scispace - formally typeset
Search or ask a question

Showing papers in "Acta Crystallographica Section D-biological Crystallography in 2011"


Journal ArticleDOI
TL;DR: An overview of the CCP4 software suite for macromolecular crystallography is given.
Abstract: The CCP4 (Collaborative Computational Project, Number 4) software suite is a collection of programs and associated data and software libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims. The programs are from a wide variety of sources but are connected by a common infrastructure provided by standard file formats, data objects and graphical interfaces. Structure solution by macromolecular crystallo­graphy is becoming increasingly automated and the CCP4 suite includes several automation pipelines. After giving a brief description of the evolution of CCP4 over the last 30 years, an overview of the current suite is given. While detailed descriptions are given in the accompanying articles, here it is shown how the individual programs contribute to a complete software package.

11,023 citations


Journal ArticleDOI
TL;DR: The general principles behind the macromolecular crystal structure refinement program REFMAC5 are described.
Abstract: This paper describes various components of the macromolecular crystallographic refinement program REFMAC5, which is distributed as part of the CCP4 suite. REFMAC5 utilizes different likelihood functions depending on the diffraction data employed (amplitudes or intensities), the presence of twinning and the availability of SAD/SIRAS experimental diffraction data. To ensure chemical and structural integrity of the refined model, REFMAC5 offers several classes of restraints and choices of model parameterization. Reliable models at resolutions at least as low as 4 A can be achieved thanks to low-resolution refinement tools such as secondary-structure restraints, restraints to known homologous structures, automatic global and local NCS restraints, `jelly-body' restraints and the use of novel long-range restraints on atomic displacement parameters (ADPs) based on the Kullback–Leibler divergence. REFMAC5 additionally offers TLS parameterization and, when high-resolution data are available, fast refinement of anisotropic ADPs. Refinement in the presence of twinning is performed in a fully automated fashion. REFMAC5 is a flexible and highly optimized refinement package that is ideally suited for refinement across the entire resolution spectrum encountered in macromolecular crystallography.

7,134 citations


Journal ArticleDOI
TL;DR: A new graphical user interface to the MOSFLM program has been developed to simplify the processing of macromolecular diffraction data and provides visual feedback on the progress of each stage.
Abstract: iMOSFLM is a graphical user interface to the diffraction data-integration program MOSFLM. It is designed to simplify data processing by dividing the process into a series of steps, which are normally carried out sequentially. Each step has its own display pane, allowing control over parameters that influence that step and providing graphical feedback to the user. Suitable values for integration parameters are set automatically, but additional menus provide a detailed level of control for experienced users. The image display and the interfaces to the different tasks (indexing, strategy calculation, cell refinement, integration and history) are described. The most important parameters for each step and the best way of assessing success or failure are discussed.

2,707 citations


Journal ArticleDOI
TL;DR: A summary of how to run the data-reduction programs in the CCP4 suite.
Abstract: This paper presents an overview of how to run the CCP4 programs for data reduction (SCALA, POINTLESS and CTRUNCATE) through the CCP4 graphical interface ccp4i and points out some issues that need to be considered, together with a few examples. It covers determination of the point-group symmetry of the diffraction data (the Laue group), which is required for the subsequent scaling step, examination of systematic absences, which in many cases will allow inference of the space group, putting multiple data sets on a common indexing system when there are alternatives, the scaling step itself, which produces a large set of data-quality indicators, estimation of |F| from intensity and finally examination of intensity statistics to detect crystal pathologies such as twinning. An appendix outlines the scoring schemes used by the program POINTLESS to assign probabilities to possible Laue and space groups.

