CYGD: the Comprehensive Yeast Genome Database.

The Comprehensive Yeast Genome Database (CYGD) compiles a comprehensive data resource for information on the cellular functions of the yeast Saccharomyces cerevisiae and related species, chosen as the best understood model organism for eukaryotes. The database serves as a common resource generated by a European consortium, going beyond the provision of sequence information and functional annotations on individual genes and proteins. In addition, it provides information on the physical and functional interactions among proteins as well as other genetic elements. These cellular networks include metabolic and regulatory pathways, signal transduction and transport processes as well as co-regulated gene clusters. As more yeast genomes are published, their annotation becomes greatly facilitated using S.cerevisiae as a reference. CYGD provides a way of exploring related genomes with the aid of the S.cerevisiae genome as a backbone and SIMAP, the Similarity Matrix of Proteins. The comprehensive resource is available under http://mips.gsf.de/genre/proj/yeast/.

Combining pattern discovery and discriminant analysis to predict gene co-regulation.

MOTIVATION: Several pattern discovery methods have been proposed to detect over-represented motifs in upstream sequences of co-regulated genes, and are for example used to predict cis-acting elements from clusters of co-expressed genes. The clusters to be analyzed are often noisy, containing a mixture of co-regulated and non-co-regulated genes. We propose a method to discriminate co-regulated from non-co-regulated genes on the basis of counts of pattern occurrences in their non-coding sequences. METHODS: String-based pattern discovery is combined with discriminant analysis to classify genes on the basis of putative regulatory motifs. RESULTS: The approach is evaluated by comparing the significance of patterns detected in annotated regulons (positive control), random gene selections (negative control) and high-throughput regulons (noisy data) from the yeast Saccharomyces cerevisiae. The classification is evaluated on the annotated regulons, and the robustness and rejection power is assessed with mixtures of co-regulated and random genes.

Standard atomic volumes in double-stranded DNA and packing in protein--DNA interfaces.

Standard volumes for atoms in double-stranded B-DNA are derived using high resolution crystal structures from the Nucleic Acid Database (NDB) and compared with corresponding values derived from crystal structures of small organic compounds in the Cambridge Structural Database (CSD). Two different methods are used to compute these volumes: the classical Voronoi method, which does not depend on the size of atoms, and the related Radical Planes method which does. Results show that atomic groups buried in the interior of double-stranded DNA are, on average, more tightly packed than in related small molecules in the CSD. The packing efficiency of DNA atoms at the interfaces of 25 high resolution protein-DNA complexes is determined by computing the ratios between the volumes of interfacial DNA atoms and the corresponding standard volumes. These ratios are found to be close to unity, indicating that the DNA atoms at protein-DNA interfaces are as closely packed as in crystals of B-DNA. Analogous volume ratios, computed for buried protein atoms, are also near unity, confirming our earlier conclusions that the packing efficiency of these atoms is similar to that in the protein interior. In addition, we examine the number, volume and solvent occupation of cavities located at the protein-DNA interfaces and compared them with those in the protein interior. Cavities are found to be ubiquitous in the interfaces as well as inside the protein moieties. The frequency of solvent occupation of cavities is however higher in the interfaces, indicating that those are more hydrated than protein interiors. Lastly, we compare our results with those obtained using two different measures of shape complementarity of the analysed interfaces, and find that the correlation between our volume ratios and these measures, as well as between the measures themselves, is weak. Our results indicate that a tightly packed environment made up of DNA, protein and solvent atoms plays a significant role in protein-DNA recognition.

From molecular activities and processes to biological function.

This paper describes how biological function can be represented in terms of molecular activities and processes. It presents several key features of a data model that is based on a conceptual description of the network of interactions between molecular entities within the cell and between cells. This model is implemented in the aMAZE database that presently deals with information on metabolic pathways, gene regulation, sub- or supracellular locations, and transport. It is shown that this model constitutes a useful generalisation of data representations currently implemented in metabolic pathway databases, and that it can furthermore include multiple schemes for categorising and classifying molecular entities, activities, processes and localisations. In particular, we highlight the flexibility offered by our system in representing multiple molecular activities and their control, in viewing biological function at different levels of resolution and in updating this view as our knowledge evolves.

Checking nucleic acid crystal structures.

