NONE-No attached Diamond beamline
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Abstract: Using X-ray absorption spectroscopy (XAS) at the Cu L(2,3) and Co L(2,3) edges, we demonstrate that the valence state of copper in the thiospinel carrollite (CuCo(2)S(4)) is Cu(+) with a d count of Cu d(9.8). The Co has a d count of d(6.4), so that the highly covalent mineral has an electronic formula of Cu(1.2+)(Co(2.4+))(2)(S(1.5-))(4). There is half a hole per atom in the S 3p band (per unit formula); the Co L(2,3) XAS indicates a covalent low-spin electronic structure. These data dispel the notion of the presence of Cu(2+) in carrollite.
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Oct 2008
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I04-Macromolecular Crystallography
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Abstract: Archaeal family B DNA polymerases bind tightly to template-strand uracil and stall replication on encountering the pro-mutagenic base. This article describes an X-ray crystal structure, at 2.8 Å resolution, of Thermococcus gorgonarius polymerase in complex with a DNA primer–template containing uracil in the single-stranded region. The DNA backbone is distorted to position the uracil deeply within a pocket, located in the amino-terminal domain of the polymerase. Specificity arises from a combination of hydrogen bonds between the protein backbone and uracil, with the pocket shaped to prevent the stable binding of the four standard DNA bases. Strong interactions are seen with the two phosphates that flank the uracil and the structure gives clues concerning the coupling of uracil binding to the halting of replication. The importance of key amino acids, identified by the analysis of the structure and their conservation between archaeal polymerases, was confirmed by site-directed mutagenesis. The crystal structure of V93Q, a polymerase variant that no longer recognises uracil, is also reported, explaining the V93Q phenotype by the steric exclusion of uracil from the pocket.
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Sep 2008
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I04-Macromolecular Crystallography
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Abstract: A 2-His-1-carboxylate triad of iron binding residues is present in many non-heme iron oxygenases including the Fe(II) and 2-oxoglutarate (2OG)-dependent dioxygenases. Three variants (D201A, D201E, and D201G) of the iron binding Asp-201 residue of an asparaginyl hydroxylase, factor inhibiting HIF (FIH), were made and analyzed. FIH-D201A and FIH-D201E did not catalyze asparaginyl hydroxylation, but in the presence of a reducing agent, they displayed enhanced 2OG turnover when compared with wild-type FIH. Turnover of 2OG by FIH-D201A was significantly stimulated by the addition of HIF-1?786–826 peptide. Like FIH-D201A and D201E, the D201G variant enhanced 2OG turnover but rather unexpectedly catalyzed asparaginyl hydroxylation. Crystal structures of the FIH-D201A and D201G variants in complex with Fe(II)/Zn(II), 2OG, and HIF-1?786–826/788–806 implied that only two FIH-based residues (His-199 and His-279) are required for metal binding. The results indicate that variation of 2OG-dependent dioxygenase iron-ligating residues as a means of functional assignment should be treated with caution. The results are of mechanistic interest in the light of recent biochemical and structural analyses of non-heme iron and 2OG-dependent halogenases that are similar to the FIH-D201A/G variants in that they use only two His-residues to ligate iron.
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Sep 2008
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[614]
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Sep 2008
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I03-Macromolecular Crystallography
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Open Access
Abstract: Post?translational modification of protein serines/threonines with N?acetylglucosamine (O?GlcNAc) is dynamic, inducible and abundant, regulating many cellular processes by interfering with protein phosphorylation. O?GlcNAcylation is regulated by O?GlcNAc transferase (OGT) and O?GlcNAcase, both encoded by single, essential, genes in metazoan genomes. It is not understood how OGT recognises its sugar nucleotide donor and performs O?GlcNAc transfer onto proteins/peptides, and how the enzyme recognises specific cellular protein substrates. Here, we show, by X?ray crystallography and mutagenesis, that OGT adopts the (metal?independent) GT?B fold and binds a UDP?GlcNAc analogue at the bottom of a highly conserved putative peptide?binding groove, covered by a mobile loop. Strikingly, the tetratricopeptide repeats (TPRs) tightly interact with the active site to form a continuous 120 Å putative interaction surface, whereas the previously predicted phosphatidylinositide?binding site locates to the opposite end of the catalytic domain. On the basis of the structure, we identify truncation/point mutants of the TPRs that have differential effects on activity towards proteins/peptides, giving first insights into how OGT may recognise its substrates.
