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K S Wilson

Publications and source records attributed to K S Wilson.

At least 19 recordsLinked to original sources
PubMed2006

Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens.

The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.

Animals↗
PubMed2006

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

The Structural Proteomics In Europe (SPINE) consortium contained a workpackage to address the automated X-ray analysis of macromolecules. The aim of this workpackage was to increase the throughput of three-dimensional structures while maintaining the high quality of conventional analyses. SPINE was able to bring together developers of software with users from the partner laboratories. Here, the results of a workshop organized by the consortium to evaluate software developed in the member laboratories against a set of bacterial targets are described. The major emphasis was on molecular-replacement suites, where automation was most advanced. Data processing and analysis, use of experimental phases and model construction were also addressed, albeit at a lower level.

Algorithms↗
PubMed2006

Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis.

A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining ;low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs.

Bacillus anthracis↗
PubMed2006

ACORN: a review.

The ACORN system was originally developed as a means of ab initio solution of protein structures when atomic resolution data were available. The first step is to obtain a starting set of phases, which must be at least slightly better than random. These may be calculated from a fragment of the structure, which can be anything from a single metal atom to a complete molecular-replacement model. A number of standard procedures are available in ACORN to orientate and position such a fragment. The fragment provides initial phases that give the first of a series of maps that are iteratively refined by a dynamic density-modification (DDM) process. Another FFT-based procedure is Sayre-equation refinement (SER), which modifies phases better to satisfy the Sayre equation. With good-quality atomic resolution data, the final outcome of applying DDM and SER is a map similar in appearance to that found from a refined structure, which is readily interpreted by automated procedures. Further development of ACORN now enables structures to be solved with less than atomic resolution data. A critical part of this development is the artificial extension of the data from the observed limit to 1 A resolution. These extended reflections are allocated unit normalized structure amplitudes and then treated in a similar way to observed reflections except that they are down-weighted in the calculation of maps. ACORN maps, especially at low resolution, tend to show C atoms less well, in particular C(alpha) atoms which fall within the first diffraction minimum of their three neighbours. Two new density-modification procedures (DDM1 and DDM2) and a density-enhancement procedure (ENH) have been devised to counter this problem. It is demonstrated that high-quality maps showing individual atoms can be produced with the new ACORN. ACORN has also been demonstrated to be very effective in refining phase sets derived from physical processes such as those using anomalous scattering or isomorphous derivative data. Future work will be directed towards applying ACORN to resolutions down to 2 A.

Computer Simulation↗
PubMed2006

Molecular methods for virus discovery.

The potential use of novel cell substrates from diverse animal species raises concerns about the transmission of hitherto unknown viral agents. Viruses that do not cause a cytopathic effect in cell culture may escape detection by conventional methods and molecular methods may therefore prove useful for screening for hitherto unknown viruses. This review describes currently used molecular methods for virus discovery, including degenerate PCR assays, representational difference analysis and rolling circle amplification, and summarises the advantages and disadvantages of each technique.

Amino Acid Sequence↗
PubMed2005

A modified ACORN to solve protein structures at resolutions of 1.7 A or better.

ACORN has previously been shown to provide an efficient density-modification procedure for the solution of protein structures using diffraction data to better than 1.3 A. The initial phase set could be obtained from a variety of sources such as the position of a heavy atom, a set of scatterers such as S that had been positioned from anomalous dispersion measurements, a fragment or a very low homology model placed from a molecular-replacement search. Several structures solved using the early version of ACORN have been reported in the literature. Here, the effect of applying the original ACORN procedures at lower resolution is reported and new procedures that yield good-quality maps with data sets of resolution down to 1.7 A are described. These new procedures involve the artificial extension of data to atomic resolution and new density-modification processes that develop density at atomic positions that was previously suppressed. The test calculations were aimed firstly towards a proof of principle using a small fragment of a known structure to demonstrate that the procedure could generate correct density and a derived model in initially empty regions of the cell. Further tests addressed the use of more realistic starting models.

Algorithms↗
PubMed2003

Structure of the human S100A12-copper complex: implications for host-parasite defence.

S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins. Together with S100A8 and S100A9, it belongs to the calgranulin subfamily, i.e. it is mainly expressed in granulocytes, although there is an increasing body of evidence of expression in keratinocytes and psoriatic lesions. As well as being linked to inflammation, allergy and neuritogenesis, S100A12 is involved in host-parasite response, as are the other two calgranulins. Recent data suggest that the function of the S100-family proteins is modulated not only by calcium, but also by other metals such as zinc and copper. Previously, the structure of human S100A12 in low-calcium and high-calcium structural forms, crystallized in space groups R3 and P2(1), respectively, has been reported. Here, the structure of S100A12 in complex with copper (space group P2(1)2(1)2; unit-cell parameters a = 70.6, b = 119.0, c = 90.2 A) refined at 2.19 A resolution is reported. Comparison of anomalous difference electron-density maps calculated with data collected with radiation of wavelengths 1.37 and 1.65 A shows that each monomer binds a single copper ion. The copper binds at an equivalent site to that at which another S100 protein, S100A7, binds zinc. The results suggest that copper binding may be essential for the functional role of S100A12 and probably the other calgranulins in the early immune response.

