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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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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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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.

Open Access

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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.

Open Access

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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.