1,269 citations


Journal ArticleDOI
TL;DR: Typical topics and problems encountered during data processing of diffraction experiments are discussed and the tools provided in the autoPROC software are described.
Abstract: A typical diffraction experiment will generate many images and data sets from different crystals in a very short time. This creates a challenge for the high-throughput operation of modern synchrotron beamlines as well as for the subsequent data processing. Novice users in particular may feel overwhelmed by the tables, plots and numbers that the different data-processing programs and software packages present to them. Here, some of the more common problems that a user has to deal with when processing a set of images that will finally make up a processed data set are shown, concentrating on difficulties that may often show up during the first steps along the path of turning the experiment (i.e. data collection) into a model (i.e. interpreted electron density). Difficulties such as unexpected crystal forms, issues in crystal handling and suboptimal choices of data-collection strategies can often be dealt with, or at least diagnosed, by analysing specific data characteristics during processing. In the end, one wants to distinguish problems over which one has no immediate control once the experiment is finished from problems that can be remedied a posteriori. A new software package, autoPROC, is also presented that combines third-party processing programs with new tools and an automated workflow script that is intended to provide users with both guidance and insight into the offline processing of data affected by the difficulties mentioned above, with particular emphasis on the automated treatment of multi-sweep data sets collected on multi-axis goniostats.

1,239 citations


Journal ArticleDOI
TL;DR: The CCP4 molecular-graphics program now uses the Qt framework to provide a modern look and feel and there are many new features including rendering for publication-quality images and sequence alignment.
Abstract: CCP4mg is a molecular-graphics program that is designed to give rapid access to both straightforward and complex static and dynamic representations of macromolecular structures. It has recently been updated with a new interface that provides more sophisticated atom-selection options and a wizard to facilitate the generation of complex scenes. These scenes may contain a mixture of coordinate-derived and abstract graphical objects, including text objects, arbitrary vectors, geometric objects and imported images, which can enhance a picture and eliminate the need for subsequent editing. Scene descriptions can be saved to file and transferred to other molecules. Here, the substantially enhanced version 2 of the program, with a new underlying GUI toolkit, is described. A built-in rendering module produces publication-quality images.

1,112 citations



Journal ArticleDOI
TL;DR: The molecular-replacement model-improvement program Sculptor is described, with an analysis of the algorithms used.
Abstract: In molecular replacement, the quality of models can be improved by transferring information contained in sequence alignment to the template structure. A family of algorithms has been developed that make use of the sequence-similarity score calculated from residue-substitution scores smoothed over nearby residues to delete or downweight parts of the model that are unreliable. These algorithms have been implemented in the program Sculptor, together with well established methods that are in common use for model improvement. An analysis of the new algorithms has been performed by studying the effect of algorithm parameters on the quality of models. Benchmarking against existing tech­niques shows that models from Sculptor compare favourably, especially if the alignment is unreliable. Carrying out multiple trials using alternative models created from the same structure but using different algorithm parameters can significantly improve the success rate.

203 citations


Journal ArticleDOI
TL;DR: The crystal structure of PERK's kinase domain suggests conservation in the mode of activation of eIF2α kinases and is consistent with a `line-up' model for PERK activation triggered by oligomerization of its luminal domain.
Abstract: The endoplasmic reticulum (ER) unfolded protein response (UPR) is comprised of several intracellular signaling pathways that alleviate ER stress. The ER-localized transmembrane kinase PERK is one of three major ER stress transducers. Oligomerization of PERK's N-terminal ER luminal domain by ER stress promotes PERK trans-autophosphorylation of the C-terminal cytoplasmic kinase domain at multiple residues including Thr980 on the kinase activation loop. Activated PERK phosphorylates Ser51 of the α-subunit of translation initiation factor 2 (eIF2α), which inhibits initiation of protein synthesis and reduces the load of unfolded proteins entering the ER. The crystal structure of PERK's kinase domain has been determined to 2.8 A resolution. The structure resembles the back-to-back dimer observed in the related eIF2α kinase PKR. Phosphorylation of Thr980 stabilizes both the activation loop and helix αG in the C-terminal lobe, preparing the latter for eIF2α binding. The structure suggests conservation in the mode of activation of eIF2α kinases and is consistent with a `line-up' model for PERK activation triggered by oligomerization of its luminal domain.

