crystal visual inspection; diffraction data collection
The emergence of drug-resistant bacteria highlights the importance of identifying potential drug targets. Dihydrodipicolinate synthase (DHDPS) is a valid but as yet unexploited antimicrobial target that functions in the biosynthesis of (S)-lysine. In this study, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DHDPS from S. pneumoniae are described.
Dihydrodipicolinate synthase (DHDPS; EC 188.8.131.52) catalyzes the rate-limiting step in the (S)-lysine biosynthesis pathway of bacteria and plants. Here, the cloning of the DHDPS gene from a clinical isolate of Streptococcus pneumoniae (OXC141 strain) and the strategy used to express, purify and crystallize the recombinant enzyme are described. Diffracting crystals were grown in high-molecular-weight PEG precipitants using the hanging-drop vapour-diffusion method. The best crystal, from which data were collected, diffracted to beyond 2.0 Å resolution. Initially, the crystals were thought to belong to space group P42212, with unit-cell parameters a = 105.5, b = 105.5, c = 62.4 Å. However, the R factors remained high following initial processing of the data. It was subsequently shown that the data set was twinned and it was thus reprocessed in space group P2, resulting in a significant reduction in the R factors. Determination of the structure will provide insight into the design of novel antimicrobial agents targeting this important enzyme from S. pneumoniae.
antimicrobials; antibiotic resistance; dihydrodipicolinate synthase; lysine biosynthesis; Streptococcus pneumoniae
Diffraction data collection parameters leading to optimal data quality are discussed in the context of different applications of these data.
Diffraction data collection is the last experimental stage in structural crystallography. It has several technical and theoretical aspects and a compromise usually has to be found between various parameters in order to achieve optimal data quality. The influence and importance of various experimental parameters and their consequences are discussed in the context of different data applications, such as molecular replacement, anomalous phasing, high-resolution refinement or searching for ligands.
diffraction data collection; data-collection strategies; diffraction experiments
Radiation-induced decay of crystal diffraction and additional specific chemical changes of macromolecules forming the crystal lattice are currently two of the main limiting factors in the acquisition of macromolecular diffraction data and macromolecular structure determination. Data-processing and phasing protocols are discussed in the context of radiation-induced changes.
In macromolecular crystallography, the acquisition of a complete set of diffraction intensities typically involves a high cumulative dose of X-ray radiation. In the process of data acquisition, the irradiated crystal lattice undergoes a broad range of chemical and physical changes. These result in the gradual decay of diffraction intensities, accompanied by changes in the macroscopic organization of crystal lattice order and by localized changes in electron density that, owing to complex radiation chemistry, are specific for a particular macromolecule. The decay of diffraction intensities is a well defined physical process that is fully correctable during scaling and merging analysis and therefore, while limiting the amount of diffraction, it has no other impact on phasing procedures. Specific chemical changes, which are variable even between different crystal forms of the same macromolecule, are more difficult to predict, describe and correct in data. Appearing during the process of data collection, they result in gradual changes in structure factors and therefore have profound consequences in phasing procedures. Examples of various combinations of radiation-induced changes are presented and various considerations pertinent to the determination of the best strategies for handling diffraction data analysis in representative situations are discussed.
radiation-induced specific changes; relative B factor; scaling B factor; experimental phasing; synchrotron radiation
The Microcapillary Protein Crystallization System (MPCS) is a new protein-crystallization technology used to generate nanolitre-sized crystallization experiments for crystal screening and optimization. Using the MPCS, diffraction-ready crystals were grown in the plastic MPCS CrystalCard and were used to solve the structure of methionine-R-sulfoxide reductase.
The Microcapillary Protein Crystallization System (MPCS) embodies a new semi-automated plug-based crystallization technology which enables nanolitre-volume screening of crystallization conditions in a plasticware format that allows crystals to be easily removed for traditional cryoprotection and X-ray diffraction data collection. Protein crystals grown in these plastic devices can be directly subjected to in situ X-ray diffraction studies. The MPCS integrates the formulation of crystallization cocktails with the preparation of the crystallization experiments. Within microfluidic Teflon tubing or the microfluidic circuitry of a plastic CrystalCard, ∼10–20 nl volume droplets are generated, each representing a microbatch-style crystallization experiment with a different chemical composition. The entire protein sample is utilized in crystallization experiments. Sparse-matrix screening and chemical gradient screening can be combined in one comprehensive ‘hybrid’ crystallization trial. The technology lends itself well to optimization by high-granularity gradient screening using optimization reagents such as precipitation agents, ligands or cryoprotectants.
protein crystallization; Microcapillary Protein Crystallization System
A modified Laue technique suitable for time-resolved diffraction is described in which profile-independent integration is used, the RATIO method is applied and multi-crystal data are normalized to a common scale. The method is applied in single-pulse pump–probe studies of a binuclear Rh complex, showing Rh—Rh bond shortening of 0.136 (8) Å on excitation.