The program SFCHECK [Vaguine et al. (1999), Acta Cryst. D55, 191-205] is used to survey the quality of the structure-factor data and the agreement of those data with the atomic coordinates in 105 nucleic acid crystal structures for which structure-factor amplitudes have been deposited in the Nucleic Acid Database [NDB; Berman et al. (1992), Biophys. J. 63, 751-759]. Nucleic acid structures present a particular challenge for structure-quality evaluations. The majority of these structures, and DNA molecules in particular, have been solved by molecular replacement of the double-helical motif, whose high degree of symmetry can lead to problems in positioning the molecule in the unit cell. In this paper, the overall quality of each structure was evaluated using parameters such as the R factor, the correlation coefficient and various atomic error estimates. In addition, each structure is characterized by the average values of several local quality indicators, which include the atomic displacement, the density correlation, the B factor and the density index. The latter parameter measures the relative electron-density level at the atomic position. In order to assess the quality of the model in specific regions, the same local quality indicators are also surveyed for individual groups of atoms in each structure. Several of the global quality indicators are found to vary linearly with resolution and less than a dozen structures are found to exhibit values significantly different from the mean for these indicators, showing that the quality of the nucleic acid structures tends to be rather uniform. Analysis of the mutual dependence of the values of different local quality indicators, computed for individual residues and atom groups, reveals that these indicators essentially complement each other and are not redundant with the B factor. Using several of these indicators, it was found that the atomic coordinates of the nucleic acid bases tend to be better defined than those of the backbone. One of the local indicators, the density index, is particularly useful in spotting regions of the model that fit poorly in the electron density. Using this parameter, the quality of crystallographic water positions in the analyzed structures was surveyed and it was found that a sizable fraction of these positions have poorly defined electron density and may therefore not be reliable. The possibility that cases of poorly positioned water molecules are symptomatic of more widespread problems with the structure as a whole is also raised.

A conserved Asn in transmembrane helix 7 is an on/off switch in the activation of the thyrotropin receptor.

The thyrotropin (TSH) receptor is an interesting model to study G protein-coupled receptor activation as many point mutations can significantly increase its basal activity. Here, we identified a molecular interaction between Asp(633) in transmembrane helix 6 (TM6) and Asn(674) in TM7 of the TSHr that is crucial to maintain the inactive state through conformational constraint of the Asn. We show that these residues are perfectly conserved in the glycohormone receptor family, except in one case, where they are exchanged, suggesting a direct interaction. Molecular modeling of the TSHr, based on the high resolution structure of rhodopsin, strongly favors this hypothesis. Our approach combining site-directed mutagenesis with molecular modeling shows that mutations disrupting this interaction, like the D633A mutation in TM6, lead to high constitutive activation. The strongly activating N674D (TM7) mutation, which in our modeling breaks the TM6-TM7 link, is reverted to wild type-like behavior by an additional D633N mutation (TM6), which would restore this link. Moreover, we show that the Asn of TM7 (conserved in most G protein-coupled receptors) is mandatory for ligand-induced cAMP accumulation, suggesting an active role of this residue in activation. In the TSHr, the conformation of this Asn residue of TM7 would be constrained, in the inactive state, by its Asp partner in TM6.

Solution structure of the main alpha-amylase inhibitor from amaranth seeds.

The most abundant alpha-amylase inhibitor (AAI) present in the seeds of Amaranthus hypochondriacus, a variety of the Mexican crop plant amaranth, is the smallest polypeptide (32 residues) known to inhibit alpha-amylase activity of insect larvae while leaving that of mammals unaffected. In solution, 1H NMR reveals that AAI isolated from amaranth seeds adopts a major trans (70%) and minor cis (30%) conformation, resulting from slow cis-trans isomerization of the Val15-Pro16 peptide bond. Both solution structures have been determined using 2D 1H-NMR spectroscopy and XPLOR followed by restrained energy refinement in the consistent-valence force field. For the major isomer, a total of 563 distance restraints, including 55 medium-range and 173 long-range ones, were available from the NOESY spectra. This rather large number of constraints from a protein of such a small size results from a compact fold, imposed through three disulfide bridges arranged in a cysteine-knot motif. The structure of the minor cis isomer has also been determined using a smaller constraint set. It reveals a different backbone conformation in the Pro10-Pro20 segment, while preserving the overall global fold. The energy-refined ensemble of the major isomer, consisting of 20 low-energy conformers with an average backbone rmsd of 0.29 +/- 0.19 A and no violations larger than 0.4 A, represents a considerable improvement in precision over a previously reported and independently performed calculation on AAI obtained through solid-phase synthesis, which was determined with only half the number of medium-range and long-range restraints reported here, and featured the trans isomer only. The resulting differences in ensemble precision have been quantified locally and globally, indicating that, for regions of the backbone and a good fraction of the side chains, the conformation is better defined in the new solution structure. Structural comparison of the solution structure with the X-ray structure of the inhibitor when bound to its alpha-amylase target in Tenebrio molitor shows that the backbone conformation is only slightly adjusted on complexation, while that of the side chains involved in protein-protein contacts is similar to those present in solution. Therefore, the overall conformation of AAI appears to be predisposed to binding to its target alpha-amylase, confirming the view that it acts as a lid on top of the alpha-amylase active site.

