Showing papers in "Acta Crystallographica Section D-biological Crystallography in 2004"

Coot: model-building tools for molecular graphics.

Abstract: CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for X-ray structure solution, structure comparison and analysis, and publication-quality graphics. The map-fitting tools are available as a stand-alone package, distributed as `Coot'.

PRODRG: a tool for high-throughput crystallography of protein–ligand complexes

Abstract: The small-molecule topology generator PRODRG is described, which takes input from existing coordinates or various two-dimensional formats and automatically generates coordinates and molecular topologies suitable for X-ray refinement of protein–ligand complexes. Test results are described for automatic generation of topologies followed by energy minimization for a subset of compounds from the Cambridge Structural Database, which shows that, within the limits of the empirical GROMOS87 force field used, structures with good geometries are generated. X-ray refinement in X-­PLOR/CNS, REFMAC and SHELX using PRODRG-generated topologies produces results comparable to refinement with topologies from the standard libraries. However, tests with distorted starting coordinates show that PRODRG topologies perform better, both in terms of ligand geometry and of crystallographic R factors.

Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions

Abstract: The present paper describes the SSM algorithm of protein structure comparison in three dimensions, which includes an original procedure of matching graphs built on the protein's secondary-structure elements, followed by an iterative three-dimensional alignment of protein backbone C_\alpha atoms. The SSM results are compared with those obtained from other protein comparison servers, and the advantages and disadvantages of different scores that are used for structure recognition are discussed. A new score, balancing the r.m.s.d. and alignment length N_{\rm align}, is proposed. It is found that different servers agree reasonably well on the new score, while showing considerable differences in r.m.s.d. and N_{\rm align}.

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

Abstract: 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. The dictionary stores information about monomers which represent the constitutive building blocks of biological macromolecules (amino acids, nucleic acids and saccharides) and about numerous organic/inorganic compounds commonly found in macromolecular crystallography. It also describes the modifications the building blocks undergo as a result of chemical reactions and the links required for polymer formation. More than 2000 monomer entries, 100 modification entries and 200 link entries are currently available. Algorithms and tools for updating and adding new entries to the dictionary have also been developed and are presented here. In many cases, the REFMAC5 dictionary allows entirely automatic generation of restraints within REFMAC5 refinement runs.

Likelihood-enhanced fast rotation functions.

Abstract: Experiences with the molecular-replacement program Beast have shown that maximum-likelihood rotation targets are more sensitive to the correct orientation than traditional targets. However, this comes at a high computational cost: brute-force rotation searches can take hours or even days of computation time on current desktop computers. Series approximations to the full likelihood target have been developed that can be computed by fast Fourier transforms in minutes. These likelihood-enhanced targets are more sensitive to the correct orientation than the Crowther fast rotation function and they take advantage of information from partial solutions. The likelihood-enhanced rotation targets have been implemented in the program Phaser.

Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT.

Abstract: BUSTER–TNT is a maximum-likelihood macromolecular refinement package. BUSTER assembles the structural model, scales observed and calculated structure-factor amplitudes and computes the model likelihood, whilst TNT handles the stereochemistry and NCS restraints/constraints and shifts the atomic coordinates, B factors and occupancies. In real space, in addition to the traditional atomic and bulk-solvent models, BUSTER models the parts of the structure for which an atomic model is not yet available (`missing structure') as low-resolution probability distributions for the random positions of the missing atoms. In reciprocal space, the BUSTER structure-factor distribution in the complex plane is a two-dimensional Gaussian centred around the structure factor calculated from the atomic, bulk-solvent and missing-structure models. The errors associated with these three structural components are added to compute the overall spread of the Gaussian. When the atomic model is very incomplete, modelling of the missing structure and the consistency of the BUSTER statistical model help structure building and completion because (i) the accuracy of the overall scale factors is increased, (ii) the bias affecting atomic model refinement is reduced by accounting for some of the scattering from the missing structure, (iii) the addition of a spatial definition to the source of incompleteness improves on traditional Luzzati and σA-based error models and (iv) the program can perform selective density modification in the regions of unbuilt structure alone.

