PDBe: Protein Data Bank in Europe.

The Protein Data Bank in Europe (PDBe; pdbe.org) is a partner in the Worldwide PDB organization (wwPDB; wwpdb.org) and as such actively involved in managing the single global archive of biomacromolecular structure data, the PDB. In addition, PDBe develops tools, services and resources to make structure-related data more accessible to the biomedical community. Here we describe recently developed, extended or improved services, including an animated structure-presentation widget (PDBportfolio), a widget to graphically display the coverage of any UniProt sequence in the PDB (UniPDB), chemistry- and taxonomy-based PDB-archive browsers (PDBeXplore), and a tool for interactive visualization of NMR structures, corresponding experimental data as well as validation and analysis results (Vivaldi).

PDBe: Protein Data Bank in Europe.

The Protein Data Bank in Europe (PDBe) (http://www.ebi.ac.uk/pdbe/) is actively working with its Worldwide Protein Data Bank partners to enhance the quality and consistency of the international archive of bio-macromolecular structure data, the Protein Data Bank (PDB). PDBe also works closely with its collaborators at the European Bioinformatics Institute and the scientific community around the world to enhance its databases and services by adding curated and actively maintained derived data to the existing structural data in the PDB. We have developed a new database infrastructure based on the remediated PDB archive data and a specially designed database for storing information on interactions between proteins and bound molecules. The group has developed new services that allow users to carry out simple textual queries or more complex 3D structure-based queries. The newly designed 'PDBeView Atlas pages' provide an overview of an individual PDB entry in a user-friendly layout and serve as a starting point to further explore the information available in the PDBe database. PDBe's active involvement with the X-ray crystallography, Nuclear Magnetic Resonance spectroscopy and cryo-Electron Microscopy communities have resulted in improved tools for structure deposition and analysis.

CAALIGN: a program for pairwise and multiple protein-structure alignment.

Coordinate superposition of proteins provides a structural basis to protein similarity and therefore complements the technique of sequence alignment. Methods that carry out structure alignment are faced with the problem of the large number of trials necessary to determine the optimal alignment solution. This article presents a method of carrying out rapid (subsecond) protein-structure alignment between pairs of proteins based on a maximal C(alpha)-atom superposition. The algorithm can return alignments of 12 or more residues in length as multiple non-overlapping solutions of alignment between a pair of proteins which are independent of the fold connectivity and secondary-structure content. The algorithm is equally effective for all protein fold types and can align proteins containing no secondary-structure elements such as is the case when searching for common turn structures in proteins. It has high sensitivity and returns the set of true positive results before any false positives as judged by SCOP classification. It can find alignments between topologically different folds and returns information about sequence alignment based on structure alignment. Additionally, this algorithm has been extended to carry out multiple structure alignment to determine common structures within groups of proteins, including the nondegenerate set of proteins in the PDB. The algorithm has been implemented within the program CAALIGN and this article presents results from pairwise structure alignment, multiple structure alignment and the generation of common structure fragments found within the PDB using multiple structure alignment.

E-MSD: an integrated data resource for bioinformatics.

The Macromolecular Structure Database (MSD) group (http://www.ebi.ac.uk/msd/) continues to enhance the quality and consistency of macromolecular structure data in the Protein Data Bank (PDB) and to work towards the integration of various bioinformatics data resources. We have implemented a simple form-based interface that allows users to query the MSD directly. The MSD 'atlas pages' show all of the information in the MSD for a particular PDB entry. The group has designed new search interfaces aimed at specific areas of interest, such as the environment of ligands and the secondary structures of proteins. We have also implemented a novel search interface that begins to integrate separate MSD search services in a single graphical tool. We have worked closely with collaborators to build a new visualization tool that can present both structure and sequence data in a unified interface, and this data viewer is now used throughout the MSD services for the visualization and presentation of search results. Examples showcasing the functionality and power of these tools are available from tutorial webpages (http://www. ebi.ac.uk/msd-srv/docs/roadshow_tutorial/).

Field sports and conservation in the United Kingdom.

Many natural habitats exist on privately owned land outside protected areas, but few governments can afford to enforce or subsidize conservation of this biodiversity. Even in some developed countries, conservation subsidy schemes have only achieved limited success. Fortunately, some landowners may be willing to accept management costs in return for other benefits, although this remains controversial when it involves the killing of charismatic species. For example, participants in British field sports, such as fox hunting and game-bird shooting, may voluntarily conserve important habitats that are required by quarry species. Here we report results from a multidisciplinary study that addressed this issue by focusing on three sites across central England. We found that landowners participating in field sports maintained the most established woodland and planted more new woodland and hedgerows than those who did not, despite the equal availability of subsidies. Therefore, voluntary habitat management appears to be important for biodiversity conservation in Britain. Current debates on the future of field sports in Britain, and similar activities globally, may benefit from considering their utility as incentives to conserve additional habitat on private land.