The TXP motif in the second transmembrane helix of CCR5. A structural determinant of chemokine-induced activation.

CCR5 is a G-protein-coupled receptor activated by the chemokines RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein 1alpha and 1beta, and monocyte chemotactic protein 2 and is the main co-receptor for the macrophage-tropic human immunodeficiency virus strains. We have identified a sequence motif (TXP) in the second transmembrane helix of chemokine receptors and investigated its role by theoretical and experimental approaches. Molecular dynamics simulations of model alpha-helices in a nonpolar environment were used to show that a TXP motif strongly bends these helices, due to the coordinated action of the proline, which kinks the helix, and of the threonine, which further accentuates this structural deformation. Site-directed mutagenesis of the corresponding Pro and Thr residues in CCR5 allowed us to probe the consequences of these structural findings in the context of the whole receptor. The P84A mutation leads to a decreased binding affinity for chemokines and nearly abolishes the functional response of the receptor. In contrast, mutation of Thr-82(2.56) into Val, Ala, Cys, or Ser does not affect chemokine binding. However, the functional response was found to depend strongly on the nature of the substituted side chain. The rank order of impairment of receptor activation is P84A > T82V > T82A > T82C > T82S. This ranking of impairment parallels the bending of the alpha-helix observed in the molecular simulation study.

Automatic procedures for protein design.

This review describes computational procedures for deriving the amino acid sequences that are compatible with a given protein backbone structure. Such procedures can be used to gain insight into the constraints imposed by the 3D structure of the protein sequence, or to design proteins that are likely to adopt a given backbone conformation. We start by presenting a short overview of the various types of approaches to protein design developed over more than a decade. This is followed by a more detailed presentation of a recently developed sequence selection procedure DESIGNER. This latter presentation illustrates the basic principles underlying this type of procedures, described what they may teach us when applied to small proteins, and highlights issues that need to be addressed in order to go forward.

Representing and analysing molecular and cellular function using the computer.

Determining the biological function of a myriad of genes, and understanding how they interact to yield a living cell, is the major challenge of the post genome-sequencing era. The complexity of biological systems is such that this cannot be envisaged without the help of powerful computer systems capable of representing and analysing the intricate networks of physical and functional interactions between the different cellular components. In this review we try to provide the reader with an appreciation of where we stand in this regard. We discuss some of the inherent problems in describing the different facets of biological function, give an overview of how information on function is currently represented in the major biological databases, and describe different systems for organising and categorising the functions of gene products. In a second part, we present a new general data model, currently under development, which describes information on molecular function and cellular processes in a rigorous manner. The model is capable of representing a large variety of biochemical processes, including metabolic pathways, regulation of gene expression and signal transduction. It also incorporates taxonomies for categorising molecular entities, interactions and processes, and it offers means of viewing the information at different levels of resolution, and dealing with incomplete knowledge. The data model has been implemented in the database on protein function and cellular processes 'aMAZE' (http://www.ebi.ac.uk/research/pfbp/), which presently covers metabolic pathways and their regulation. Several tools for querying, displaying, and performing analyses on such pathways are briefly described in order to illustrate the practical applications enabled by the model.

Automatic protein design with all atom force-fields by exact and heuristic optimization.