115 citations


Journal ArticleDOI
TL;DR: SAD data can be used in Phaser to solve novel structures, supplement molecular-replacement phase information or identify anomalous scatterers from a final refined model.
Abstract: Phaser is a program that implements likelihood-based methods to solve macromolecular crystal structures, currently by molecular replacement or single-wavelength anomalous diffraction (SAD). SAD phasing is based on a likelihood target derived from the joint probability distribution of observed and calculated pairs of Friedel-related structure factors. This target combines information from the total structure factor (primarily non-anomalous scattering) and the difference between the Friedel mates (anomalous scattering). Phasing starts from a substructure, which is usually but not necessarily a set of anomalous scatterers. The substructure can also be a protein model, such as one obtained by molecular replacement. Additional atoms are found using a log-likelihood gradient map, which shows the sites where the addition of scattering from a particular atom type would improve the likelihood score. An automated completion algorithm adds new sites, choosing optionally among different atom types, adds anisotropic B-factor parameters if appropriate and deletes atoms that refine to low occupancy. Log-likelihood gradient maps can also identify which atoms in a refined protein structure are anomalous scatterers, such as metal or halide ions. These maps are more sensitive than conventional model-phased anomalous difference Fouriers and the iterative completion algorithm is able to find a significantly larger number of convincing sites.

110 citations


Journal ArticleDOI
TL;DR: This work provides an objective test that can contribute to making decisions in both focused and structural genomics crystallography projects and proposes a simple method for estimation of the crystallization likelihood of biological samples.
Abstract: The identification of crystallization conditions for biological molecules largely relies on a trial-and-error process in which a number of parameters are explored in large screening experiments. Currently, construct design and sample formulation are recognized as critical variables in this process and often a number of protein variants are assayed for crystallization either sequentially or in parallel, which adds complexity to the screening process. Significant effort is dedicated to sample characterization and quality-control experiments in order to identify at an early stage and prioritize those samples which would be more likely to crystallize. However, large-scale studies relating crystallization success to sample properties are generally lacking. In this study, the thermal stability of 657 samples was estimated using a simplified Thermofluor assay. These samples were also subjected to automated vapour-diffusion crystallization screening under a constant protocol. Analysis of the data shows that samples with an apparent melting temperature (T(m)) of 318 K or higher crystallized in 49% of cases, while the crystallization success rate decreased rapidly for samples with lower T(m). Only 23% of samples with a T(m) below 316 K produced crystals. Based on this analysis, a simple method for estimation of the crystallization likelihood of biological samples is proposed. This method is easy, rapid and consumes very small amounts of sample. The results of this assay can be used to determine optimal incubation temperatures for crystallization experiments or to prioritize certain constructs. More generally, this work provides an objective test that can contribute to making decisions in both focused and structural genomics crystallography projects.

Journal ArticleDOI
TL;DR: In this article, the crystal structures of human nucleosomes containing either H3.2 or H 3.3 have been solved, and the structures were essentially the same as that of the H1.1 nucleosome.
Abstract: The nucleosome is the fundamental repeating unit of chromatin, via which genomic DNA is packaged into the nucleus in eukaryotes. In the nucleosome, two copies of each core histone, H2A, H2B, H3 and H4, form a histone octamer which wraps 146 base pairs of DNA around itself. All of the core histones except for histone H4 have nonallelic isoforms called histone variants. In humans, eight histone H3 variants, H3.1, H3.2, H3.3, H3T, H3.5, H3.X, H3.Y and CENP-A, have been reported to date. Previous studies have suggested that histone H3 variants possess distinct functions in the formation of specific chromosome regions and/or in the regulation of transcription and replication. H3.1, H3.2 and H3.3 are the most abundant H3 variants. Here, crystal structures of human nucleosomes containing either H3.2 or H3.3 have been solved. The structures were essentially the same as that of the H3.1 nucleosome. Since the amino-acid residues specific for H3.2 and H3.3 are located on the accessible surface of the H3/H4 tetramer, they may be potential interaction sites for H3.2- and H3.3-specific chaperones.

