Biomedical subjects

Gerard J Kleywegt

Publications and source records attributed to Gerard J Kleywegt.

18 recordsLinked to original sources

PubMed2023

EMPIAR: the Electron Microscopy Public Image Archive.

Public archiving in structural biology is well established with the Protein Data Bank (PDB; wwPDB.org) catering for atomic models and the Electron Microscopy Data Bank (EMDB; emdb-empiar.org) for 3D reconstructions from cryo-EM experiments. Even before the recent rapid growth in cryo-EM, there was an expressed community need for a public archive of image data from cryo-EM experiments for validation, software development, testing and training. Concomitantly, the proliferation of 3D imaging techniques for cells, tissues and organisms using volume EM (vEM) and X-ray tomography (XT) led to calls from these communities to publicly archive such data as well. EMPIAR (empiar.org) was developed as a public archive for raw cryo-EM image data and for 3D reconstructions from vEM and XT experiments and now comprises over a thousand entries totalling over 2 petabytes of data. EMPIAR resources include a deposition system, entry pages, facilities to search, visualize and download datasets, and a REST API for programmatic access to entry metadata. The success of EMPIAR also poses significant challenges for the future in dealing with the very fast growth in the volume of data and in enhancing its reusability.

Imaging, Three-Dimensional↗

PubMed2007

Quality control and validation.

This chapter discusses two important aspects of the structure determination process that are related to the accuracy and reliability of the crystallographic model under investigation. Quality control is defined as the analysis of an intermediate model to identify aspects of it that are unusual in some sense and that could, therefore, be a result of errors in the model building or refinement process. Any such errors need to be fixed, if at all possible, prior to analysis and publication of the model. Validation is the process of assessing the reliability of the final model (or certain aspects of it, e.g., the active site residues) that is about to be analyzed, published, deposited, and possibly used in follow-up studies.

Algorithms↗

PubMed2006

Crystallographic refinement of ligand complexes.

Model building and refinement of complexes between biomacromolecules and small molecules requires sensible starting coordinates as well as the specification of restraint sets for all but the most common non-macromolecular entities. Here, it is described why this is necessary, how it can be accomplished and what pitfalls need to be avoided in order to produce chemically plausible models of the low-molecular-weight entities. A number of programs, servers, databases and other resources that can be of assistance in the process are also discussed.

Animals↗

PubMed2006

New crystal structures of human glutathione transferase A1-1 shed light on glutathione binding and the conformation of the C-terminal helix.

Human glutathione transferase A1-1 is a well studied enzyme, but despite a wealth of structural and biochemical data a number of aspects of its catalytic function are still poorly understood. Here, five new crystal structures of this enzyme are described that provide several insights. Firstly, the structure of a complex of the wild-type human enzyme with glutathione was determined for the first time at 2.0 angstroms resolution. This reveals that glutathione binds in the G site in a very similar fashion as the glutathione portion of substrate analogues in other structures and also that glutathione binding alone is sufficient to stabilize the C-terminal helix of the protein. Secondly, we have studied the complex with a decarboxylated glutathione conjugate that is known to dramatically decrease the activity of the enzyme. The T68E mutant of human glutathione transferase A1-1 recovers some of the activity that is lost with the decarboxylated glutathione, but our structures of this mutant show that none of the earlier explanations of this phenomenon are likely to be correct. Thirdly, and serendipitously, the apo structures also reveal the conformation of the crucial C-terminal region that is disordered in all previous apo structures. The C-terminal region can adopt an ordered helix-like structure even in the apo state, but shows a strong tendency to unwind. Different conformations of the C-terminal regions were observed in the apo states of the two monomers, which suggests that cooperativity could play a role in the activity of the enzyme.

Binding Sites↗

PubMed2005

Structural basis of the suppressed catalytic activity of wild-type human glutathione transferase T1-1 compared to its W234R mutant.

The crystal structures of wild-type human theta class glutathione-S-transferase (GST) T1-1 and its W234R mutant, where Trp234 was replaced by Arg, were solved both in the presence and absence of S-hexyl-glutathione. The W234R mutant was of interest due to its previously observed enhanced catalytic activity compared to the wild-type enzyme. GST T1-1 from rat and mouse naturally contain Arg in position 234, with correspondingly high catalytic efficiency. The overall structure of GST T1-1 is similar to that of GST T2-2, as expected from their 53% sequence identity at the protein level. Wild-type GST T1-1 has the side-chain of Trp234 occupying a significant portion of the active site. This bulky residue prevents efficient binding of both glutathione and hydrophobic substrates through steric hindrance. The wild-type GST T1-1 crystal structure, obtained from co-crystallization experiments with glutathione and its derivatives, showed no electron density for the glutathione ligand. However, the structure of GST T1-1 mutant W234R showed clear electron density for S-hexyl-glutathione after co-crystallization. In contrast to Trp234 in the wild-type structure, the side-chain of Arg234 in the mutant does not occupy any part of the substrate-binding site. Instead, Arg234 is pointing in a different direction and, in addition, interacts with the carboxylate group of glutathione. These findings explain our earlier observation that the W234R mutant has a markedly improved catalytic activity with most substrates tested to date compared to the wild-type enzyme. GST T1-1 catalyzes detoxication reactions as well as reactions that result in toxic products, and our findings therefore suggest that humans have gained an evolutionary advantage by a partially disabled active site.