A fully automatic procedure for predicting the amino acid sequences compatible with a given target structure is described. It is based on the CHARMM package, and uses an all atom force-field and rotamer libraries to describe and evaluate side-chain types and conformations. Sequences are ranked by a quantity akin to the free energy of folding, which incorporates hydration effects. Exact (Branch and Bound) and heuristic optimisation procedures are used to identifying highly scoring sequences from an astronomical number of possibilities. These sequences include the minimum free energy sequence, as well as all amino acid sequences whose free energy lies within a specified window from the minimum. Several applications of our procedure are illustrated. Prediction of side-chain conformations for a set of ten proteins yields results comparable to those of established side-chain placement programs. Applications to sequence optimisation comprise the re-design of the protein cores of c-Crk SH3 domain, the B1 domain of protein G and Ubiquitin, and of surface residues of the SH3 domain. In all calculations, no restrictions are imposed on the amino acid composition and identical parameter settings are used for core and surface residues. The best scoring sequences for the protein cores are virtually identical to wild-type. They feature no more than one to three mutations in a total of 11-16 variable positions. Tests suggest that this is due to the balance between various contributions in the force-field rather than to overwhelming influence from packing constraints. The effectiveness of our force-field is further supported by the sequence predictions for surface residues of the SH3 domain. More mutations are predicted than in the core, seemingly in order to optimise the network of complementary interactions between polar and charged groups. This appears to be an important energetic requirement in absence of the partner molecules with which the SH3 domain interacts, which were not included in the calculations. Finally, a detailed comparison between the sequences generated by the heuristic and exact optimisation algorithms, commends a note of caution concerning the efficiency of heuristic procedures in exploring sequence space.

Pathways of ligand clearance in acetylcholinesterase by multiple copy sampling.

The clearance of seven different ligands from the deeply buried active-site of Torpedo californica acetylcholinesterase is investigated by combining multiple copy sampling molecular dynamics simulations, with the analysis of protein-ligand interactions, protein motion and the electrostatic potential sampled by the ligand copies along their journey outwards. The considered ligands are the cations ammonium, methylammonium, and tetramethylammonium, the hydrophobic methane and neopentane, and the anionic product acetate and its neutral form, acetic acid. We find that the pathways explored by the different ligands vary with ligand size and chemical properties. Very small ligands, such as ammonium and methane, exit through several routes. One involves the main exit through the mouth of the enzyme gorge, another is through the so-called back door near Trp84, and a third uses a side door at a direction of approximately 45 degrees to the main exit. The larger polar ligands, methylammonium and acetic acid, leave through the main exit, but the bulkiest, tetramethylammonium and neopentane, as well as the smaller acetate ion, remain trapped in the enzyme gorge during the time of the simulations. The pattern of protein-ligand contacts during the diffusion process is highly non-random and differs for different ligands. A majority is made with aromatic side-chains, but classical H-bonds are also formed. In the case of acetate, but not acetic acid, the anionic and neutral form, respectively, of one of the reaction products, specific electrostatic interactions with protein groups, seem to slow ligand motion and interfere with protein flexibility; protonation of the acetate ion is therefore suggested to facilitate clearance. The Poisson-Boltzmann formalism is used to compute the electrostatic potential of the thermally fluctuating acetylcholinesterase protein at positions actually visited by the diffusing ligand copies. Ligands of different charge and size are shown to sample somewhat different electrostatic potentials during their migration, because they explore different microscopic routes. The potential along the clearance route of a cation such as methylammonium displays two clear minima at the active and peripheral anionic site. We find moreover that the electrostatic energy barrier that the cation needs to overcome when moving between these two sites is small in both directions, being of the order of the ligand kinetic energy. The peripheral site thus appears to play a role in trapping inbound cationic ligands as well as in cation clearance, and hence in product release.

Effect of mutations within the peripheral anionic site on the stability of acetylcholinesterase.

Torpedo acetylcholinesterase is irreversibly inactivated by modifying a buried free cysteine, Cys231, with sulfhydryl reagents. The stability of the enzyme, as monitored by measuring the rate of inactivation, was reduced by mutating a leucine, Leu282, to a smaller amino acid residue. Leu282 is located within the "peripheral" anionic site, at the entrance to the active-site gorge. Thus, loss of activity was due to the increased reactivity of Cys231. This was paralleled by an increased susceptibility to thermal denaturation, which was shown to be due to a large decrease in the activation enthalpy. Similar results were obtained when either of two other residues in contact with Leu282 in Torpedo acetylcholinesterase, Trp279 and Ser291, was replaced by an amino acid with a smaller side chain. We studied the effects of various ligands specific for either the active or peripheral sites on both thermal inactivation and on inactivation by 4,4'-dithiodipyridine. The wild-type and mutated enzymes could be either protected or sensitized. In some cases, opposite effects of the same ligand were observed for chemical modification and thermal denaturation. The mutated residues are within a conserved loop, W279-S291, at the top of the active-site gorge, that contributes to the peripheral anionic site. Theoretical analysis showed that Torpedo acetylcholinesterase consists of two structural domains, each comprising one contiguous polypeptide segment. The W279-S291 loop, located in the first domain, makes multiple contacts with the second domain across the active-site gorge. We postulate that the mutations to residues with smaller side chains destabilize the conserved loop, thus disrupting cross-gorge interactions and, ultimately, the entire structure.

