NONE-No attached Diamond beamline
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Abstract: The human pathogen Streptococcus pneumoniae expresses neuraminidase proteins that cleave sialic acids from complex carbohydrates. The pneumococcus genome encodes up to three neuraminidase proteins that have been shown to be important virulence factors. Here, we report the first structure of a neuraminidase from S. pneumoniae: the crystal structure of NanB in complex with its reaction product 2,7-anhydro-Neu5Ac. Our structural data, together with biochemical analysis, establish NanB as an intramolecular trans-sialidase with strict specificity towards ?2-3 linked sialic acid substrates. In addition, we show that NanB differs in its substrate specificity from the other pneumococcal neuraminidase NanA.
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Sep 2008
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I04-Macromolecular Crystallography
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Abstract: Radiation damage limits the amount of time that a macromolecular crystal diffracts
when exposed to X-ray irradiation. Various aspects of this problem were investigated in
this work along with methods development for quantitative determination of the metal
content of proteins and cells. In particular, the iron storage protein ferritin was used to
extend current understanding of the changes that occur in macromolecular crystals held at
100 K during exposure to X-rays. The characteristics of the expansion in unit cell volume
with absorbed dose and with temperature were examined and found to be distinguishable. A
comparison between the iron loaded (holo) and the iron void (apo) forms of ferritin allowed
the contribution to the damage of the absorbing iron core to be determined.
Glutaraldehyde cross-linking was also tested as a method to increase radiation
resistance.
Experiments were carried out to establish the room temperature dose limit of protein
crystals and compare this with the dose limit measured at cryotemperatures. Surprisingly, it
was found that there was a significant decrease in radiation damage at higher dose rates.
An online microspectrophotometer at the European Synchrotron Radiation Facility was
used to screen large numbers of potential radioprotectants at 100 K by monitoring the
absorbance spectra of the 400 nm peak associated with a disulphide radical anion formed
by X-ray cleavage of a disulphide bond. Ascorbate, quinone, TEMP and DTT were
identified as effective radioprotectants. The most promising radioprotectant (ascorbate) was
put into co-crystallisation and soaking trials with lysozyme to measure its protective effect
against X-ray induced damage at 100 K and room temperature.
For high throughput trace element detection, XRF was investigated, but calibration for
proteins proved problematic.
MicroPIXE was used for trace element mapping of wild type and Niemann Pick Type-
C (a neurodegenerative disorder) cells to identify alterations in trace element composition.
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Sep 2008
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NONE-No attached Diamond beamline
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Abstract: Helical oligomers of beta-peptides represent a particularly promising type of building block for directed assembly of organic nanostructures because the helical secondary structure can be designed to be very stable and because control of the beta-amino acid sequence can lead to precise patterning of chemical functional groups over the helix surfaces. In this paper, we report the use of small angle x-ray scattering measurements (SAXS) to characterize nanostructures formed by the directed assembly of beta-peptide A with sequence H(2)N-beta(3)hTyr-beta(3)hLys-beta(3)hPhe-ACHC-beta(3)hPhe-ACHC-beta(3)hPhe-beta(3)hLys-ACHC-ACHC-beta(3)hPhe-beta(3)hLys-CONH(2). Whereas prior cryo-TEM studies have revealed the presence of nanofibers in aqueous solutions of beta-peptide A, SAXS measurements from the nanofibers were not well-fit by a form factor model describing solid nanofibers. An improved fit to the scattering data at high q was obtained by using a form factor model describing a cylinder with a hollow center and radial polydispersity. When combined with a structure factor calculated from the polymer reference interaction site model (PRISM) theory, the scattered intensity of x-rays measured over the entire q range was well described by the model. Analysis of our SAXS data suggests a model in which individual beta-peptides assemble to form long cylindrical nanofibers with a hollow core radius of 15 A (polydispersity of 21%) and a shell thickness of 20 A. This model is supported by negative stain transmission electron microscopy. (C) 2008 American Institute of Physics.