Developments in the CCP4 molecular-graphics project

Abstract: Progress towards structure determination that is both high-throughput and high-value is dependent on the development of integrated and automatic tools for electron-density map interpretation and for the analysis of the resulting atomic models. Advances in map-interpretation algorithms are extending the resolution regime in which fully automatic tools can work reliably, but at present human intervention is required to interpret poor regions of macromolecular electron density, particularly where crystallographic data is only available to modest resolution [for example, I/σ(I) < 2.0 for minimum resolution 2.5 A]. In such cases, a set of manual and semi-manual model-building molecular-graphics tools is needed. At the same time, converting the knowledge encapsulated in a molecular structure into understanding is dependent upon visualization tools, which must be able to communicate that understanding to others by means of both static and dynamic representations. CCP4mg is a program designed to meet these needs in a way that is closely integrated with the ongoing development of CCP4 as a program suite suitable for both low- and high-intervention computational structural biology. As well as providing a carefully designed user interface to advanced algorithms of model building and analysis, CCP4mg is intended to present a graphical toolkit to developers of novel algorithms in these fields.

The Uppsala Electron-Density Server

Abstract: 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 ∼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.

Automated and accurate deposition of structures solved by X-ray diffraction to the Protein Data Bank.

Abstract: The RCSB Protein Data Bank (PDB) has a number of options for deposition of structural data and has developed software tools to facilitate the process. In addition to ADIT and the PDB Validation Suite, a new software application, pdb_extract, has been designed to promote automatic data deposition of structures solved by X-ray diffraction. The pdb_extract software can extract information about data reduction, phasing, molecular replacement, density modification and refinement from the output files produced by many X-ray crystallographic applications. The options, procedures and tools for accurate and automated PDB data deposition are described here.

The architecture of metal coordination groups in proteins.

Abstract: A set of tables is presented and a survey given of the architecture of metal coordination groups in a representative set of protein structures from the Protein Data Bank [Bernstein et al. (1977), J. Mol. Biol. 112, 535–542; Berman et al. (2000), Nucleic Acids Res. 28, 235–242]. The structures have been determined to a resolution of 2.5 A or better; the metals considered are Ca, Mg, Mn, Fe, Cu, Zn, Na and K, with particular emphasis on Ca and Zn and the exclusion of haem groups and Fe/S clusters; the proteins are a representative set in which none has more than 30% sequence identity with any other. In them the metal is coordinated by several donor groups from different amino-acid residues in the protein chain and often also by water or other small molecules. The tables, for ∼600 metal coordination groups, include information on the conformations of the protein chain in the region around the metal and reliability indicators. They illustrate the wide variety of coordination numbers, chelate-loop sizes and other properties and the different characteristics of different metals. They show that glycine has a particular significance in the position adjacent to a donor residue, especially in Ca coordination groups. They also show that metal coordination does not appear to lead to significant distortions of the torsion angles φ, ψ from their normally allowed values. Very few metal coordination groups occur more than once in the representative set and when they do they are usually related in fold and function; they have similar but not necessarily identical conformations. However, individual chelate loops, for example Zn(—C—X—X′—C—), in which both cysteines are coordinated to Zn through S, and X and X′ are any amino acids, are repeated frequently in many different and unrelated proteins. Not all chelate loops with the same composition have the same conformation, but for smaller loops there are usually one or two strongly preferred and well defined conformations. Quite frequently more than one metal coordination group is associated with one protein chain; these proteins are identified.

The importance of alignment accuracy for molecular replacement.