Identification of structural domains in proteins by a graph heuristic.

A novel automatic procedure for identifying domains from protein atomic coordinates is presented. The procedure, termed STRUDL (STRUctural Domain Limits), does not take into account information on secondary structures and handles any number of domains made up of contiguous or non-contiguous chain segments. The core algorithm uses the Kernighan-Lin graph heuristic to partition the protein into residue sets which display minimum interactions between them. These interactions are deduced from the weighted Voronoi diagram. The generated partitions are accepted or rejected on the basis of optimized criteria, representing basic expected physical properties of structural domains. The graph heuristic approach is shown to be very effective, it approximates closely the exact solution provided by a branch and bound algorithm for a number of test proteins. In addition, the overall performance of STRUDL is assessed on a set of 787 representative proteins from the Protein Data Bank by comparison to domain definitions in the CATH protein classification. The domains assigned by STRUDL agree with the CATH assignments in at least 81% of the tested proteins. This result is comparable to that obtained previously using PUU (Holm and Sander, Proteins 1994;9:256-268), the only other available algorithm designed to identify domains with any number of non-contiguous chain segments. A detailed discussion of the structures for which our assignments differ from those in CATH brings to light some clear inconsistencies between the concept of structural domains based on minimizing inter-domain interactions and that of delimiting structural motifs that represent acceptable folding topologies or architectures. Considering both concepts as complementary and combining them in a layered approach might be the way forward.

SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model.

In this paper we present SFCHECK, a stand-alone software package that features a unified set of procedures for evaluating the structure-factor data obtained from X-ray diffraction experiments and for assessing the agreement of the atomic coordinates with these data. The evaluation is performed completely automatically, and produces a concise PostScript pictorial output similar to that of PROCHECK [Laskowski, MacArthur, Moss & Thornton (1993). J. Appl. Cryst. 26, 283-291], greatly facilitating visual inspection of the results. The required inputs are the structure-factor amplitudes and the atomic coordinates. Having those, the program summarizes relevant information on the deposited structure factors and evaluates their quality using criteria such as data completeness, structure-factor uncertainty and the optical resolution computed from the Patterson origin peak. The dependence of various parameters on the nominal resolution (d spacing) is also given. To evaluate the global agreement of the atomic model with the experimental data, the program recomputes the R factor, the correlation coefficient between observed and calculated structure-factor amplitudes and Rfree (when appropriate). In addition, it gives several estimates of the average error in the atomic coordinates. The local agreement between the model and the electron-density map is evaluated on a per-residue basis, considering separately the macromolecule backbone and side-chain atoms, as well as solvent atoms and heterogroups. Among the criteria are the normalized average atomic displacement, the local density correlation coefficient and the polymer chain connectivity. The possibility of computing these criteria using the omit-map procedure is also provided. The described software should be a valuable tool in monitoring the refinement procedure and in assessing structures deposited in databases.

Structural features of protein-nucleic acid recognition sites.

We analyzed the atomic models of 75 X-ray structures of protein-nucleic acid complexes with the aim of uncovering common properties. The interface area measured the extent of contact between the protein and nucleic acid. It was found to vary between 1120 and 5800 A2. Despite this wide variation, the interfaces in complexes of transcription factors with double-stranded DNA could be broken up into recognition modules where 12 +/- 3 nucleotides on the DNA side contact 24 +/- 6 amino acids on the protein side, with interface areas in the range 1600 +/- 400 A2. For enzymes acting on DNA, the recognition module is on average 600 A2 larger, due to the requirement of making an active site. As judged by its chemical and amino acid composition, the average protein surface in contact with the DNA is more polar than the solvent accessible surface or the typical protein-protein interface. The protein side is rich in positively charged groups from lysine and arginine side chains; on the DNA side the negative charges from phosphate groups dominate. Hydrogen bonding patterns were also analyzed, and we found one intermolecular hydrogen bond per 125 A2 of interface area in high-resolution structures. An equivalent number of polar interactions involved water molecules, which are generally abundant at protein-DNA interfaces. Calculations of Voronoi atomic volumes, performed in the presence and absence of water molecules, showed that protein atoms buried at the interface with DNA are on average as closely packed as in the protein interior. Water molecules contribute to the close packing, thereby mediating shape complementarity. Finally, conformational changes accompanying association were analyzed in 24 of the complexes for which the structure of the free protein was also available. On the DNA side the extent of deformation showed some correlation with the size of the interface area. On the protein side the type and size of the structural changes spanned a wide spectrum. Disorder-to-order transitions, domain movements, quaternary and tertiary changes were observed, and the largest changes occurred in complexes with large interfaces.