Journal ArticleDOI
TL;DR: Analysis of the anisotropic displacement parameters included in the crystallographic models is consistent with the variation found in the loop structures and the view that the dynamic nature of the protein structure contributes to the ability of theprotein to bind biotin so tightly.
Abstract: Atomic resolution crystallographic studies of streptavidin and its biotin complex have been carried out at 1.03 and 0.95 A, respectively. The wild-type protein crystallized with a tetramer in the asymmetric unit, while the crystals of the biotin complex contained two subunits in the asymmetric unit. Comparison of the six subunits shows the various ways in which the protein accommodates ligand binding and different crystal-packing environments. Conformational variation is found in each of the polypeptide loops connecting the eight strands in the β-­sandwich subunit, but the largest differences are found in the flexible binding loop (residues 45–52). In three of the unliganded subunits the loop is in an ‘open’ conformation, while in the two subunits binding biotin, as well as in one of the unliganded subunits, this loop ‘closes’ over the biotin–binding site. The ‘closed’ loop contributes to the protein’s high affinity for biotin. Analysis of the anisotropic displacement parameters included in the crystallographic models is consistent with the variation found in the loop structures and the view that the dynamic nature of the protein structure contributes to the ability of the protein to bind biotin so tightly.

Journal ArticleDOI
TL;DR: The CRANK algorithm as mentioned in this paper has been shown to effectively detect and phase anomalous scatterers from single-wavelength anomalous diffraction data, and many more structures can be built automatically with single- or multiple-waspectral or single isomorphous replacement with anomalous scattering data.
Abstract: For its first release in 2004, CRANK was shown to effectively detect and phase anomalous scatterers from single-wavelength anomalous diffraction data. Since then, CRANK has been significantly improved and many more structures can be built automatically with single- or multiple-wavelength anomalous diffraction or single isomorphous replacement with anomalous scattering data. Here, the new algorithms that have been developed that have led to these substantial improvements are discussed and CRANK's performance on over 100 real data sets is shown. The latest version of CRANK is freely available for download at http://www.bfsc.leidenuniv.nl/software/crank/ and from CCP4 (http://www.ccp4.ac.uk/).

Journal ArticleDOI
TL;DR: These results demonstrate a possible method for automation of ligand screening by X-ray crystallography, and use of dedicated plates in connection with an optimal parametrization of the G-rob robot allows efficient data collection.
Abstract: X-ray crystallography is now a recognized technique for ligand screening, especially for fragment-based drug design. However, protein crystal handling is still tedious and limits further automation. An alternative method for the solution of crystal structures of proteins in complex with small ligands is proposed. Crystallization drops are directly exposed to an X-ray beam after cocrystallization or soaking with the desired ligands. The use of dedicated plates in connection with an optimal parametrization of the G-rob robot allows efficient data collection. Three proteins currently under study in our laboratory for ligand screening by X-ray crystallography were used as validation test cases. The protein crystals belonged to different space groups, including a challenging monoclinic case. The resulting diffraction data can lead to clear ligand recognition, including indication of alternating conformations. These results demonstrate a possible method for automation of ligand screening by X-ray crystallography.

Journal ArticleDOI
TL;DR: The crystal structure of the phosphorylated-state mimic T172D mutant kinase domain from the human AMPK α2 subunit is reported in the apo form and in complex with a selective inhibitor, compound C, to help guide drug discovery.
Abstract: AMP-activated protein kinase (AMPK) is a serine/threonine kinase that functions as a sensor to maintain energy balance at both the cellular and the whole-body levels and is therefore a potential target for drug design against metabolic syndrome, obesity and type 2 diabetes. Here, the crystal structure of the phosphorylated-state mimic T172D mutant kinase domain from the human AMPK α2 subunit is reported in the apo form and in complex with a selective inhibitor, compound C. The AMPK α2 kinase domain exhibits a typical bilobal kinase fold and exists as a monomer in the crystal. Like the wild-type apo form, the T172D mutant apo form adopts the autoinhibited structure of the `DFG-out' conformation, with the Phe residue of the DFG motif anchored within the putative ATP-binding pocket. Compound C binding dramatically alters the conformation of the activation loop, which adopts an intermediate conformation between DFG-out and DFG-in. This induced fit forms a compound-C binding pocket composed of the N-lobe, the C-lobe and the hinge of the kinase domain. The pocket partially overlaps with the putative ATP-binding pocket. These three-dimensional structures will be useful to guide drug discovery.