Abstract: Many crystallographic protein structures are being determined using molecular replacement (MR), a model-based phasing method that has become increasingly important with the steady growth of the PDB. While there are several highly automated software packages for MR, the methods for preparing optimal search models for MR are relatively unexplored. Recent advances in sequence-comparison methods allow the detection of more distantly related homologs and more accurate alignment of their sequences. It was investigated whether simple homology models (without modeling of unaligned regions) based on alignments from these improved methods are able to increase the potential of MR. 27 crystal structures were determined using a highly parallelized MR pipeline that facilitates all steps including homology detection, model preparation, MR searches, automated refinement and rebuilding. Several types of search models prepared with standard sequence–sequence alignment (BLAST) and more accurate profile–sequence and profile–profile methods (PSI-BLAST, FFAS) were compared in MR trials. The analysis shows that models based on more accurate alignments have a higher success rate in cases where the unknown structure and the search model share less than 35% sequence identity. It is concluded that by using different types of simple models based on accurate alignments, the success rate of MR can be significantly increased.

A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases.

Abstract: A high-throughput robotic system has been developed for crystallizing membrane proteins using lipidic mesophases. It incorporates commercially available components and is relatively inexpensive. The crystallization robot uses standard automated liquid-handlers and a specially built device for accurately and reproducibly delivering nanolitre volumes of highly viscous protein/lipid mesophases. Under standard conditions, the robot uses just 20 nl protein solution, 30 nl lipid and 1 µl precipitant solution. 96 wells can be set up using the robot in 13 min. Trials are performed in specially designed 96-well glass plates. The slim (<2 mm high) plates have exquisite optical properties and are well suited for the detection of microcrystals and for birefringence-free imaging between crossed polarizers. Quantitative evaluation of the crystallization progress is performed using an automated imaging system. The optics, in combination with the slim crystallization plates, enables in-focus imaging of the entire well volume in a single shot such that a 96-well plate can be imaged in just 4.5 min. The performance characteristics of the robotic system and the versatility of the crystallization robot in performing vapor-diffusion, microbatch and bicelle crystallizations of membrane and soluble proteins are described.

Structure of bovine carbonic anhydrase II at 1.95 A resolution.

Abstract: Carbonic anhydrase (CA) is a zinc-containing enzyme that catalyzes the reversible hydration of CO2 to HCO_{3}^{-}. In eukaryotes, the enzyme plays a role in various physiological functions, including interconversion between CO2 and HCO_{3}^{-} in intermediary metabolism, facilitated diffusion of CO2, pH homeostasis and ion transport. The structure of bovine carbonic anhydrase II (BCA II) has been determined by molecular replacement and refined to 1.95 A resolution by simulated-annealing and individual B-factor refinement. The final R factor for the BCA II structure was 19.4%. BCA II has a C-terminal knot structure similar to that observed in human CA II. It contains one zinc ion in the active site coordinated to three histidines and one putative water molecule in a tetrahedral geometry. The structure of BCA II reveals a probable alternative proton-wire pathway that differs from that of HCA II.

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

Abstract: 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.

Direct incorporation of experimental phase information in model refinement

Abstract: The incorporation of prior phase information into a maximum-likelihood formalism has been shown to strengthen model refinement. However, the currently available likelihood refinement target using prior phase information has shortcomings; the `phased' refinement target considers experimental phase information indirectly and statically in the form of Hendrickson–Lattman coefficients. Furthermore, the current refinement target implicitly assumes that the prior phase information is independent of the calculated model structure factor. This paper describes the derivation of a multivariate likelihood function that overcomes these shortcomings and directly incorporates experimental phase information from a single-wavelength anomalous diffraction (SAD) experiment. This function, which simultaneously refines heavy-atom and model parameters, has been implemented in the refinement program REFMAC5. The SAD function used in conjunction with the automated model-building procedures of ARP/wARP leads to a successful solution when current likelihood functions fail in a test case shown.

Novel buffer systems for macromolecular crystallization.

Abstract: In protein crystallization, screening is initially performed to obtain an indication of the conditions under which a macromolecule might crystallize. These preliminary conditions are then optimized to produce (in a perfect world) well diffracting crystals; this process of optimization often involves fine grid screening around the initial conditions. An issue in optimization is to find factors which are independent, so as to simplify the analysis of the results of optimization trials. This is necessarily difficult with buffers, as a buffer and its pH range tend to be very highly correlated. Multi-buffer systems for pH modulation are presented which enable a broad pH range to be sampled without changing the chemical composition of the buffering component.