Conformational analysis of GpA and GpAp in aqueous solution by molecular dynamics and statistical methods.

Barnase, an extracellular endoribonuclease from Bacillus amyloliquefaciens, hydrolyses single-stranded RNA. Its very low catalytic activity toward GpN dinucleotides, where N stands for any nucleoside, is markedly increased when a phosphate is added to the 3'-end, as in GpNp. Here we investigate the conformational properties of GpA and GpAp in solution, in order to determine whether differences in these properties may be related to the changes in enzymatic activity. Two independent 1.3 ns molecular dynamics trajectories are generated for each dinucleotide in the presence of explicit water molecules and counter ions. These trajectories are analysed by monitoring molecular properties, such as the solvent accessible surface area, the distance and orientation between the bases, the behaviour of torsion angles and formation of intramolecular H-bonds. To identify relevant correlations between these parameters, statistical techniques, comprising multiple regression, clustering and discriminant analysis are used. Results show that GpA has a significant propensity to form folded conformations (approximately 50%), fostered by a small number of intramolecular H-bonds, whereas GpAp remains essentially extended. The latter behaviour seems to be due to an H-bond between the terminal phosphate and adenosine ribose group, which restricts rotation about the adenine Agamma angle. We also find that GpA folding is induced by a concerted motion of specific torsion angles, which is closely coupled to the formation of a network of flexible hydrogen bonds. Finally, on the basis of an expression for barnase KM, which incorporates the folded/extended conformational equilibria of the dinucleotide substrates, it is argued that our findings on the differences between these equilibria, can qualitatively rationalize the experimentally measured differences in enzymatic properties.

Typical interaction patterns in alphabeta and betaalpha turn motifs.

A fully automatic classification procedure of short protein fragments is applied to identify connections between alpha-helices and beta-strands in a dataset of 141 protein chains. It yields 15 structural families of alphabeta turns and 15 families of betaalpha turns with at least five members. The sequence and structural features of these turn motifs are analysed with the focus on the local interactions located at alpha-helix and beta-strand ends. This analysis reveals specific interaction patterns that occur frequently among the members of many of the identified turn motifs. For the beta-strands, novel patterns are identified at the strands' entry and exit; they involve side chain/side chain contacts and beta-turns, generally of type I or II. For the alpha-helices, the interaction patterns consist of several backbone/backbone or backbone/side chain hydrogen bonds and of hydrophobic contacts; they generalize the well known N-terminal capping and C-terminal Schellman motifs. The interaction patterns at both ends of alpha-helices and beta-strands are found to constitute favourable structure motifs with low amino acid sequence specificity; their possible stabilizing role is discussed. Finally, the robustness of our classification procedure and of the description of N- and C-cap interaction patterns is validated by repeating our analysis on a larger dataset of 381 protein chains and showing that the results are maintained.

Analysis of zinc binding sites in protein crystal structures.

The geometrical properties of zinc binding sites in a dataset of high quality protein crystal structures deposited in the Protein Data Bank have been examined to identify important differences between zinc sites that are directly involved in catalysis and those that play a structural role. Coordination angles in the zinc primary coordination sphere are compared with ideal values for each coordination geometry, and zinc coordination distances are compared with those in small zinc complexes from the Cambridge Structural Database as a guide of expected trends. We find that distances and angles in the primary coordination sphere are in general close to the expected (or ideal) values. Deviations occur primarily for oxygen coordinating atoms and are found to be mainly due to H-bonding of the oxygen coordinating ligand to protein residues, bidentate binding arrangements, and multi-zinc sites. We find that H-bonding of oxygen containing residues (or water) to zinc bound histidines is almost universal in our dataset and defines the elec-His-Zn motif. Analysis of the stereochemistry shows that carboxyl elec-His-Zn motifs are geometrically rigid, while water elec-His-Zn motifs show the most geometrical variation. As catalytic motifs have a higher proportion of carboxyl elec atoms than structural motifs, they provide a more rigid framework for zinc binding. This is understood biologically, as a small distortion in the zinc position in an enzyme can have serious consequences on the enzymatic reaction. We also analyze the sequence pattern of the zinc ligands and residues that provide elecs, and identify conserved hydrophobic residues in the endopeptidases that also appear to contribute to stabilizing the catalytic zinc site. A zinc binding template in protein crystal structures is derived from these observations.