I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12346]
Open Access
Abstract: Crystallization is the bottleneck in macromolecular crystallography; even when a protein crystallises, crystal packing often influences ligand-binding and protein–protein interaction interfaces, which are the key points of interest for functional and drug discovery studies. The human hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) readily crystallises as a homotrimer, but with a sterically blocked active site. We explored strategies aimed at altering PHD2 crystal packing by protein modification and molecules that bind at its active site and elsewhere. Following the observation that, despite weak inhibition/binding in solution, succinamic acid derivatives readily enable PHD2 crystallization, we explored methods to induce crystallization without active site binding. Cyclic peptides obtained via mRNA display bind PHD2 tightly away from the active site. They efficiently enable PHD2 crystallization in different forms, both with/without substrates, apparently by promoting oligomerization involving binding to the C-terminal region. Although our work involves a specific case study, together with those of others, the results suggest that mRNA display-derived cyclic peptides may be useful in challenging protein crystallization cases.
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Dec 2020
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I13-1-Coherence
I18-Microfocus Spectroscopy
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Peter
Martin
,
Christopher P.
Jones
,
Stuart
Bartlett
,
Konstantin
Ignatyev
,
Dave
Megson-smith
,
Yukihiko
Sato
,
Silvia
Cipiccia
,
Darren J.
Batey
,
Christoph
Rau
,
Keisuke
Sueki
,
Tatsuya
Ishii
,
Junya
Igarashi
,
Kazuhiko
Ninomiya
,
Atsushi
Shinohara
,
Alison
Rust
,
Thomas B.
Scott
Diamond Proposal Number(s):
[24769, 19881]
Open Access
Abstract: The structural form and elemental distribution of material originating from different Fukushima Daiichi Nuclear Power Plant reactors (Units 1 and 3) is hereby examined to elucidate their contrasting release dynamics and the current in-reactor conditions to influence future decommissioning challenges. Complimentary computed X-ray absorption tomography and X-ray fluorescence data show that the two suites of Si-based material sourced from the different reactor Units have contrasting internal structure and compositional distribution. The known event and condition chronology correlate with the observed internal and external structures of the particulates examined, which suggest that Unit 1 ejecta material sustained a greater degree of melting than that likely derived from reactor Unit 3. In particular, we attribute the near-spherical shape of Unit 1 ejecta and their internal voids to there being sufficient time for surface tension to round these objects before the hot (and so relatively low viscosity) silicate melt cooled to form glass. In contrast, a more complex internal form associated with the sub-mm particulates invoked to originate from Unit 3 suggest a lower peak temperature, over a longer duration. Using volcanic analogues, we consider the structural form of this material and how it relates to its environmental particulate stability and the bulk removal of residual materials from the damaged reactors. We conclude that the brittle and angular Unit 3 particulate are more susceptible to further fragmentation and particulate generation hazard than the round, higher-strength, more homogenous Unit 1 material.
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Dec 2020
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[14263]
Open Access
Abstract: Nora virus, a virus of Drosophila, encapsidates one of the largest single-stranded RNA virus genomes known. Its taxonomic affinity is uncertain as it has a picornavirus-like cassette of enzymes for virus replication, but the capsid structure was at the time for genome publication unknown. By solving the structure of the virus, and through sequence comparison, we clear up this taxonomic ambiguity in the invertebrate RNA virosphere. Despite the lack of detectable similarity in the amino acid sequences, the 2.7 Å resolution cryoEM map showed Nora virus to have T = 1 symmetry with the characteristic capsid protein β-barrels found in all the viruses in the Picornavirales order. Strikingly, α-helical bundles formed from the extended C-termini of capsid protein VP4B and VP4C protrude from the capsid surface. They are similar to signalling molecule folds and implicated in virus entry. Unlike other viruses of Picornavirales, no intra-pentamer stabilizing annulus was seen, instead the intra-pentamer stability comes from the interaction of VP4C and VP4B N-termini. Finally, intertwining of the N-termini of two-fold symmetry-related VP4A capsid proteins and RNA, provides inter-pentamer stability. Based on its distinct structural elements and the genetic distance to other picorna-like viruses we propose that Nora virus, and a small group of related viruses, should have its own family within the order Picornavirales.