Journal ArticleDOI
TL;DR: 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, suggesting that such multi-crystal strategies may be broadly useful when only weak anomalous signals are available.
Abstract: 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 (dmin = 3.5 A) 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.

Journal ArticleDOI
TL;DR: An introduction to the proceedings of the CCP4 study weekend is given.
Abstract: The CCP4 Study Weekend 2010 was held at the University of Nottingham. Macromolecular crystallography is increasingly being performed by scientists who do not regard themselves as dedicated crystallographers, with more than half of the structures deposited in the Protein Data Bank having a first author who is submitting for the first or second time. Regular attendants to the CCP4 Study Weekend know that the audience has become larger and is composed of scientists with wider biological interests who use crystallography as but one of a number of tools. With this in mind, the 2010 Study Weekend was organised to provide an overview of macromolecular crystallography techniques with an emphasis on software and the CCP4 Suite in particular. Emphasis was placed on teaching students rather than fundamental research in software and tech­niques. The CCP4 Suite has been at the forefront of macromolecular software for the last 30 years. The meeting was opened with a history of the Suite by one of its founders, Eleanor Dodson, followed by Martyn Winn who gave an overview of its current state and on-going developments. Zygmunt Derewenda then reviewed the current best practices in protein crystallization. This was followed by a presentation on the PiMS software by Chris Morris, replacing Rob Esnouf who was prevented from reaching the meeting due to the severe weather conditions. The next session focussed on data acquisition, and the first speaker, Gwyndaf Evans, introduced the problems faced in designing the best experiment. In a highly relevant session for inexpert users, Andrew Leslie spoke about data integration and initial processing with iMOSFLM. Phil Evans introduced data reduction and the information on data quality it can provide, and Clemens Vonrhein gave valuable advice on handling routine and problematic cases. The third session covered molecular replacement (MR). Gabor Bunkoczi spoke about automation of MR for complex cases in the Phaser software. Ronan Keegan described the database-driven automation of MR in the MrBump and Balbes pipelines. Bert Janssen gave a case study for a hard problem beyond current automation approaches. Gerard Kleywegt closed the session with an overview of changes at the PDBe. The fourth session dealt with experimental phasing, with Raj Pannu talking about automation of experimental phasing from various sources in the CRANK pipeline. Randy Read talked about recent experience of SAD phasing in Phaser, and Pavol Skubak described some new developments in density modification to reduce the problems of bias in this method. The fifth session covered refinement and model building. Dale Tronrud gave an overview of the different types of maps used during structure solution. Garib Murshudov introduced the latest features of the Refmac refinement software. Henry van den Bedem described the experience of the JCSG with different model building software in automated pipelines. Jane Richardson gave a guide on the use of MolProbity in the validation of macromolecular structures. The final session included an introduction to the identification of protein complexes by Eugene Krissinel, and talks on CCP4mg by Stuart McNicholas and Coot visualization and validation software by Paul Emsley. It now seems clear that continuing developments in integration and automation of the powerful software here described will soon lead to routine generation of high-quality (but perhaps not at the moment ‘fully polished’) novel structures from molecular replacement or experimental phasing generated within an hour of starting the data collection while at the synchrotron. Some of the presentations provided excellent teaching material but were less suitable for publication in this issue. The reader is encouraged to view the available presentations online at the CCP4 website: http://www.ccp4.ac.uk/courses/stwk10/talks.html. We thank the speakers for their contribution to the Study Weekend and the proceedings articles. We thank Shirley Miller, Damian Jones and Laura Johnston for organisational help and support.