A modified Laue method is shown to produce excited-state structures at atomic resolution of a quality competitive with those from monochromatic experiments. The much faster data collection allows the use of only one or a few X-ray pulses per data frame, which minimizes crystal damage caused by laser exposure of the samples and optimizes the attainable time resolution. The method has been applied to crystals of the α-modification of Rh2(μ-PNP)2(PNP)2 (BPh4)2 [PNP = CH3N(P(OCH3)2)2, Ph = phenyl]. The experimental results show a shortening of the Rh—Rh distance in the organometallic complex of 0.136 (8) Å on excitation and are quantitatively supported by quantum-mechanical (QM)/molecular-mechanics (MM) theoretical calculations which take into account the confining effect of the crystal environment, but not by theoretical results on the isolated complex, demonstrating the defining effect of the crystal matrix.
Laue techniques; single-pulse diffraction; quantum-mechanical/molecular-mechanics calculations; QM/MM calculations; time-resolved X-ray crystallography
A shutterless continuous rotation method using an X-ray complementary metal-oxide semiconductor (CMOS) detector has been developed for high-speed, precise data collection in protein crystallography. The new method and detector were applied to the structure determination of three proteins by multi- and single-wavelength anomalous diffraction phasing and have thereby been proved to be applicable in protein crystallography.
A new shutterless continuous rotation method using an X-ray complementary metal-oxide semiconductor (CMOS) detector has been developed for high-speed, precise data collection in protein crystallography. The principle of operation and the basic performance of the X-ray CMOS detector (Hamamatsu Photonics KK C10158DK) have been shown to be appropriate to the shutterless continuous rotation method. The data quality of the continuous rotation method is comparable to that of the conventional oscillation method using a CCD detector and, furthermore, the combination with fine ϕ slicing improves the data accuracy without increasing the data-collection time. The new method is more sensitive to diffraction intensity because of the narrow dynamic range of the CMOS detector. However, the strong diffraction spots were found to be precisely measured by recording them on successive multiple images by selecting an adequate rotation step. The new method has been used to successfully determine three protein structures by multi- and single-wavelength anomalous diffraction phasing and has thereby been proved applicable in protein crystallography. The apparatus and method may become a powerful tool at synchrotron protein crystallography beamlines with important potential across a wide range of X-ray wavelengths.
protein crystallography; shutterless continuous rotation method; X-ray CMOS detectors; X-ray wavelength capabilities
Native zinc-containing ATP sulfurylase from D. desulfuricans ATCC 27774 was purified to homogeneity and crystallized. Diffraction data were collected to 2.5 Å resolution.
Native zinc/cobalt-containing ATP sulfurylase (ATPS; EC 184.108.40.206; MgATP:sulfate adenylyltransferase) from Desulfovibrio desulfuricans ATCC 27774 was purified to homogeneity and crystallized. The orthorhombic crystals diffracted to beyond 2.5 Å resolution and the X-ray data collected should allow the determination of the structure of the zinc-bound form of this ATPS. Although previous biochemical studies of this protein indicated the presence of a homotrimer in solution, a dimer was found in the asymmetric unit. Elucidation of this structure will permit a better understanding of the role of the metal in the activity and stability of this family of enzymes.
ATP sulfurylases; zinc; cobalt; sulfate-reducing bacteria
A fully automated procedure for solving MIR and MAD structures has been developed using a scoring scheme to convert the structure-solution process into an optimization problem.
Obtaining an electron-density map from X-ray diffraction data can be difficult and time-consuming even after the data have been collected, largely because MIR and MAD structure determinations currently require many subjective evaluations of the qualities of trial heavy-atom partial structures before a correct heavy-atom solution is obtained. A set of criteria for evaluating the quality of heavy-atom partial solutions in macromolecular crystallography have been developed. These have allowed the conversion of the crystal structure-solution process into an optimization problem and have allowed its automation. The SOLVE software has been used to solve MAD data sets with as many as 52 selenium sites in the asymmetric unit. The automated structure-solution process developed is a major step towards the fully automated structure-determination, model-building and refinement procedure which is needed for genomic scale structure determinations.
MAD; MIR; automated structure solution