Simple algorithm for a maximum-likelihood SAD function

Abstract: Recently, the multivariate complex normal distribution has been used to develop a maximum-likelihood probability function for single-wavelength anomalous diffraction phasing and refinement of heavy-atom parameters [Pannu & Read (2004), Acta Cryst D60, 22–27] The function accounts explicitly for the correlations between the observed and calculated Friedel mates and their errors However, the method of derivation of the equation described by Pannu & Read (2004) leads to a complicated likelihood expression that suffers from a number of algorithmic limitations Here, an alternative derivation of the PSAD function is described that leads to simplified algorithmic requirements and that allows an intuitive understanding of the expression

Short hydrogen bonds in photoactive yellow protein

Abstract: Eight high-resolution crystal structures of the ground state of photoactive yellow protein (PYP) solved under a variety of conditions reveal that its chromophore is stabilized by two unusually short hydrogen bonds Both Tyr42 Oη and Glu46 O∊ are separated from the chromophore phenolate oxygen by less than the sum of their atomic van der Waals radii, 26 A This is characteristic of strong hydrogen bonding, in which hydrogen bonds acquire significant covalent character The hydrogen bond from the protonated Glu46 to the negatively charged phenolate oxygen is 258 ± 001 A in length, while that from Tyr42 is considerably shorter, 249 ± 001 A The E46Q mutant was solved to 095 A resolution; the isosteric mutation increased the length of the hydrogen bond from Glx46 to the chromophore by 029 ± 001 A to that of an average hydrogen bond, 288 ± 001 A The very short hydrogen bond from Tyr42 explains why mutating this residue has such a severe effect on the ground-state structure and PYP photocycle The effect of isosteric mutations on the photocycle can be largely explained by the alterations to the length and strength of these hydrogen bonds

On the potential of normal-mode analysis for solving difficult molecular-replacement problems

Abstract: Molecular replacement (MR) is the method of choice for X-ray crystallographic data phasing when structural data of suitable homologues are available. However, MR may fail even in cases of high sequence homology when conformational changes arising for example from ligand binding or different crystallogenic conditions come into play. In this work, the potential of normal-mode analysis as an extension to MR to allow recovery from such drawbacks is demonstrated. Three examples are presented in which screening for MR solutions with templates perturbed in the direction of one or two normal modes allows a valid MR solution to be found where MR using the original template failed to yield a model that could ultimately be refined. It has been shown recently that half of the known protein movements can be modelled by displacing the studied structure using at most two low-frequency normal modes. This suggests that normal-mode analysis has the potential to break tough MR problems in up to 50% of cases. Moreover, even in cases where an MR solution is available, this method can be used to further improve the starting model prior to refinement, eventually reducing the time spent on manual model construction (in particular for low-resolution data sets).

Optimum solubility (OS) screening: an efficient method to optimize buffer conditions for homogeneity and crystallization of proteins.

Abstract: One of the most critical steps in the preparation of protein samples for structural studies by X-ray crystallography is to obtain biochemically pure and conformationally homogenous protein samples. Very often, the purified sample does not meet these qualifications and therefore does not crystallize. A screening method, Optimum Solubility Screen, has been developed that consists of two steps. The first step selects a better buffer than that used during purification. 24 different buffers ranging from pH 3 to pH 10 are screened using a vapor-diffusion method and very small amounts of protein. The solubility of the protein is first determined by visual examination using a light microscope and those drops that remain clear after 24 h are further evaluated using dynamic light scattering. If the results from the first step are still not satisfactory, a second step explores a variety of chemical additives in order to improve the monodispersity of the protein sample. In 64% of the cases, crystallization was successful from proteins that had initially shown high levels of aggregation. This screen can be configured to perform in an automated high-throughput mode and can be expanded for additional buffers and additives.