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Sep 2008
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NONE-No attached Diamond beamline
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Abstract: Homoleptic gallium tris(alkoxides) [Ga(OR)3]2 were prepared by the reaction of [Ga(NMe2)3]2 (1) and excess ROH (R = CH2CH2NMe2 (2), CH(CH3)CH2NMe2 (3), C(CH3)2CH2OMe (4), CH2CH2OMe (5)) in toluene at room temperature. Compounds 2–5 were isolated as colourless oils. The side-products, [Ga(OCH2CH2NMe2)2Cl] (6), [Ga(OCH(CH3)CH2NMe2)2Cl] (7) and [Ga(OC(CH3)2CH2OMe)Cl2]2 (8) were also isolated in low yield during the synthesis of 2, 3 and 4, respectively. However, compounds 6 and 7 were also prepared directly from the reaction of [Ga(NMe2)2Cl] and 2 equivalents of ROH (6, R = CH2CH2NMe2; 7, R = CH(CH3)CH2NMe2). Similarly, compound 8 was isolated from the reaction of [Ga(NMe2)Cl2] and 1 equivalent of HOC(CH3)2CH2OMe. Single crystal X-ray crystallography showed that the gallium bis(alkoxides) (6 and 7) are monomeric in the solid state with the gallium centre adopting a distorted trigonal bipyramidal geometry. In contrast, the gallium mono(alkoxide) 8 is dimeric
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Sep 2008
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NONE-No attached Diamond beamline
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Abstract: The major chlorophyll (Chl) a/b complexes of photosystem II (LHCIIb), in addition to their primary light-harvesting function, play key roles in the organization of the granal ultrastructure of the thylakoid membranes and in various regulatory processes. These functions depend on the structural stability and flexibility of the complexes. The lumenal side of LHCIIb is exposed to broadly variable pH environments, due to the build-up and decay of the pH gradient during photosynthesis. Therefore, the negatively charged amino acids in the lumenal loop might be of paramount importance for adjusting the structure and functions of LHCIIb. In order to clarify the structural roles of these residues, we investigated the pigment stoichiometries, absorption, linear and circular dichroism spectra of the reconstituted LHCIIb complexes, in which the negatively charged amino acids in the lumenal loop were exchanged to neutral ones (E94G, E107V and D111V). The mutations influenced the pigment binding and the molecular architecture of the complexes. Exchanging E94 to G destabilized the 310 helix in the lumenal loop structure and led to an acquired pH sensitivity of the LHCIIb structure. We conclude that these amino acids are important not only for pigment binding in the complexes, but also in stabilizing the conformation of LHCIIb at different pHs.
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Sep 2008
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NONE-No attached Diamond beamline
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Abstract: We have performed a resonant x-ray scattering RXS study near the Co K edge on a single crystal of Ca3Co2O6. In the magnetically ordered phase a new class of weak reflections appears at the magnetic propagation vector 1, 3 , 1, 3 , 1, 3. These new reflections allow direct access to the dipolar-quadrupolar E1E2 scattering channel. The theoretical possibility of observing isolated E1E2 electromagnetic multipoles has attracted a lot of interest in recent years. Unfortunately in many systems of interest, parity even and parity odd tensor contributions occur at the same positions in reciprocal space. We demonstrate that in Ca3Co2O6 it is possible to completely separate the parity even from the parity odd terms. The possibility of observing such terms even in globally centrosymmetric systems using RXS has been investigated theoretically; Ca3Co2O6 allows a symmetry based separation of this contribution.
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Sep 2008
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NONE-No attached Diamond beamline
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Abstract: A previous paper [Nave & Hill (2005). J. Synchrotron Rad. 12, 299-303] examined the possibility of reduced radiation damage for small crystals (10 µm and below in size) under conditions where the photoelectrons could escape from the sample. The conclusion of this paper was that higher-energy radiation (e.g. 40 keV) could offer an advantage as the photoelectron path length was greater and less energy would be deposited in the crystal. This paper refines these calculations further by including the effects of energy deposited owing to Compton scattering and the energy difference between the incident photon and the emitted photoelectron. An estimate is given for the optimum wavelength for collecting data from a protein crystal of a given size and composition. Another way of reducing radiation damage from a protein crystal is to collect data with a very short pulsed X-ray source where a single image can be obtained before subsequent radiation damage occurs. A comparison of this approach compared with the use of shorter wavelengths is made.
