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Abstract: Synchrotron light sources create intense x-ray beams. Many synchrotron beamlines require an x-ray spot with an intensity profile as specified by the user. Moreover, many researchers are striving for x-ray spots of width <1?m. Bimorph mirrors, first tested at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France,1, 2 are now increasingly used to focus x-rays at synchrotron beamlines to achieve these goals. Because Diamond Light Source (the UK's national synchrotron facility, in Oxfordshire) uses more bimorph mirrors than any other synchrotron, we have automated the optimization of these complex optics.

Open Access

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Abstract: DNA polymerase theta (Polθ) has been identified as a crucial alternative non-homologous end-joining factor in mammalian cells. Polθ is upregulated in a range of cancer cell types defective in homologous recombination, and knockdown has been shown to inhibit cell survival in a subset of these, making it an attractive target for cancer treatment. We present crystal structures of the helicase domain of human Polθ in the presence and absence of bound nucleotides, and a characterization of its DNA-binding and DNA-stimulated ATPase activities. Comparisons with related helicases from the Hel308 family identify several unique features. Polθ exists as a tetramer both in the crystals and in solution. We propose a model for DNA binding to the Polθ helicase domain in the context of the Polθ tetramer, which suggests a role for the helicase domain in strand annealing of DNA templates for subsequent processing by the polymerase domain.

Open Access

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Abstract: Streptococcus groups A and B cause serious infections, including early onset sepsis and meningitis in newborns. Rib domain-containing surface proteins are found associated with invasive strains and elicit protective immunity in animal models. Yet, despite their apparent importance in infection, the structure of the Rib domain was previously unknown. Structures of single Rib domains of differing length reveal a rare case of domain atrophy through deletion of 2 core antiparallel strands, resulting in the loss of an entire sheet of the β-sandwich from an immunoglobulin-like fold. Previously, observed variation in the number of Rib domains within these bacterial cell wall-attached proteins has been suggested as a mechanism of immune evasion. Here, the structure of tandem domains, combined with molecular dynamics simulations and small angle X-ray scattering, suggests that variability in Rib domain number would result in differential projection of an N-terminal host-colonization domain from the bacterial surface. The identification of 2 further structures where the typical B-D-E immunoglobulin β-sheet is replaced with an α-helix further confirms the extensive structural malleability of the Rib domain.

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Abstract: The cation-independent mannose 6-phosphate/ Insulin-like growth factor-2 receptor (CI-MPR/ IGF2R) is a ~300 kDa transmembrane glycoprotein that is critical for intracellular protein trafficking, lysosome biogenesis and regulation of cell growth. The extracellular region consists of fifteen domains homologous to one another including mannose 6-phosphate (M6P) binding domains (D) 3, 5, 9 and 15, and IGF2 binding domain 11. To date, high-resolution structures have been determined for human D1-5 and D11-14. Although low resolution cryoEM structures of bovine CI-MPR have recently been determined at pH 4.5 and 7.4, a structure of the full extracellular region of human CI-MPR has yet to be determined. This thesis details structural studies on the central, uncharacterised region of human CI-MPR with particular focus on the elusive, specific and high-affinity M6P binding domain, D9. A modular approach has resulted in crystal structures of human CI-MPR D8, D9-10 and D7-11. D9-10 forms a rigid homodimer stabilised by a bridging N-linked glycan and maintained in D7-11, whereby two penta-domains intertwine to form a dimeric helical-type coil. Remarkably the D7-11 structure closely matches an IGF2 bound state of the receptor, suggesting this may be an intrinsically stable conformation at neutral pH. Interdomain clusters of histidine and proline residues at the D9-10 and D11-12 interfaces may impart receptor rigidity and play a role in cargo dissociation and structural rearrangement at low pH. A parallel project took an iterative, structure-based approach to engineer a synthetic lectin. The hydrophobic IGF2 binding site of CI-MPR D11 was mutated by site-directed mutagenesis to resemble the positively charged M6P binding sites of D3 and 9. Following preparation, D11 mutants were screened by 1H-15N HSQC NMR for binding to monosaccharides. Although chemical shift perturbations were observed following addition of M6P, further work is required to validate these preliminary results.

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Abstract: Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water–air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.