E-MSD: the European Bioinformatics Institute Macromolecular Structure Database.

The E-MSD macromolecular structure relational database (http://www.ebi.ac.uk/msd) is designed to be a single access point for protein and nucleic acid structures and related information. The database is derived from Protein Data Bank (PDB) entries. Relational database technologies are used in a comprehensive cleaning procedure to ensure data uniformity across the whole archive. The search database contains an extensive set of derived properties, goodness-of-fit indicators, and links to other EBI databases including InterPro, GO, and SWISS-PROT, together with links to SCOP, CATH, PFAM and PROSITE. A generic search interface is available, coupled with a fast secondary structure domain search tool.

LigandFit: a novel method for the shape-directed rapid docking of ligands to protein active sites.

We present a new shape-based method, LigandFit, for accurately docking ligands into protein active sites. The method employs a cavity detection algorithm for detecting invaginations in the protein as candidate active site regions. A shape comparison filter is combined with a Monte Carlo conformational search for generating ligand poses consistent with the active site shape. Candidate poses are minimized in the context of the active site using a grid-based method for evaluating protein-ligand interaction energies. Errors arising from grid interpolation are dramatically reduced using a new non-linear interpolation scheme. Results are presented for 19 diverse protein-ligand complexes. The method appears quite promising, reproducing the X-ray structure ligand pose within an RMS of 2A in 14 out of the 19 complexes. A high-throughput screening study applied to the thymidine kinase receptor is also presented in which LigandFit, when combined with LigScore, an internally developed scoring function, yields very good hit rates for a ligand pool seeded with known actives.

Data mining the protein data bank: residue interactions.

The protein databank contains a vast wealth of structural and functional information. The analysis of this macromolecular information has been the subject of considerable work in order to advance knowledge beyond the collection of molecular coordinates. This article presents a method that determines local structural information within proteins using mathematical data mining techniques. The mine program described returns many known configurations of residues such as the catalytic triad, metal binding sites and the N-linked glycosylation site; as well as many other multiple residue interactions not previously categorized. Because mathematical constructs are used as targets, this method can identify new information not previously known, and also provide unbiased results of typical structure and their expected deviations. Because the results are defined mathematically, they cannot indicate the biological implications of the results. Therefore two support programs are described that provide insight into the biological context for the mine results. The first allows a weighted RMSD search between a template set of coordinates and a list of PDB files, and the second allows the labeling of a protein with the template results from mining to aid in the classification of this protein.

Creating structure features by data mining the PDB to use as molecular-replacement models.

Mathematical data-mining techniques to generate a representative set of protein fragments are described. Protein fragments are used as search models within the macromolecular phasing method of molecular replacement to attempt to phase protein data without a homologous model correctly. Preliminary investigations using these fragments indicate that molecular replacement with AMoRe is not sensitive enough to phase myoglobin or insulin data sufficiently for successful refinement. The results suggest that more advanced molecular replacement techniques may be successful, though at present these are not computationally practical.

X-LIGAND: an application for the automated addition of flexible ligands into electron density.

With the advent of drug-design experiments where the interaction between a protein and a ligand is determined using X-ray crystallography, the use of automated methods for modelling the ligand into electron density represents a powerful tool. Once the protein structure has been determined by crystallography it is normal that subsequent ligand-complex structures are isomorphous, or nearly so, with the original structure and it is necessary only to determine the fit of ligand to any unsatisfied electron density. The X-LIGAND application was designed with this protocol in mind and provides a tool that searches for unsatisfied electron density and then fits flexible ligands to this within minutes without user intervention.

A number of real-space torsion-angle refinement techniques for proteins, nucleic acids, ligands and solvent.

This paper describes the implementation of real-space torsion-angle refinement as a tool for model (re)building. The algorithmic details and parameterization for a number of different protocols are presented, as well as the handling of special conditions. Examples illustrating the use of the algorithms show that these tools provide a great advantage over traditional methods for building macromolecular structures. All these algorithms have been implemented in QUANTA (MSI), currently available as version QUANTA98.

EXTRACT: a program to extract three-dimensional coordinates from stereo diagrams of proteins.

The program EXTRACT has been developed to extract accurate three-dimensional coordinates from published stereo alpha-carbon diagrams of protein structures. The approach is based on the display of scanned images of the left and right eye views of the diagram on a stereo-equipped workstation, allowing construction of a molecular model using the diagram as a guide. A number of structural checks assess the building, including probability maps derived for alpha-carbon geometry in protein structures. The procedure has also been extended to produce less accurate models from mono images.

Analysis of C alpha geometry in protein structures.