Amino Acid Sequence↗
PubMed2003

Multiple structural states of S100A12: A key to its functional diversity.

S100A12 is a member of the S100 family of EF-hand calcium-binding proteins. Together with two other calgranulins, S100A8 and S100A9, it is mostly expressed in human granulocytes, although there is increasing evidence of expression in keratinocytes and psoriatic lesions. It is involved in host-parasite response, and linked to corneal autoimmune diseases connected with filarial parasite infestation. Interaction of S100A12 with a multiligand receptor for advanced glycation end products (RAGE) mediates inflammation. Human recombinant S100A12 was found to induce neuritogenesis of cultured hippocampal cells, similar to two other S100 proteins, S100B and S100A4. X-ray structure of S100A12 has been solved in two crystal forms: R3 and P2(1). In the R3 crystal form S100A12 is a dimer, and in the P2(1) crystal form the dimers are arranged as a hexamer. The hexameric form suggests its role in receptor oligomerisation. S100A12 binds copper at the predicted zinc/copper binding site, which is located close to the surface of the protein. We propose copper-mediated generation of reactive oxygen species by S100A12 as its function in host-parasite response.

Amino Acid Sequence↗
PubMed2002

The structure of S100A12 in a hexameric form and its proposed role in receptor signalling.

S100A12 is a member of the S100 subfamily of EF-hand calcium-binding proteins; it has been shown to be one of the ligands of the 'receptor for advanced glycation end products' (RAGE) that belongs to the immunoglobulin superfamily and is involved in diabetes, Alzheimer's disease, inflammation and tumour invasion. The structure of the dimeric form of native S100A12 from human granulocytes in the presence of calcium in space group R3 has previously been reported. Here, the structure of a second crystal form in space group P2(1) (unit-cell parameters a = 53.9, b = 100.5, c = 112.7A, beta = 94.6 degrees) solved at 2.7A resolution by molecular replacement using the R3 structure as a search model is reported. Like most S100 proteins, S100A12 is a dimer. However, in the P2(1) crystal form dimers of S100A12 are arranged in a spherical hexameric assembly with an external diameter of about 55 A stabilized by calcium ions bound between adjacent dimers. The putative target-binding sites of S100A12 are located at the outer surface of the hexamer, making it possible for the hexamer to bind several targets. It is proposed that the S100A12 hexameric assembly might interact with three extracellular domains of the receptor, bringing them together into large trimeric assemblies.

Binding Sites↗
PubMed2002

Atomic resolution structures of ribonuclease A at six pH values.

The diffraction pattern of protein crystals extending to atomic resolution guarantees a very accurate picture of the molecular structure and enables the study of subtle phenomena related to protein functionality. Six structures of bovine pancreatic ribonuclease at the pH* values 5.2, 5.9, 6.3, 7.1, 8.0 and 8.8 and at resolution limits in the range 1.05-1.15A have been refined. An overall description of the six structures and several aspects, mainly regarding pH-triggered conformational changes, are described here. Since subtle variations were expected, a thorough validation assessment of the six refined models was first carried out. Some stereochemical parameters, such as the N[bond]C(alpha)[bond]C angle and the pyramidalization at the carbonyl C atoms, indicate that the standard target values and their weights typically used in refinement may need revision. A detailed comparison of the six structures has provided experimental evidence on the role of Lys41 in catalysis. Furthermore, insights are given into the structural effects related to the pH-dependent binding of a sulfate anion, which mimics the phosphate group of RNA, in the active site. Finally, the results support a number of thermodynamic and kinetic experimental data concerning the role of the disulfide bridge between Cys65 and Cys72 in the folding of RNase A.

Animals↗
PubMed2002

Structural diversity in metal ion chelation and the structure of uroporphyrinogen III synthase.

All tetrapyrroles are synthesized through a branched pathway, and although each tetrapyrrole receives unique modifications around the ring periphery, they all share the unifying feature of a central metal ion. Each pathway maintains a unique metal ion chelatase, and several tertiary structures have been determined, including those of the protoporphyrin ferrochelatase from both human and Bacillus subtilus, and the cobalt chelatase CbiK. These enzymes exhibit strong structural similarity and appear to function by a similar mechanism. Met8p, from Saccharomyces cerevisiae, catalyses ferrochelation during the synthesis of sirohaem, and the structure reveals a novel chelatase architecture whereby both ferrochelation and NAD(+)-dependent dehydrogenation take place in a single bifunctional active site. Asp-141 appears to participate in both catalytic reactions. The final common biosynthetic step in tetrapyrrole biosynthesis is the generation of uroporphyrinogen by uroporphyrinogen III synthase, whereby the D ring of hydroxymethylbilane is flipped during ring closure to generate the asymmetrical structure of uroporphyrinogen III. The recently derived structure of uroporphyrinogen III synthase reveals a bi-lobed structure in which the active site lies between the domains.