Journal ArticleDOI
TL;DR: The structure of the bacterial microcompartment protein PduT, which has a tandem structural repeat within the subunit and forms trimers with pseudo-hexagonal symmetry, is reported.
Abstract: Propanediol metabolism in Citrobacter freundii occurs within a metabolosome, a subcellular proteinaceous bacterial microcompartment. The propanediol-utilization (Pdu) microcompartment shell is constructed from thousands of hexagonal-shaped protein complexes made from seven different types of protein subunit. Here, the structure of the bacterial microcompartment protein PduT, which has a tandem structural repeat within the subunit and forms trimers with pseudo-hexagonal symmetry, is reported. This trimeric assembly forms a flat approximately hexagonally shaped disc with a central pore that is suitable for a 4Fe–4S cluster. The essentially cubic shaped 4Fe–4S cluster conforms to the threefold symmetry of the trimer with one free iron, the role of which could be to supply electrons to an associated microcompartment enzyme, PduS.

Journal ArticleDOI
TL;DR: Methods for the analysis of the relationship between macromolecular complexes and interactions and their manifestation in crystal packing are described and discussed.
Abstract: This paper presents a discussion of existing methods for the analysis of macromolecular interactions and complexes in crystal packing. Typical situations and conditions where wrong answers may be obtained in the course of ordinary procedures are presented and discussed. The more general question of what the relationship is between natural (in-solvent) and crystallized assemblies is discussed and researched. A computational analysis suggests that weak interactions with Kd ≥ 100 µM have a considerable chance of being lost during the course of crystallization. In such instances, crystal packing misrepresents macromolecular complexes and interactions. For as many as 20% of protein dimers in the PDB the likelihood of misrepresentation is estimated to be higher than 50%. Given that weak macromolecular interactions play an important role in many biochemical processes, these results suggest that a complementary noncrystallographic study should be always conducted when inferring structural aspects of weakly bound complexes.

Journal ArticleDOI
TL;DR: These structures provide a detailed picture of the drug-binding domain of FKBP51 and the molecular binding mode of its ligand as a starting point for the rational design of improved inhibitors.
Abstract: Steroid hormone receptors are key components of mammalian stress and sex hormone systems. Many of them rely on the Hsp90 chaperone system for full function and are further fine-tuned by Hsp90-associated peptidyl–prolyl isomerases such as FK506-binding proteins 51 and 52. FK506-binding protein 51 (FKBP51) has been shown to reduce glucocorticoid receptor signalling and has been genetically associated with human stress resilience and with numerous psychiatric disorders. The peptidyl–prolyl isomerase domain of FKBP51 contains a high-affinity binding site for the natural products FK506 and rapamycin and has further been shown to convey most of the inhibitory activity on the glucocorticoid receptor. FKBP51 has therefore become a prime new target for the treatment of stress-related affective disorders that could be amenable to structure-based drug design. Here, a series of high-resolution structures of the peptidyl–prolyl isomerase domain of FKBP51 as well as a cocrystal structure with the prototypic ligand FK506 are described. These structures provide a detailed picture of the drug-binding domain of FKBP51 and the molecular binding mode of its ligand as a starting point for the rational design of improved inhibitors.

Journal ArticleDOI
TL;DR: This novel XCC4471(GGDEF)-c-di-GMP complex structure may serve as a general model for the design of lead compounds to block the DGC activity of GGDEF-domain-containing proteins in X. campestris or other microorganisms that contain multiple GGdef-domain proteins.
Abstract: Cyclic diguanosine monophosphate (c-di-GMP) is a key signalling molecule involved in regulating many important biological functions in bacteria. The synthesis of c-­di-GMP is catalyzed by the GGDEF-domain-containing diguanylate cyclase (DGC), the activity of which is regulated by the binding of product at the allosteric inhibitory (I) site. However, a significant number of GGDEF domains lack the RxxD motif characteristic of the allosteric I site. Here, the structure of XCC4471GGDEF, the GGDEF domain of a DGC from Xanthomonas campestris, in complex with c-di-GMP has been solved. Unexpectedly, the structure of the complex revealed a GGDEF-domain dimer cross-linked by two molecules of c-di-GMP at the strongly conserved active sites. In the complex (c-­di-GMP)2 adopts a novel partially inter­calated form, with the peripheral guanine bases bound to the guanine-binding pockets and the two central bases stacked upon each other. Alteration of the residues involved in specific binding to c-di-GMP led to dramatically reduced Kd values between XCC4471GGDEF and c-di-GMP. In addition, these key residues are strongly conserved among the many thousands of GGDEF-domain sequences identified to date. These results indicate a new product-bound form for GGDEF-domain-containing proteins obtained via (c-di-GMP)2 binding at the active site. This novel XCC4471GGDEF–c-di-GMP complex structure may serve as a general model for the design of lead compounds to block the DGC activity of GGDEF-domain-containing proteins in X. campestris or other microorganisms that contain multiple GGDEF-domain proteins.