Binding Sites↗

PubMed2005

Structure of human semicarbazide-sensitive amine oxidase/vascular adhesion protein-1.

Semicarbazide-sensitive amine oxidase (SSAO) belongs to a ubiquitous family of copper-containing amine oxidases (CuAOs). SSAO is also known as vascular adhesion protein-1 (VAP-1) and has been identified as one of the adhesion molecules involved in the leukocyte-extravasation process. The structure of a truncated soluble form of human SSAO has been solved and refined to 2.5 A. As expected, SSAO is a homodimer with a fold typical of the CuAO family. The topaquinone (TPQ) cofactor and a copper ion characteristic of CuAOs are present in the active site, with the TPQ in the active ;off-copper' conformation. The structure reveals that a leucine residue (Leu469) located adjacent to the active site could function as a gate controlling its accessibility. An RGD motif is displayed on the surface, where it could be involved in integrin binding and possibly play a role in the shedding of SSAO from the membrane. Carbohydrate moieties are observed at five of six potential N-glycosylation sites. Carbohydrates attached to Asn232 flank the active-site entrance and might influence substrate specificity. The structure of an adduct of SSAO and the irreversible inhibitor 2-hydrazinopyridine has been solved and refined to 2.9 A resolution. Together, these structures will aid efforts to identify natural substrates, provide valuable information for the design of specific inhibitors and direct further studies.

Amine Oxidase (Copper-Containing)↗

PubMed2005

A survey of left-handed helices in protein structures.

All naturally occurring amino acids with the exception of glycine contain one or more chiral carbon atoms and can therefore occur in two different configurations, L (levo, left-handed) and D (dextro, right-handed). Proteins are almost exclusively built from L-amino acids. The stereochemical bias of nature is further reflected at the secondary structure level where right-handed helices are strongly preferred over left-handed helices. The handedness of helices has not received much attention in the past and is often overlooked during the analysis, description and deposition of experimentally solved protein structures. Therefore, an extensive survey of left-handed helices in the Protein Data Bank (PDB) was undertaken to analyse their frequency of occurrence, length, amino acid composition, conservation and possible structural or functional role. All left-handed helices (of four or more residues) in a non-redundant subset of the PDB, were identified using hydrogen-bonding analysis, comparison of related structures, and experimental electron density assessment to filter out likely spurious and artefactual hits. This analysis yielded 31 verified left-handed helices in a set of 7284 proteins. The phi angles of the residues in the left-handed helices lie between 30 degrees and 130 degrees and the psi angles lie between -50 degrees and 100 degrees . Most of the helices are short (four residues) and for 87% of them, it was possible to determine that they are important for the stability of the protein, for ligand binding, or as part of the active site. This suggests that, even though left-handed helices are rare, when they do occur, they are structurally or functionally significant. Four secondary structure assignment programs were tested for their ability to identify the handedness of the helices. Of these programs, only DSSP correctly assigns the handedness.

Amino Acid Sequence↗

PubMed2005

Crystallization of a truncated soluble human semicarbazide-sensitive amine oxidase.

Human semicarbazide-sensitive amine oxidase (SSAO) is a homodimeric copper-containing monoamine oxidase that occurs in both a membrane-bound and a soluble form. SSAO is also known as vascular adhesion protein-1 (VAP-1). A truncated soluble form of human SSAO (comprising residues 29-763) was expressed in human embryonic kidney 293 cells and purified to homogeneity. Tetragonal crystals were obtained and a data set extending to 2.5 A was collected. The crystals are merohedrally twinned and the estimation of the twinning fraction was complicated by pseudo-symmetry and the anisotropic character of the crystals. Using a recently developed method for twinning detection that is insensitive to phenomena such as anisotropy or pseudo-symmetry [Padilla & Yeates (2003), Acta Cryst. D59, 1124-1130], the twinning fraction was estimated to be 0.3. The structure was eventually solved by molecular replacement in space group P4(3).

Amine Oxidase (Copper-Containing)↗

PubMed2004

The Uppsala Electron-Density Server.