Bacillus subtilis↗
PubMed2001

A quick solution: ab initio structure determination of a 19 kDa metalloproteinase using ACORN.

A data set from the metalloproteinase deuterolysin was collected at atomic resolution (1.0 A) with synchrotron radiation. The high resolution allowed the structure to be solved with the new direct-methods program ACORN using the coordinates of the Zn atom as a starting point. The phases obtained from ACORN were of sufficient quality to allow automated building to be carried out in ARP/wARP. Minimal manual rebuilding of the model was required and the structure determination was completed using the maximum-likelihood refinement program REFMAC. The whole process, starting from the processed and merged data and ending with a refined model, required less than 6 h of computational time.

Aspergillus oryzae↗
PubMed2001

Crystal structures of penicillin acylase enzyme-substrate complexes: structural insights into the catalytic mechanism.

The crystal structure of penicillin G acylase from Escherichia coli has been determined to a resolution of 1.3 A from a crystal form grown in the presence of ethylene glycol. To study aspects of the substrate specificity and catalytic mechanism of this key biotechnological enzyme, mutants were made to generate inactive protein useful for producing enzyme-substrate complexes. Owing to the intimate association of enzyme activity and precursor processing in this protein family (the Ntn hydrolases), most attempts to alter active-site residues lead to processing defects. Mutation of the invariant residue Arg B263 results in the accumulation of a protein precursor form. However, the mutation of Asn B241, a residue implicated in stabilisation of the tetrahedral intermediate during catalysis, inactivates the enzyme but does not prevent autocatalytic processing or the ability to bind substrates. The crystal structure of the Asn B241 Ala oxyanion hole mutant enzyme has been determined in its native form and in complex with penicillin G and penicillin G sulphoxide. We show that Asn B241 has an important role in maintaining the active site geometry and in productive substrate binding, hence the structure of the mutant protein is a poor model for the Michaelis complex. For this reason, we subsequently solved the structure of the wild-type protein in complex with the slowly processed substrate penicillin G sulphoxide. Analysis of this structure suggests that the reaction mechanism proceeds via direct nucleophilic attack of Ser B1 on the scissile amide and not as previously proposed via a tightly H-bonded water molecule acting as a "virtual" base.

Amino Acid Substitution↗
PubMed2001

ARP/wARP and molecular replacement.

The aim of ARP/wARP is improved automation of model building and refinement in macromolecular crystallography. Once a molecular-replacement solution has been obtained, it is often tedious to refine and rebuild the initial (search) model. ARP/wARP offers three options to automate that task to varying extents: (i) autobuilding of a completely new model based on phases calculated from the molecular-replacement solution, (ii) updating of the initial model by atom addition and deletion to obtain an improved map and (iii) docking of a structure onto a new (or mutated) sequence, followed by rebuilding and refining the side chains in real space. A few examples are presented where ARP/wARP made a considerable difference in the speed of structure solution and/or made possible refinement of otherwise difficult or uninterpretable maps. The resolution range allowing complete autobuilding of protein structures is currently 2.0 A, but for map improvement considerable advances over more conventional refinement techniques are evident even at 3.2 A spacing.

Alu Elements↗
PubMed2001

Oligosaccharide binding to family 11 xylanases: both covalent intermediate and mutant product complexes display (2,5)B conformations at the active centre.

The glycoside hydrolase sequence-based classification reveals two families of enzymes which hydrolyse the beta-1,4-linked backbone of xylan, xylanases, termed families GH-10 and GH-11. Family GH-11 xylanases are intriguing in that catalysis is performed via a covalent intermediate adopting an unusual (2,5)B (boat) conformation, a conformation which also fulfils the stereochemical constraints of the oxocarbenium ion-like transition state. Here, the 1.9 A structure of a nucleophile, E94A, mutant of the Xyn11 from Bacillus agaradhaerens in complex with xylotriose is presented. Intriguingly, this complex also adopts the (2,5)B conformation in the -1 subsite, with the vacant space provided by the Glu-->Ala mutation allowing the sugar to adopt the alpha-configuration at C1. The structure of the covalent 2-deoxy-2-fluoroxylobiosyl-enzyme intermediate has been extended to atomic (1.1 A) resolution.

Amino Acid Substitution↗
PubMed2001

Optimization of Met8p crystals through protein-storage buffer manipulation.

Sirohaem, the prosthetic group of assimilatory sulfite and nitrite reductases, is a modified tetrapyrrole that belongs to the same fraternity of metallo-prosthetic groups as haem, chlorophyll, cobalamin and coenzyme F430 [Warren & Scott (1990), Trends Biochem Sci. 15, 486-491]. In Saccharomyces cerevisiae, the last step in the biosynthesis of sirohaem involves Met8p, a bifunctional enzyme responsible for both the NAD(+)-dependent dehydrogenation of the corrin ring and ferrochelation. Optimization of the protein storage buffer according to the results of crystallization trials resulted in a more monodisperse protein solution. Crystals were grown that diffracted to 2.1 A.

Buffers↗