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Muse Oke,
Lester G Carter,
Kenneth A Johnson,
Huanting Liu,
Stephen A McMahon,
Xuan Yan,
Melina Kerou,
Nadine D Weikart,
Nadia Kadi,
Md Arif Sheikh, [......],
David Prangishvili,
Catherine H Botting,
Peter J Coote,
David T F Dryden,
Geoffrey J Barton,
Ulrich Schwarz-Linek,
Gregory L Challis,
Garry L Taylor,
Malcolm F White,
James H Naismith
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ABSTRACT: The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology.
Journal of Structural and Functional Genomics 04/2010; 11(2):167-80.
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Jaldappagari Seetharamappa,
Muse Oke,
Huanting Liu,
Stephen A McMahon,
Kenneth A Johnson,
Lester Carter,
Mark Dorward, Michal Zawadzki,
Ian M Overton,
C A Johannes van Niekirk,
Shirley Graham,
Catherine H Botting,
Garry L Taylor,
Malcolm F White,
Geoffrey J Barton,
Peter J Coote,
James H Naismith
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ABSTRACT: Sar2676, a pantothenate synthetase with a molecular weight of 31 419 Da from methicillin-resistant Staphylococcus aureus, has been expressed, purified and crystallized at 293 K. The protein crystallizes in a primitive triclinic lattice, with unit-cell parameters a = 45.3, b = 60.5, c = 117.6 A, alpha = 87.2, beta = 81.2, gamma = 68.4 degrees . A complete data set has been collected to 2.3 A resolution at the ESRF. Consideration of the likely solvent content suggested the asymmetric unit to contain four molecules. This has been confirmed by molecular-replacement phasing calculations, which give a solution with four monomers using a monomer of pantothenate synthetase from Escherichia coli (PDB code 1iho), which is 41% identical to Sar2676, as a search model.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 07/2007; 63(Pt 6):488-91. · 0.51 Impact Factor
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Jaldappagari Seetharamappa,
Muse Oke,
Huanting Liu,
Stephen A McMahon,
Kenneth A Johnson,
Lester Carter,
Mark Dorward, Michal Zawadzki,
Ian M Overton,
C A Johannes van Niekirk,
Shirley Graham,
Catherine H Botting,
Garry L Taylor,
Malcolm F White,
Geoffrey J Barton,
Peter J Coote,
James H Naismith
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ABSTRACT: Sar2028, an aspartate/tyrosine/phenylalanine pyridoxal-5'-phosphate-dependent aminotransferase with a molecular weight of 48,168 Da, was overexpressed in methicillin-resistant Staphylococcus aureus compared with a methicillin-sensitive strain. The protein was expressed in Escherichia coli, purified and crystallized. The protein crystallized in a primitive orthorhombic Laue group with unit-cell parameters a = 83.6, b = 91.3, c = 106.0 A, alpha = beta = gamma = 90 degrees. Analysis of the systematic absences along the three principal axes indicated the space group to be P2(1)2(1)2(1). A complete data set was collected to 2.5 A resolution.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 06/2007; 63(Pt 5):452-6. · 0.51 Impact Factor
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ABSTRACT: A strategy of rationally engineering protein surfaces with the aim of obtaining mutants that are distinctly more susceptible to crystallization than the wild-type protein has previously been suggested. The strategy relies on replacing small clusters of two to three surface residues characterized by high conformational entropy with alanines. This surface entropy reduction (or SER) method has proven to be an effective salvage pathway for proteins that are difficult to crystallize. Here, a systematic comparison of the efficacy of using Ala, His, Ser, Thr and Tyr to replace high-entropy residues is reported. A total of 40 mutants were generated and screened using two different procedures. The results reaffirm that alanine is a particularly good choice for a replacement residue and identify tyrosines and threonines as additional candidates that have considerable potential to mediate crystal contacts. The propensity of these mutants to form crystals in alternative screens in which the normal crystallization reservoir solutions were replaced with 1.5 M NaCl was also examined. The results were impressive: more than half of the mutants yielded a larger number of crystals with salt as the reservoir solution. This method greatly increased the variety of conditions that yielded crystals. Taken together, these results suggest a powerful crystallization strategy that combines surface engineering with efficient screening using standard and alternate reservoir solutions.
Acta Crystallographica Section D Biological Crystallography 06/2007; 63(Pt 5):636-45. · 12.62 Impact Factor
