The CCP4 suite: integrative software for macromolecular crystallography.

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces.

Virulence regulation with Venus flytrap domains: structure and function of the periplasmic moiety of the sensor-kinase BvgS.

Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT) domains.

JLigand: a graphical tool for the CCP4 template-restraint library.

Biological macromolecules are polymers and therefore the restraints for macromolecular refinement can be subdivided into two sets: restraints that are applied to atoms that all belong to the same monomer and restraints that are associated with the covalent bonds between monomers. The CCP4 template-restraint library contains three types of data entries defining template restraints: descriptions of monomers and their modifications, both used for intramonomer restraints, and descriptions of links for intermonomer restraints. The library provides generic descriptions of modifications and links for protein, DNA and RNA chains, and for some post-translational modifications including glycosylation.

Crystal structure of D-serine dehydratase from Escherichia coli.

D-Serine dehydratase from Escherichia coli is a member of the β-family (fold-type II) of the pyridoxal 5'-phosphate-dependent enzymes, catalyzing the conversion of D-serine to pyruvate and ammonia. The crystal structure of monomeric D-serine dehydratase has been solved to 1.97Å-resolution for an orthorhombic data set by molecular replacement.

Structural flexibility of the macrophage dengue virus receptor CLEC5A: implications for ligand binding and signaling.

The human C-type lectin-like molecule CLEC5A is a critical macrophage receptor for dengue virus. The binding of dengue virus to CLEC5A triggers signaling through the associated adapter molecule DAP12, stimulating proinflammatory cytokine release. We have crystallized an informative ensemble of CLEC5A structural conformers at 1.

REFMAC5 for the refinement of macromolecular crystal structures.

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.

Evaluating the solution from MrBUMP and BALBES.

Molecular replacement is one of the key methods used to solve the problem of determining the phases of structure factors in protein structure solution from X-ray image diffraction data. Its success rate has been steadily improving with the development of improved software methods and the increasing number of structures available in the PDB for use as search models. Despite this, in cases where there is low sequence identity between the target-structure sequence and that of its set of possible homologues it can be a difficult and time-consuming chore to isolate and prepare the best search model for molecular replacement.

Overview of the CCP4 suite and current developments.

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.

Molecular replacement with MOLREP.

MOLREP is an automated program for molecular replacement that utilizes a number of original approaches to rotational and translational search and data preparation. Since the first publication describing the program, MOLREP has acquired a variety of features that include weighting of the X-ray data and search models, multi-copy search, fitting the model into electron density, structural superposition of two models and rigid-body refinement. The program can run in a fully automatic mode using optimized parameters calculated from the input data.

BALBES: a molecular-replacement pipeline.

The number of macromolecular structures solved and deposited in the Protein Data Bank (PDB) is higher than 40 000. Using this information in macromolecular crystallography (MX) should in principle increase the efficiency of MX structure solution. This paper describes a molecular-replacement pipeline, BALBES, that makes extensive use of this repository.

ARP/wARP and molecular replacement: the next generation.

Automatic iterative model (re-)building, as implemented in ARP/wARP and its new control system flex-wARP, is particularly well suited to follow structure solution by molecular replacement. More than 100 molecular-replacement solutions automatically solved by the BALBES software were submitted to three standard protocols in flex-wARP and the results were compared with final models from the PDB. Standard metrics were gathered in a systematic way and enabled the drawing of statistical conclusions on the advantages of each protocol.

Model preparation in MOLREP and examples of model improvement using X-ray data.

The success of molecular replacement is critically dependent on the quality of the search model. Several model-preparation procedures are integrated in the molecular-replacement program MOLREP. These include model modification on the basis of amino-acid sequence alignment and model correction based on analysis of the solvent-accessibility of the atoms.

Structural framework for DNA translocation via the viral portal protein.

Tailed bacteriophages and herpesviruses load their capsids with DNA through a tunnel formed by the portal protein assembly. Here we describe the X-ray structure of the bacteriophage SPP1 portal protein in its isolated 13-subunit form and the pseudoatomic structure of a 12-subunit assembly. The first defines the DNA-interacting segments (tunnel loops) that pack tightly against each other forming the most constricted part of the tunnel; the second shows that the functional dodecameric state must induce variability in the loop positions.

SPINE workshop on automated X-ray analysis: a progress report.

The Structural Proteomics In Europe (SPINE) consortium contained a workpackage to address the automated X-ray analysis of macromolecules. The aim of this workpackage was to increase the throughput of three-dimensional structures while maintaining the high quality of conventional analyses. SPINE was able to bring together developers of software with users from the partner laboratories.

Structural bases of feed-back control of arginine biosynthesis, revealed by the structures of two hexameric N-acetylglutamate kinases, from Thermotoga maritima and Pseudomonas aeruginosa.