Dynamite: a simple way to gain insight into protein motions

Abstract: A public web-based facility to infer, analyse and graphically represent the likely modes of a protein motion, starting from a static structure, is presented. This facility is based on the use of CONCOORD to generate an ensemble of feasible protein structures that are subsequently analysed by principal component analysis to identify probable concerted motions. The user is returned the ensemble of feasible structures, together with associated analyses, including animations and graphical representations of both the principal component of the ensemble covariance and indicators of strongly correlated pairwise atomic motions. Whilst users are warned that completely reliable inferences about protein motion may be beyond even substantially more rigorous tools for exploring configurational space, it is hoped that the service will allow a much wider community to benefit from the insights that simple dynamic data may offer.

Structure of a feruloyl esterase from Aspergillus niger

Abstract: The crystallographic structure of feruloyl esterase from Aspergillus niger has been determined to a resolution of 1.5 A by molecular replacement. The protein has an α/β-hydrolase structure with a Ser-His-Asp catalytic triad; the overall fold of the protein is very similar to that of the fungal lipases. The structure of the enzyme–product complex was determined to a resolution of 1.08 A and reveals dual conformations for the serine and histidine residues at the active site.

Complexed and ligand-free high-resolution structures of urate oxidase (Uox) from Aspergillus flavus: a reassignment of the active-site binding mode.

Abstract: High-resolution X-ray structures of the complexes of Aspergillus Oavus urate oxidase (Uox) with three inhibitors, 8-azaxanthin (AZA), 9-methyl uric acid (MUA) and oxonic acid (OXC), were determined in an orthorhombic space group (I222). In addition, the ligand-free enzyme was also crystallized in a monoclinic form (P21) and its structure determined. Higher accuracy in the three new enzyme±inhibitor complex structures (Uox±AZA, Uox±MUA and Uox±OXC) with respect to the previously determined structure of Uox±AZA (PDB code 1uox) leads to a reversed position of the inhibitor in the active site of the enzyme. The corrected anchoring of the substrate (uric acid) allows an improvement in the understanding of the enzymatic mechanism of urate oxidase.

An improved protocol for rapid freezing of protein samples for long-term storage

Abstract: Freezing of purified protein drops directly in liquid nitrogen is a convenient technique for the long-term storage of protein samples. Although this enhances reproducibility in follow-up crystallization experiments, some protein samples are not amenable to this technique. It has been discovered that plunging PCR tubes containing protein samples into liquid nitrogen results in more rapid freezing of the samples and can safely preserve some proteins that are damaged by drop-freezing. The PCR-tube method can also be adapted to a PCR-plate freezing method with applications for high-throughput and structural genomics projects.

Interactive electron-density map interpretation: from INTER to O

Abstract: A short review of the author's computer-graphics developments since 1976 is presented. Some of the major developments in these programs are reviewed and a description is provided of some of the more recent tools that can be used for electron-density map interpretation. These tools include a secondary-structure template-building system that works in conjunction with a new sequence-decoration system.

Structural analysis of neprilysin with various specific and potent inhibitors.

Abstract: Neutral endopeptidase (NEP) is the major enzyme involved in the metabolic inactivation of a number of bioactive peptides including the enkephalins, substance P, endothelin, bradykinin and atrial natriuretic factor Owing to the physiological importance of NEP in the modulation of nociceptive and pressor responses, there is considerable interest in inhibitors of this enzyme as novel analgesics and antihypertensive agents Here, the crystal structures of the soluble extracellular domain of human NEP (residues 52–749) complexed with various potent and competitive inhibitors are described The structures unambiguously reveal the binding mode of the different zinc-chelating groups and the subsite specificity of the enzyme

Structure of an aryl esterase from Pseudomonas fluorescens.