The polypeptide of a protein molecule can be considered as a chain of C alpha atoms linked by pseudobonds between the C alpha atoms of successive amino acid residues. This paper presents an analysis of the angle and dihedral angles made by these pseudobonds in protein structures determined at high resolution by X-ray crystallography. This analysis reveals a strong correlation between C alpha geometry and the protein fold. The regular features of protein secondary structure such as alpha-helix and beta-sheet are very clearly defined. In addition, it is possible to identify with some confidence the discrete populations of particular conformations of beta-turn. Comparison with the traditional Ramachandran type of plot demonstrates that an analysis of protein structure on the basis of C alpha geometry provides a richer description of protein conformation. In addition, the characteristics of this geometry could be a useful guide in model building of protein structure.

Model structures and action of interleukin 1 and its antagonist.

A comparison has been made between the homology and hydrophobicity profiles of six interleukin amino acid sequences and that of the human interleukin 1 beta (IL-1 beta) for which a crystal structure exists. The resulting sequence alignment was used to build model structures for the sequences for three IL-1 alpha, two IL-1 beta and an interleukin receptor antagonist. Analysis of these structures demonstrates that the interleukin molecule has a strong electric dipole which is generated by the topological position of the amino acids in the sequence. Electrostatic surface calculations implicate a particular residues (Lys145) as being fundamental to interleukin activity and this supports site-directed mutation evidence that this residue is required for activity.

SQUID: a program for the analysis and display of data from crystallography and molecular dynamics.

SQUID is a flexible computer program that allows the analysis and display of molecular coordinates from crystallography, NMR, and molecular dynamics. The program can also display two-dimensional and three-dimensional data using many graph types, as well as perform array processing of data with numerous intrinsic functions. Graphics are based on the use of "move" and "draw" instructions, allowing easy development of new device drivers, including vector plotters.

High-resolution X-ray structures of pig metmyoglobin and two CD3 mutants: Mb(Lys45----Arg) and Mb(Lys45----Ser).

The structure of pig aquometmyoglobin has been refined to a crystallographic R-factor of 19.8% against X-ray diffraction data between 10- and 1.75-A spacing. The final structural model comprises two molecules of pig myoglobin, 233 water molecules, and two sulfate ions. A water molecule is coordinated to each of the heme iron atoms with an average Fe-OH2 bond distance of 2.19 A, and the mean Fe-N epsilon (proximal histidine-93) distance is 2.20 A. In contrast to the structure of sperm whale metmyoglobin, the iron is not significantly displaced from the plane of the heme. At the entrance to the heme pocket, the side-chain amino group of lysine-45 (CD3) is well-defined in the electron density map and forms salt-bridging interactions with the heme 6-propionate and with a sulfate ion. Serine and arginine replacements have been made previously at position 45 to examine the proposal that the CD3 side chain acts as a barrier to ligand entry into the protein. Crystal structures of the arginine-45 and serine-45 mutant metmyoglobins have been solved to 1.9 and 2.0 A resolution, respectively. In both cases the structural changes are confined to the site of mutation. Arginine-45 takes up a conformation closely similar to that observed for this residue in wild-type sperm whale myoglobin, in which it makes more extensive charge-charge and charge-dipole interactions and appears to restrict the movement of the distal histidine away from the ligand. The hydroxyl group of serine-45 is disordered, but it is clear that the effect of the mutation is to open up the solvent-exposed face of the heme pocket.

Determination of the crystal structure of recombinant pig myoglobin by molecular replacement and its refinement.

As part of a protein engineering study, the X-ray crystal structure of recombinant pig myoglobin, prepared and crystallized from E. coli, has been determined. Diffraction data were collected to 2.5 A spacing using a synchrotron X-ray source. The structure was solved using the molecular-replacement method and refined using least-squares minimization procedures to a crystallographic R factor of 18.5% using 14,481 reflections between 10 and 2.5 A. A preliminary comparison of the structure of pig myoglobin with other myoglobin structures is presented.

Apomyoglobin as a molecular recognition surface: expression, reconstitution and crystallization of recombinant porcine myoglobin in Escherichia coli.

Recombinant porcine myoglobin has been produced in Escherichia coli using the lambda cII fusion expression system of Nagai and Thøgersen [Nature, 309, 810-812 (1984)]. After processing and reconstitution with haem, the protein is gel-electrophoretically and spectrophotometrically indistinguishable from native pig myoglobin. Large crystals of both native and recombinant porcine myoglobin were grown from 50 mM sodium phosphate, pH 7.1, 80% ammonium sulphate. The crystals belong to space group C2 (a = 156.9 A, b = 42.0 A, c = 92.2 A, beta = 127.9 degrees) and diffract to a nominal 2.5 A resolution. We plan to explore apomyoglobin as a binding surface in studies combining site-directed mutagenesis and X-ray analysis. These experiments will be extended by studying the binding of haem analogues to the mutant apoproteins.