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Sep 2008
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I04-Macromolecular Crystallography
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Open Access
Abstract: Adhesive PfEMP1 proteins are displayed on the surface of malaria-infected red blood cells. They play a critical role in the disease, tethering infected cells away from destruction by the spleen and causing many severe symptoms. A molecular understanding of how these domains maintain their binding properties while evading immune detection will be important in developing therapeutics. In malaria of pregnancy, domains from the var2csa-encoded PfEMP1 protein interact with chondroitin sulfate on the placenta surface. This causes accumulation of infected red blood cells, leading to placental inflammation and block of blood flow to the developing fetus. This is associated with maternal anemia, low birth weight, and premature delivery and can lead to the death of mother and child. Here I present the structure of the chondroitin sulfate-binding DBL3X domain from a var2csa-encoded PfEMP1 protein. The domain adopts a fold similar to malarial invasion proteins, with extensive loop insertions. One loop is flexible in the unliganded structure but observed in the presence of sulfate or disaccharide, where it completes a sulfate-binding site. This loop, and others surrounding this putative carbohydrate-binding site, are flexible and polymorphic, perhaps protecting the binding site from immune detection. This suggests a model for how the domain maintains ligand binding while evading the immune response and will guide future drug and vaccine development.
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Aug 2008
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I03-Macromolecular Crystallography
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Abstract: Schizosaccharomyces pombe Crb2 is a checkpoint mediator required for the cellular response to DNA damage. Like human 53BP1 and Saccharomyces cerevisiae Rad9 it contains Tudor2 and BRCT2 domains. Crb2-Tudor2 domain interacts with methylated H4K20 and is required for recruitment to DNA dsDNA breaks. The BRCT2 domain is required for dimerization, but its precise role in DNA damage repair and checkpoint signaling is unclear. The crystal structure of the Crb2–BRCT2 domain, alone and in complex with a phosphorylated H2A.1 peptide, reveals the structural basis for dimerization and direct interaction with ?-H2A.1 in ionizing radiation-induced foci (IRIF). Mutational analysis in vitro confirms the functional role of key residues and allows the generation of mutants in which dimerization and phosphopeptide binding are separately disrupted. Phenotypic analysis of these in vivo reveals distinct roles in the DNA damage response. Dimerization mutants are genotoxin sensitive and defective in checkpoint signaling, Chk1 phosphorylation, and Crb2 IRIF formation, while phosphopeptide-binding mutants are only slightly sensitive to IR, have extended checkpoint delays, phosphorylate Chk1, and form Crb2 IRIF. However, disrupting phosphopeptide binding slows formation of ssDNA-binding protein (Rpa1/Rad11) foci and reduces levels of Rad22(Rad52) recombination foci, indicating a DNA repair defect.
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Aug 2008
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I03-Macromolecular Crystallography
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Open Access
Abstract: Vaccinia virus (VACV), the prototype poxvirus, encodes numerous proteins that modulate the host response to infection. Two such proteins, B14 and A52, act inside infected cells to inhibit activation of NF-κB, thereby blocking the production of pro-inflammatory cytokines. We have solved the crystal structures of A52 and B14 at 1.9 Å and 2.7 Å resolution, respectively. Strikingly, both these proteins adopt a Bcl-2–like fold despite sharing no significant sequence similarity with other viral or cellular Bcl-2–like proteins. Unlike cellular and viral Bcl-2–like proteins described previously, A52 and B14 lack a surface groove for binding BH3 peptides from pro-apoptotic Bcl-2–like proteins and they do not modulate apoptosis. Structure-based phylogenetic analysis of 32 cellular and viral Bcl-2–like protein structures reveals that A52 and B14 are more closely related to each other and to VACV N1 and myxoma virus M11 than they are to other viral or cellular Bcl-2–like proteins. This suggests that a progenitor poxvirus acquired a gene encoding a Bcl-2–like protein and, over the course of evolution, gene duplication events have allowed the virus to exploit this Bcl-2 scaffold for interfering with distinct host signalling pathways.
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Aug 2008
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