Abstract: The structure of PFE, an aryl esterase from Pseudomonas fluorescens, has been solved to a resolution of 1.8 A by X-ray diffraction and shows a characteristic α/β-hydrolase fold. In addition to catalyzing the hydrolysis of esters in vitro, PFE also shows low bromoperoxidase activity. PFE shows highest structural similarity, including the active-site environment, to a family of non-heme bacterial haloperoxidases, with an r.m.s. deviation in 271 Cα atoms between PFE and its five closest structural neighbors averaging 0.8 A. PFE has far less similarity (r.m.s. deviation in 218 Cα atoms of 5.0 A) to P. fluorescens carboxyl esterase. PFE favors activated esters with small acyl groups, such as phenyl acetate. The X-ray structure of PFE reveals a significantly occluded active site. In addition, several residues, including Trp28 and Met95, limit the size of the acyl-binding pocket, explaining its preference for small acyl groups.

Using prime-and-switch phasing to reduce model bias in molecular replacement

Abstract: Atomic models are commonly used to calculate phases in macromolecular crystallography. When combined with measured amplitudes, model-based phases yield electron-density maps with features of the correct structure but with a significant bias towards features of the model. The present contribution shows applications of the technique of prime-and-switch phasing to reduce this bias. An atomic model is used to generate phases that are close to the correct set but that may be biased. An unbiased source of phase information, an estimate of the probability that the electron-density map corresponds to a macromolecule, is then used to select a set of phases that are near the biased set, without further reference to the biased phases. The probability that the electron-density map corresponds to a macromolecule is based on agreement of the map with expectations such as a flat solvent region. Prime-and-switch phasing can be useful even for crystals with low solvent content and may reduce errors in interpretation of electron density in a wide range of applications of macromolecular crystallography, including molecular replacement, model building, ligand-binding and conformation-change studies, refinement and structure validation.

The new CCP4 Coordinate Library as a toolkit for the design of coordinate-related applications in protein crystallography.

Abstract: The new CCP4 Coordinate Library is a development aiming to provide a common layer of coordinate-related functionality to the existing applications in the CCP4 suite, as well as a variety of tools that can simplify the design of new applications where they relate to atomic coordinates. The Library comprises a wide spectrum of useful functions, ranging from parsing coordinate formats and elementary editing operations on the coordinate hierarchy of biomolecules, to high-level functionality such as calculation of secondary structure, interatomic bonds, atomic contacts, symmetry transformations, structure superposition and many others. Most of the functions are available in a C++ object interface; however, a Fortran interface is provided for compatibility with older CCP4 applications. The paper describes the general principles of the Library design and the most important functionality. The Library, together with documentation, is available under the LGPL license from the CCP4 suite version 5.0 and higher.

Structure of shikimate kinase from Mycobacterium tuberculosis reveals the binding of shikimic acid.

Abstract: Tuberculosis made a resurgence in the mid-1980s and now kills approximately 3 million people a year. The re-emergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons and the proliferation of multi-drug-resistant strains have created a need to develop new drugs. Shikimate kinase and other enzymes in the shikimate pathway are attractive targets for development of non-toxic antimicrobial agents, herbicides and anti-parasitic drugs, because the pathway is essential in these species whereas it is absent from mammals. The crystal structure of shikimate kinase from Mycobacterium tuberculosis (MtSK) complexed with MgADP and shikimic acid (shikimate) has been determined at 2.3 A resolution, clearly revealing the amino-acid residues involved in shikimate binding. This is the first three-dimensional structure of shikimate kinase complexed with shikimate. In MtSK, the Glu61 residue that is strictly conserved in shikimate kinases forms a hydrogen bond and salt bridge with Arg58 and assists in positioning the guanidinium group of Arg58 for shikimate binding. The carboxyl group of shikimate interacts with Arg58, Gly81 and Arg136 and the hydroxyl groups interact with Asp34 and Gly80. The crystal structure of MtSK-MgADP-shikimate will provide crucial information for the elucidation of the mechanism of the shikimate kinase-catalyzed reaction and for the development of a new generation of drugs against tuberculosis.