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Nov 2020
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I08-Scanning X-ray Microscopy beamline (SXM)
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Diamond Proposal Number(s):
[20900]
Open Access
Abstract: We report on the detection of primordial organic matter within the carbonaceous chondrite Maribo that is distinct from the majority of organics found in extraterrestrial samples. We have applied high-spatial resolution techniques to obtain C-N isotopic compositions, chemical, and structural information of this material. The organic matter is depleted in 15N relative to the terrestrial value at around δ15N ~ -200‰, close to compositions in the local interstellar medium. Morphological investigations by electron microscopy revealed that the material consists of µm- to sub-µm-sized diffuse particles dispersed within the meteorite matrix. Electron energy loss and synchrotron X-ray absorption near-edge structure spectroscopies show that the carbon functional chemistry is dominated by aromatic and C=O bonding environments similar to primordial organics from other carbonaceous chondrites. The nitrogen functional chemistry is characterized by C-N double and triple bonding environments distinct from what is usually found in 15N-enriched organics from aqueously altered carbonaceous chondrites. Our investigations demonstrate that Maribo represents one of the least altered CM chondrite breccias found to date and contains primordial organic matter, probably originating in the interstellar medium.
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Nov 2020
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I15-1-X-ray Pair Distribution Function (XPDF)
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Diamond Proposal Number(s):
[18638, 23164]
Open Access
Abstract: Biogenic and synthetic hydroxyapatites are confounding materials whose properties remain uncertain, even after years of study. Pair distribution function (PDF) analysis was applied to hydroxyapatites in the 1970’s and 1980’s, but this area of research has not taken full advantage of the relatively recent advances in synchrotron facilities. Here, synchrotron X-ray PDF analysis is compared to techniques commonly used to characterise hydroxyapatite (such as wide angle X-ray scattering, Fourier-transform infrared spectroscopy and thermogravimetric analysis) for a range of biogenic and synthetic hydroxyapatites with a wide range of carbonate substitution. Contributions to the pair distribution function from collagen, carbonate and finite crystallite size were examined through principal component analysis and comparison of PDFs. Noticeable contributions from collagen were observed in biogenic PDFs when compared to synthetic PDFs (namely r < 15 Å), consistent with simulated PDFs of collagen structures. Additionally, changes in local structure were observed for PDFs of synthetic hydroxyapatites with differing carbonate content, notably in features near 4 Å, 8 Å and 19 Å. Regression models were generated to predict carbonate substitution from peak position within the PDFs.
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Nov 2020
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I13-2-Diamond Manchester Imaging
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Berit
Zeller-plumhoff
,
Anna-lena
Robisch
,
Daniele
Pelliccia
,
Elena
Longo
,
Hanna
Slominska
,
Alexander
Hermann
,
Martin
Krenkel
,
Malte
Storm
,
Yuri
Estrin
,
Regine
Willumeit-römer
,
Tim
Salditt
,
Dmytro
Orlov
Diamond Proposal Number(s):
[21697]
Open Access
Abstract: Magnesium and its alloys attract increasingly wide attention in various fields, ranging from transport to medical solutions, due to their outstanding structural and degradation properties. These properties can be tailored through alloying and thermo-mechanical processing, which is often complex and multi-step, thus requiring in-depth analysis. In this work, we demonstrate the capability of synchrotron-based nanotomographic X-ray imaging methods, namely holotomography and transmission X-ray microscopy, for the quantitative 3D analysis of the evolution of intermetallic precipitate (particle) morphology and distribution in magnesium alloy Mg–5.78Zn–0.44Zr subjected to a complex multi-step processing. A rich history of variation of the intermetallic particle structure in the processed alloy provided a testbed for challenging the analytical capabilities of the imaging modalities studied. The main features of the evolving precipitate structure revealed earlier by traditional light and electron microscopy methods were confirmed by the 3D techniques of synchrotron-based X-ray imaging. We further demonstrated that synchrotron-based X-ray imaging enabled uncovering finer details of the variation of particle morphology and number density at various stages of processing—above and beyond the information provided by visible light and electron microscopy.
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Sep 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[17212]
Open Access
Abstract: Complex polyketides of bacterial origin are biosynthesised by giant assembly-line like megaenzymes of the type 1 modular polyketide synthase (PKS) class. The trans-AT family of modular PKSs, whose biosynthetic frameworks diverge significantly from those of the archetypal cis-AT type systems represent a new paradigm in natural product enzymology. One of the most distinctive enzymatic features common to trans-AT PKSs is their ability to introduce methyl groups at positions β to the thiol ester in the growing polyketide chain. This activity is achieved through the action of a five protein HCS cassette, comprising a ketosynthase, a 3-hydroxy-3-methylglutaryl-CoA synthase, a dehydratase, a decarboxylase and a dedicated acyl carrier protein. Here we report a molecular level description, achieved using a combination of X-ray crystallography, in vitro enzyme assays and site-directed mutagenesis, of the bacillaene synthase dehydratase/decarboxylase enzyme couple PksH/PksI, responsible for the final two steps in β-methyl branch installation in this trans-AT PKS. Our work provides detailed mechanistic insight into this biosynthetic peculiarity and establishes a molecular framework for HCS cassette enzyme exploitation and manipulation, which has future potential value in guiding efforts in the targeted synthesis of functionally optimised ‘non-natural’ natural products.
