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Abstract: A number of commercially available waxes in the form of thin disc samples have been investigated as possible diffraction intensity standards for macromolecular crystallography synchrotron beamlines. Synchrotron X-ray powder diffraction measurements show that beeswax offers the best performance of these waxes owing to its polycrystallinity. Crystallographic lattice parameters and diffraction intensities were examined between 281 and 309 K, and show stable and predictable thermal behaviour. Using an X-ray beam of known incident flux at [lambda] = 1 Å, the diffraction power of two strong Bragg reflections for beeswax were quantified as a function of sample thickness and normalized to 1010 photons s-1. To demonstrate its feasibility as a diffraction intensity standard, test measurements were then performed on a new third-generation macromolecular crystallography synchrotron beamline.

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Abstract: When the number of overlapping lattices ntwins present in a merohedrally twinned crystal sample is greater than 2, the twinned diffraction intensities need analysing with a formalism that goes beyond the more familiar one used in the case of hemihedral twinning (ntwins =2). The special case of ntwins=4 aka tetartohedral twinning, will be summarised and discussed, with examples taken from the recent literature. Particular attention will be given to the fact that tetartohedral twinning is almost invariably associated with 222 rotational non-crystallographic symmetry (NCS), and thus is often a special case of a general phenomenon where the twinning operators coincide with the rotational part of the NCS. The available refinement and detwinning strategies in the Refmac5 and CNS computer programs will be discussed, and the recent crystal structure determination and refinement of human Complement Factor I from tetartohedrally twinned intensities will be illustrated.

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Abstract: The class II chelatases associated with heme, siroheme, and cobalamin biosynthesis are structurally related enzymes that insert a specific metal ion (Fe2+ or Co2+) into the center of a modified tetrapyrrole (protoporphyrin or sirohydrochlorin). The structures of two related class II enzymes, CbiXS from Archaeoglobus fulgidus and CbiK from Salmonella enterica, that are responsible for the insertion of cobalt along the cobalamin biosynthesis pathway are presented in complex with their metallated product. A further structure of a CbiK from Desulfovibrio vulgaris Hildenborough reveals how cobalt is bound at the active site. The crystal structures show that the binding of sirohydrochlorin is distinctly different to porphyrin binding in the protoporphyrin ferrochelatases and provide a molecular overview of the mechanism of chelation. The structures also give insights into the evolution of chelatase form and function. Finally, the structure of a periplasmic form of Desulfovibrio vulgaris Hildenborough CbiK reveals a novel tetrameric arrangement of its subunits that are stabilized by the presence of a heme b cofactor. Whereas retaining colbaltochelatase activity, this protein has acquired a central cavity with the potential to chaperone or transport metals across the periplasmic space, thereby evolving a new use for an ancient protein subunit.

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Abstract: Complement factor H (FH) attenuates C3b molecules tethered by their thioester domains to self surfaces and thereby protects host tissues. Factor H is a cofactor for initial C3b proteolysis that ultimately yields a surface-attached fragment (C3d) corresponding to the thioester domain. We used NMR and X-ray crystallography to study the C3d–FH19–20 complex in atomic detail and identify glycosaminoglycan-binding residues in factor H module 20 of the C3d–FH19–20 complex. Mutagenesis justified the merging of the C3d–FH19–20 structure with an existing C3b–FH1–4 crystal structure. We concatenated the merged structure with the available FH6–8 crystal structure and new SAXS-derived FH1–4, FH8–15 and FH15–19 envelopes. The combined data are consistent with a bent-back factor H molecule that binds through its termini to two sites on one C3b molecule and simultaneously to adjacent polyanionic host-surface markers.

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Abstract: Raltegravir (RAL) and related HIV-1 integrase (IN) strand transfer inhibitors (INSTIs) efficiently block viral replication in vitro and suppress viremia in patients. These small molecules bind to the IN active site causing it to disengage from the deoxyadenosine at the 3' end of viral DNA. The emergence of viral strains that are highly resistant to RAL underscores the pressing need to develop INSTIs with improved resistance profiles. Herein, we show that the candidate second-generation drug Dolutegravir (DTG, S/GSK1349572) effectively inhibits a panel of HIV 1 IN variants resistant to first-generation INSTIs. To elucidate the structural basis for the increased potency of DTG against RAL resistant INs, we determined crystal structures of wild type and mutant prototype foamy virus intasomes bound to this compound. The overall IN binding mode of DTG is strikingly similar to that of the tricyclic hydroxypyrrole MK2048. Both second-generation INSTIs occupy almost the same physical space within the IN active site and make contacts with the β4-α2 loop of the catalytic core domain. The extended linker region connecting the metal chelating core and the halobenzyl group of DTG allows it to enter farther into the pocket vacated by the displaced viral DNA base and to make more intimate contacts with viral DNA, compared to those made by RAL and other INSTIs. In addition, our structures suggest that DTG has the ability to subtly readjust its position and conformation in response to structural changes in the active sites of RAL-resistant INs.