N-Acetylglutamate kinase (NAGK) catalyses the second step in the route of arginine biosynthesis. In many organisms this enzyme is inhibited by the final product of the route, arginine, and thus plays a central regulatory role. In addition, in photosynthetic organisms NAGK is the target of the nitrogen-signalling protein PII.

Intensity statistics in twinned crystals with examples from the PDB.

Entries deposited in the Protein Data Bank as of February 2004 for which both model and X-ray data were available were analysed to identify cases of twinning using such simple statistics as the R factor between potential twin-related reflections. Careful consideration of all identified twins showed that in many cases twinning was ignored during structure solution and refinement. Manual analysis of the models showed that twinning often occurs in association with rotational pseudosymmetry parallel to the twinning operator.

The structure of the oligopeptide-binding protein, AppA, from Bacillus subtilis in complex with a nonapeptide.

Besides their role as a source of amino acids for Bacillus subtilis, exogenous peptides play important roles in the signalling pathways leading to the development of competence and sporulation. B.subtilis has three peptide transport systems all belonging to the ATP-binding cassette family, a dipeptide permease (Dpp) and two oligopeptide permeases (Opp and App) with overlapping specificity.

REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use.

One of the most important aspects of macromolecular structure refinement is the use of prior chemical knowledge. Bond lengths, bond angles and other chemical properties are used in restrained refinement as subsidiary conditions. This contribution describes the organization and some aspects of the use of the flexible and human/machine-readable dictionary of prior chemical knowledge used by the maximum-likelihood macromolecular-refinement program REFMAC5.

Crystallization and preliminary X-ray diffraction studies of a fungal hydrolase from Ophiostoma novo-ulmi.

Dutch elm disease fungus Ophiostoma novo-ulmi contains a hydrolase activity which catalyses the resolution of racemic ethyl naproxen to the corresponding acid. The recombinant enzyme has been crystallized by the vapour-diffusion method in two crystal forms. The crystals of the first form belong to space group P2(1)2(1)2, with unit-cell parameters a = 115.

The crystal structure of a complex of Campylobacter jejuni dUTPase with substrate analogue sheds light on the mechanism and suggests the "basic module" for dimeric d(C/U)TPases.

The crystal structure of the dUTPase from the important gastric pathogen Campylobacter jejuni has been solved at 1.65 A spacing. This essential bacterial enzyme is the second representative of the new family of dimeric dUTPases to be structurally characterised.

Spherically averaged phased translation function and its application to the search for molecules and fragments in electron-density maps.

The molecular-replacement method has been extended to locate molecules and their fragments in an electron-density map. The approach is based on a new spherically averaged phased translation function. The position of the centre of mass of a search model is found prior to determination of its orientation.

Comparison of the decameric structure of peroxiredoxin-II by transmission electron microscopy and X-ray crystallography.

The decameric human erythrocyte protein torin is identical to the thiol-specific antioxidant protein-II (TSA-II), also termed peroxiredoxin-II (Prx-II). Single particle analysis from electron micrographs of Prx-II molecules homogeneously orientated across holes in the presence of a thin film of ammonium molybdate and trehalose has facilitated the production of a >/=20 A 3-D reconstruction by angular reconstitution that emphasises the D5 symmetry of the ring-like decamer. The X-ray structure for Prx-II was fitted into the transmission electron microscopic reconstruction by molecular replacement.

An approach to multi-copy search in molecular replacement.

The molecular-replacement method has been extended to a simultaneous search for multiple copies of the macromolecule in the unit cell. The central point of this approach is the construction of a multi-copy search model from the properly oriented monomers using a special translation function. The multi-copy search method has been implemented in the program MOLREP and successfully tested using experimental data.

The molecular basis of vancomycin resistance in clinically relevant Enterococci: crystal structure of D-alanyl-D-lactate ligase (VanA).

d-alanine-d-lactate ligase from Enterococcus faecium BM4147 is directly responsible for the biosynthesis of alternate cell-wall precursors in bacteria, which are resistant to the glycopeptide antibiotic vancomycin. The crystal structure has been determined with data extending to 2.5-A resolution.

Crystal structure of decameric 2-Cys peroxiredoxin from human erythrocytes at 1.7 A resolution.

Background: The peroxiredoxins (Prxs) are an emerging family of multifunctional enzymes that exhibit peroxidase activity in vitro, and in vivo participate in a range of cellular processes known to be sensitive to reactive oxygen species. Thioredoxin peroxidase B (TPx-B), a 2-Cys type II Prx from erythrocytes, promotes potassium efflux and down-regulates apoptosis and the recruitment of monocytes by endothelial tissue.

Results: The crystal structure of human decameric TPx-B purified from erythrocytes has been determined to 1.