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Sep 2008
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Abstract: Although LysR-type regulators (LTTRs) represent the largest family of transcriptional regulators in bacteria, the full-length structure of only one annotated LTTR (CbnR) has been deposited in the PDB. CrgA, a LTTR from pathogenic Neisseria meningitidis MC58, which is up-regulated upon bacterial cell contact with human epithelial cells, has been cloned, purified and crystallized. Crystals of full-length CrgA were obtained after buffer screening with a thermal shift assay and concentration with 0.2 M NDSB-256. Data were collected from two crystal forms of full-length CrgA belonging to space groups P212121 and P21, diffracting to 3.0 and 3.8 Å resolution and consistent with the presence of between six and ten and between ten and 20 copies of CrgA in the asymmetric unit, respectively. In addition, diffraction data were collected to 2.3 Å resolution from the selenomethionine derivative of the regulatory domain of CrgA. The crystals belonged to space group P21 and contained two molecules in the asymmetric unit.
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Sep 2008
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Vacuum
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Abstract: The Diamond storage ring has been operating with a 3 GeV electron beam since September 2006 and 190 A.h of beam dose have been accumulated. The pressure in the storage ring is 4.2 10(-10) mbar without beam, rising to 7.9 10(-10) mbar with 125 mA of stored beam. Data on the storage ring vacuum performance and experience from commissioning and beam conditioning are presented.
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Sep 2008
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Abstract: Intersubband transitions within the conduction band in semiconductor quantum wells have been widely explored, which eventually led to their application in quantum well infrared photodetectors and quantum cascade lasers (QCLs). There have been considerably fewer studies of hole intersubband transitions, which are more complicated than their electronic counterparts. They are very interesting, however, because of optical activity for both TM and TE light polarization, offering the possibility of both edge and surface-normal emission or absorption. Earlier studies have mostly focused on bound- continuum transitions in GaAs/AlGaAs and Si/SiGe systems for infrared detection.
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Sep 2008
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1219]
Abstract: Crystals of recombinant NovN, an O-carbamoyltransferase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in two different crystal forms. Crystal form I belonged to space group C2 and native data were collected to 2.9 Å resolution in-house. Crystal form II had I-centred orthorhombic symmetry and native data were recorded to a resolution of 2.3 Å at a synchrotron. NovN catalyses the final step in the biosynthesis of the aminocoumarin antibiotic novobiocin that targets the essential bacterial enzyme DNA gyrase.
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Sep 2008
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B23-Circular Dichroism
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Abstract: Principal component analysis (PCA) is a method of simplifying complex datasets to generate a lower number of parameters, while retaining the essential differences and allowing objective comparison of large numbers of datasets. Glycosaminoglycans (GAGs) are a class of linear sulfated carbohydrates with diverse sequences and consequent complex conformation and structure. Here, PCA is applied to three problems in GAG research: (i) distinguishing origins of heparin preparations, (ii) structural analysis of heparin derivatives, and (iii) classification of chondroitin sulfates (CS). The results revealed the following. (i) PCA of heparin 13C NMR spectra allowed their origins to be distinguished and structural differences were identified. (ii) Analysis of the information-rich 1H and 13C NMR spectra of a series of systematically modified heparin derivatives uncovered underlying properties. These included the presence of interactions between residues, providing evidence that a degree of degeneracy exists in linkage geometry and that a different degree of variability exists for the two types of glycosidic linkage. The relative sensitivity of each position (C or H nucleus) in the disaccharide repeating unit to changes in O-, N-sulfation and N-acetylation was also revealed. (iii) Analysis of the 1H NMR and CD spectra of a series of CS samples from different origins allowed their structural classification and highlighted the power of employing complementary spectroscopic methods in concert with PCA.
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Sep 2008
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