Journal ArticleDOI
TL;DR: Crystallographic studies suggest that pyridyl derivatives of vanillin and INN-312 may act to prevent sickling of SS cells by increasing the fraction of the soluble high-affinity Hb S and/or by stereospecific inhibition of deoxygenated HB S polymerization.
Abstract: The affiliation of one of the authors of Abdulmalik et al. (2011) [Acta Cryst. D67, 920–928] is corrected.

Journal ArticleDOI
TL;DR: It is demonstrated that binding of GK activators does not result in conformational changes of the active protein but in stabilization of theactive form of Gk.
Abstract: Glucokinase (GK) catalyses the formation of glucose 6-­phosphate from glucose and ATP. A specific feature of GK amongst hexokinases is that it can cycle between active and inactive conformations as a function of glucose concentration, resulting in a unique positive kinetic cooperativity with glucose, which turns GK into a unique key sensor of glucose metabolism, notably in the pancreas. GK is a target of antidiabetic drugs aimed at the activation of GK activity, leading to insulin secretion. Here, the first structures of a GK–glucose complex without activator, of GK–glucose–AMP-PNP and of GK–glucose–AMP-PNP with a bound activator are reported. All these structures are extremely similar, thus demonstrating that binding of GK activators does not result in conformational changes of the active protein but in stabilization of the active form of GK.

Journal ArticleDOI
TL;DR: It is demonstrated that when some crystals of macromolecules are mounted in the complete absence of surrounding liquid no crystalline ice is formed and the diffraction resolution, merging R factors and mosaic spread values are comparable to those of crystals cryocooled in the presence of a cryoprotectant.
Abstract: Over the last 20 years cryocrystallography has revolutionized the field of macromolecular crystallography, greatly reducing radiation damage and allowing the collection of complete data sets at synchrotron sources. However, in order to cool crystals to 100 K cryoprotective agents must usually be added to prevent the formation of crystalline ice, which disrupts the macromolecular crystal lattice and often results in a degradation of diffraction quality. This process can involve the extensive testing of solution compositions and soaking protocols to find suitable conditions that maintain diffraction quality. In this study, it is demonstrated that when some crystals of macromolecules are mounted in the complete absence of surrounding liquid no crystalline ice is formed and the diffraction resolution, merging R factors and mosaic spread values are comparable to those of crystals cryocooled in the presence of a cryoprotectant. This potentially removes one of the most onerous manual steps in the structure-solution pipeline and could alleviate some of the foreseen difficulties in the automation of crystal mounting.

Journal ArticleDOI
TL;DR: TPE-UVF has several advantages over conventional UVF, including insensitivity to optical scattering, allowing imaging in turbid matrices, direct compatibility with conventional optical plates and windows by using visible light for excitation, and relatively simple integration into instrumentation developed for SONICC.
Abstract: Two-photon excited ultraviolet fluorescence (TPE-UVF) microscopy is explored for sensitive protein-crystal detection as a complement to second-order nonlinear optical imaging of chiral crystals (SONICC). Like conventional ultraviolet fluorescence (UVF), TPE-UVF generates image contrast based on the intrinsic fluorescence of aromatic residues, generally producing higher fluorescence emission within crystals than the mother liquor by nature of the higher local protein concentration. However, TPE-UVF has several advantages over conventional UVF, including (i) insensitivity to optical scattering, allowing imaging in turbid matrices, (ii) direct compatibility with conventional optical plates and windows by using visible light for excitation, (iii) elimination of potentially damaging out-of-plane UV excitation, (iv) improved signal to noise through background reduction from out-of-plane excitation and (v) relatively simple integration into instrumentation developed for SONICC.