The Uppsala Electron Density Server (EDS; http://eds.bmc.uu.se/) is a web-based facility that provides access to electron-density maps and statistics concerning the fit of crystal structures and their maps. Maps are available for approximately 87% of the crystallographic Protein Data Bank (PDB) entries for which structure factors have been deposited and for which straightforward map calculations succeed in reproducing the published R value to within five percentage points. Here, an account is provided of the methods that are used to generate the information contained in the server. Some of the problems that are encountered in the map-generation process as well as some spin-offs of the project are also discussed.

Computational Biology↗

PubMed2004

Towards complete validated models in the next generation of ARP/wARP.

The design of a new versatile control system that will underlie future releases of the automated model-building package ARP/wARP is presented. A sophisticated expert system is under development that will transform ARP/wARP from a very useful model-building aid to a truly automated package capable of delivering complete, well refined and validated models comparable in quality to the result of intensive manual checking, rebuilding, hypothesis testing, refinement and validation cycles of an experienced crystallographer. In addition to the presentation of this control system, recent advances, ideas and future plans for improving the current model-building algorithms, especially for completing partially built models, are presented. Furthermore, a concept for integrating validation routines into the iterative model-building process is also presented.

Algorithms↗

PubMed2004

Incorporation of a single His residue by rational design enables thiol-ester hydrolysis by human glutathione transferase A1-1.

A strategy for rational enzyme design is reported and illustrated by the engineering of a protein catalyst for thiol-ester hydrolysis. Five mutants of human glutathione (GSH; gamma-Glu-Cys-Gly) transferase A1-1 were designed in the search for a catalyst and to provide a set of proteins from which the reaction mechanism could be elucidated. The single mutant A216H catalyzed the hydrolysis of the S-benzoyl ester of GSH under turnover conditions with a k(cat)/K(M) of 156 M(-1) x min(-1), and a catalytic proficiency of >10(7) M(-1) when compared with the first-order rate constant of the uncatalyzed reaction. The wild-type enzyme did not hydrolyze the substrate, and thus, the introduction of a single histidine residue transformed the wild-type enzyme into a turnover system for thiol-ester hydrolysis. By kinetic analysis of single, double, and triple mutants, as well as from studies of reaction products, it was established that the enzyme A216H catalyzes the hydrolysis of the thiol-ester substrate by a mechanism that includes an acyl intermediate at the side chain of Y9. Kinetic measurements and the crystal structure of the A216H GSH complex provided compelling evidence that H216 acts as a general-base catalyst. The introduction of a single His residue into human GSH transferase A1-1 created an unprecedented enzymatic function, suggesting a strategy that may be of broad applicability in the design of new enzymes. The protein catalyst has the hallmarks of a native enzyme and is expected to catalyze various hydrolytic, as well as transesterification, reactions.

Glutathione Transferase↗

PubMed2004

Evaluation of protein fold comparison servers.

When a new protein structure has been determined, comparison with the database of known structures enables classification of its fold as new or belonging to a known class of proteins. This in turn may provide clues about the function of the protein. A large number of fold comparison programs have been developed, but they have never been subjected to a comprehensive and critical comparative analysis. Here we describe an evaluation of 11 publicly available, Web-based servers for automatic fold comparison. Both their functionality (e.g., user interface, presentation, and annotation of results) and their performance (i.e., how well established structural similarities are recognized) were assessed. The servers were subjected to a battery of performance tests covering a broad spectrum of folds as well as special cases, such as multidomain proteins, Calpha-only models, new folds, and NMR-based models. The CATH structural classification system was used as a reference. These tests revealed the strong and weak sides of each server. On the whole, CE, DALI, MATRAS, and VAST showed the best performance, but none of the servers achieved a 100% success rate. Where no structurally similar proteins are found by any individual server, it is recommended to try one or two other servers before any conclusions concerning the novelty of a fold are put on paper.

Computational Biology↗

PubMed2004

Déjà vu all over again: finding and analyzing protein structure similarities.

Structure comparison is a crucial aspect of structural biology today. The field of structure comparison is developing rapidly, with the development of new algorithms, similarity scores, and statistical scores. The predicted large increase of experimental structures and structural models made possible by high-throughput efforts means that structural comparison and searching of structural databases using automated methods will become increasingly common. This Ways & Means article is meant to guide the structural biologist in the basics of structural alignment, and to provide an overview of the available software tools. The main purpose is to encourage users to gain some understanding of the strengths and limitations of structural alignment, and to take these factors into account when interpreting the results of different programs.

Algorithms↗

PubMed2003

The crystal structure of Echinococcus granulosus fatty-acid-binding protein 1.

We describe the 1.6 A crystal structure of the fatty-acid-binding protein EgFABP1 from the parasitic platyhelminth Echinococcus granulosus. E. granulosus causes hydatid disease, which is a major zoonosis. EgFABP1 has been implicated in the acquisition, storage, and transport of lipids, and may be important to the organism since it is incapable of synthesising most of its lipids de novo. Moreover, EgFABP1 is a promising candidate for a vaccine against hydatid disease. The crystal structure reveals that EgFABP1 has the expected 10-stranded beta-barrel fold typical of the family of intracellular lipid-binding proteins, and that it is structurally most similar to P2 myelin protein. We describe the comparison of the crystal structure of EgFABP1 with these proteins and with an older homology model for EgFABP1. The electron density reveals the presence of a bound ligand inside the cavity, which we have interpreted as palmitic acid. The carboxylate group of the fatty acid interacts with the protein's P2 motif, consisting of a conserved triad R em leader R-x-Y. The hydrophobic tail of the ligand assumes a fairly flat, U-shaped conformation and has relatively few interactions with the protein.We discuss some of the structural implications of the crystal structure of EgFABP1 for related platyhelminthic FABPs.

Amino Acid Motifs↗

PubMed2003

Application and limitations of X-ray crystallographic data in structure-based ligand and drug design.

Structure-based design usually focuses upon the optimization of ligand affinity. However, successful drug design also requires the optimization of many other properties. The primary source of structural information for protein-ligand complexes is X-ray crystallography. The uncertainties introduced during the derivation of an atomic model from the experimentally observed electron density data are not always appreciated. Uncertainties in the atomic model can have significant consequences when this model is subsequently used as the basis of manual design, docking, scoring, and virtual screening efforts. Docking and scoring algorithms are currently imperfect. A good correlation between observed and calculated binding affinities is usually only observed only when very large ranges of affinity are considered. Errors in the correlation often exceed the range of affinities commonly encountered during lead optimization. Some structure-based design approaches now involve screening libraries by using technologies based on NMR spectroscopy and X-ray crystallography to discover small polar templates, which are used for further optimization. Such compounds are defined as leadlike and are also sought by more traditional high-throughput screening technologies. Structure-based design and HTS technologies show important complementarity and a degree of convergence.

Crystallization↗

PubMed2003

Pound-wise but penny-foolish: How well do micromolecules fare in macromolecular refinement?

For the refinement of protein and nucleic acid structures, high-quality geometric restraint libraries are available. Unfortunately, for other compounds, such as physiological ligands, lead compounds, substrate analogs, etc., the situation is not as favorable. As a result, the structures of small molecules found in complexes with biomacromolecules are often less reliable than those of the surrounding amino or nucleic acids. Here, we briefly review the use of geometric restraints in structure refinement (be it against X-ray crystallographic or NMR-derived data) and simulation. In addition, we discuss methods to generate both restraint libraries and (idealized) coordinates for small molecules and provide some practical advice.

Computational Biology↗

PubMed2002

The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175.

Trichoderma reesei cellobiohydrolase Cel6A is an inverting glycosidase. Structural studies have established that the tunnel-shaped active site of Cel6A contains two aspartic acids, D221 and D175, that are close to the glycosidic oxygen of the scissile bond and at hydrogen-bonding distance from each other. Here, site-directed mutagenesis, X-ray crystallography, and enzyme kinetic studies have been used to confirm the role of residue D221 as the catalytic acid. D175 is shown to affect protonation of D221 and to contribute to the electrostatic stabilization of the partial positive charge in the transition state. Structural and modeling studies suggest that the single-displacement mechanism of Cel6A may not directly involve a catalytic base. The value of (D2O)(V) of 1.16 +/- 0.14 for hydrolysis of cellotriose suggests that the large direct effect expected for proton transfer from the nucleophilic water through a water chain (Grotthus mechanism) is offset by an inverse effect arising from reversibly breaking the short, tight hydrogen bond between D221 and D175 before catalysis.

Aspartic Acid↗

PubMed2002

Homo crystallographicus--quo vadis?

As macromolecular crystal structures are determined and refined in an increasingly automated fashion, careful assessment of the reliability and quality of the resulting models becomes increasingly important. Here, we analyze various issues related to the reliability and quality of macromolecular crystal structures deposited between 1991 and 2000. We find that the average resolution at which these structures are determined is essentially constant. In line with this observation, the average quality as measured by Ramachandran analysis does not improve as a function of time. On the other hand, an observed decrease of the average discrepancy between free and conventional R values suggests that the fit of model and data is improving. Finally, we present a surprising correlation between the tendency of crystallographers to deposit their experimental data and the free R values of their models.

Crystallography, X-Ray↗