Journal ArticleDOI
TL;DR: Thoughts about the decisions made in designing macromolecular X-ray crystallography experiments at synchrotron beamlines are presented.
Abstract: The measurement of X-ray diffraction data from macro­molecular crystals for the purpose of structure determination is the convergence of two processes: the preparation of diffraction-quality crystal samples on the one hand and the construction and optimization of an X-ray beamline and end station on the other. Like sample preparation, a macromolecular crystallography beamline is geared to obtaining the best possible diffraction measurements from crystals provided by the synchrotron user. This paper describes the thoughts behind an experiment that fully exploits both the sample and the beamline and how these map into everyday decisions that users can and should make when visiting a beamline with their most precious crystals.

Journal ArticleDOI
TL;DR: The ways in which BluIce was combined with EPICS and converted into the Java-based JBluIce are described, the solutions aimed at streamlining and speeding up operations are discussed and an overview of the tools that are provided by this new open-source control system for facilitating crystallographic experiments is given.
Abstract: The trio of macromolecular crystallography beamlines constructed by the General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) in Sector 23 of the Advanced Photon Source (APS) have been in growing demand owing to their outstanding beam quality and capacity to measure data from crystals of only a few micrometres in size. To take full advantage of the state-of-the-art mechanical and optical design of these beamlines, a significant effort has been devoted to designing fast, convenient, intuitive and robust beamline controls that could easily accommodate new beamline developments. The GM/CA-CAT beamline controls are based on the power of EPICS for distributed hardware control, the rich Java graphical user interface of Eclipse RCP and the task-oriented philosophy as well as the look and feel of the successful SSRL BluIce graphical user interface for crystallography. These beamline controls feature a minimum number of software layers, the wide use of plug-ins that can be written in any language and unified motion controls that allow on-the-fly scanning and optimization of any beamline com­ponent. This paper describes the ways in which BluIce was combined with EPICS and converted into the Java-based JBluIce, discusses the solutions aimed at streamlining and speeding up operations and gives an overview of the tools that are provided by this new open-source control system for facilitating crystallo­graphic experiments, especially in the field of microcrystallo­graphy.

Journal ArticleDOI
TL;DR: 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.
Abstract: 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.

Journal ArticleDOI
TL;DR: The same reactivity that makes some scavengers effective radioprotectors in protein solutions may explain their sensitizing effect in the protein-dense environment of a crystal.
Abstract: Recent studies have defined a data-collection protocol and a metric that provide a robust measure of global radiation damage to protein crystals. Using this protocol and metric, 19 small-molecule compounds (introduced either by cocrystalliz­ation or soaking) were evaluated for their ability to protect lysozyme crystals from radiation damage. The compounds were selected based upon their ability to interact with radiolytic products (e.g. hydrated electrons, hydrogen, hydroxyl and perhydroxyl radicals) and/or their efficacy in protecting biological molecules from radiation damage in dilute aqueous solutions. At room temperature, 12 compounds had no effect and six had a sensitizing effect on global damage. Only one compound, sodium nitrate, appeared to extend crystal lifetimes, but not in all proteins and only by a factor of two or less. No compound provided protection at T = 100 K. Scavengers are ineffective in protecting protein crystals from global damage because a large fraction of primary X-ray-induced excitations are generated in and/or directly attack the protein and because the ratio of scavenger molecules to protein molecules is too small to provide appreciable competitive protection. The same reactivity that makes some scavengers effective radioprotectors in protein solutions may explain their sensitizing effect in the protein-dense environment of a crystal. A more productive focus for future efforts may be to identify and eliminate sensitizing compounds from crystallization solutions.