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Sep 2020
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I22-Small angle scattering & Diffraction
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Shaocheng
Ma
,
En Lin
Goh
,
Tabitha
Tay
,
Crispin C.
Wiles
,
Oliver
Boughton
,
John H.
Churchwell
,
Yong
Wu
,
Angelo
Karunaratne
,
Rajarshi
Bhattacharya
,
Nick
Terrill
,
Justin P.
Cobb
,
Ulrich
Hansen
,
Richard L.
Abel
Diamond Proposal Number(s):
[17664]
Open Access
Abstract: Nanoscale mineralized collagen fibrils may be important determinants of whole-bone mechanical properties and contribute to the risk of age-related fractures. In a cross-sectional study nano- and tissue-level mechanics were compared across trabecular sections from the proximal femora of three groups (n = 10 each): ageing non-fractured donors (Controls); untreated fracture patients (Fx-Untreated); bisphosphonate-treated fracture patients (Fx-BisTreated). Collagen fibril, mineral and tissue mechanics were measured using synchrotron X-Ray diffraction of bone sections under load. Mechanical data were compared across groups, and tissue-level data were regressed against nano. Compared to controls fracture patients exhibited significantly lower critical tissue strain, max strain and normalized strength, with lower peak fibril and mineral strain. Bisphosphonate-treated exhibited the lowest properties. In all three groups, peak mineral strain coincided with maximum tissue strength (i.e. ultimate stress), whilst peak fibril strain occurred afterwards (i.e. higher tissue strain). Tissue strain and strength were positively and strongly correlated with peak fibril and mineral strains. Age-related fractures were associated with lower peak fibril and mineral strain irrespective of treatment. Indicating earlier mineral disengagement and the subsequent onset of fibril sliding is one of the key mechanisms leading to fracture. Treatments for fragility should target collagen-mineral interactions to restore nano-scale strain to that of healthy bone.
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Aug 2020
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Silvia
Boccato
,
Raffaella
Torchio
,
Simone
Anzellini
,
Eglantine
Boulard
,
François
Guyot
,
Tetsuo
Irifune
,
Marion
Harmand
,
Innokenty
Kantor
,
Francesca
Miozzi
,
Paraskevas
Parisiades
,
Angelika D.
Rosa
,
Daniele
Antonangeli
,
Guillaume
Morard
Open Access
Abstract: X-ray absorption spectroscopy (XAS) is a widely used technique to probe the local environment around specific atomic species. Applied to samples under extreme pressure and temperature conditions, XAS is sensitive to phase transitions, including melting, and allows gathering insights on compositional variations and electronic changes occurring during such transitions. These characteristics can be exploited for studies of prime interest in geophysics and fundamental high-pressure physics. Here, we investigated the melting curve and the eutectic composition of four geophysically relevant iron binary systems: Fe–C, Fe–O, Fe–S and Fe–Si. Our results show that all these systems present the same spectroscopic signatures upon melting, common to those observed for other pure late 3d transition metals. The presented melting criterion seems to be general for late 3d metals bearing systems. Additionally, we demonstrate the suitability of XAS to extract melt compositional information in situ, such as the evolution of the concentration of light elements with increasing temperature. Diagnostics presented in this work can be applied to studies over an even larger pressure range exploiting the upgraded synchrotron machines, and directly transferred to time-resolved extreme condition studies using dynamic compression (ns) or fast laser heating (ms).
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Jul 2020
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
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Open Access
Abstract: Synaptic adhesion molecules play an important role in the formation, maintenance and refinement of neuronal connectivity. Recently, several leucine rich repeat (LRR) domain containing neuronal adhesion molecules have been characterized including netrin G-ligands, SLITRKs and the synaptic adhesion-like molecules (SALMs). Dysregulation of these adhesion molecules have been genetically and functionally linked to various neurological disorders. Here we investigated the molecular structure and mechanism of ligand interactions for the postsynaptic SALM3 adhesion protein with its presynaptic ligand, receptor protein tyrosine phosphatase σ (PTPσ). We solved the crystal structure of the dimerized LRR domain of SALM3, revealing the conserved structural features and mechanism of dimerization. Furthermore, we determined the complex structure of SALM3 with PTPσ using small angle X-ray scattering, revealing a 2:2 complex similar to that observed for SALM5. Solution studies unraveled additional flexibility for the complex structure, but validated the uniform mode of action for SALM3 and SALM5 to promote synapse formation. The relevance of the key interface residues was further confirmed by mutational analysis with cellular binding assays and artificial synapse formation assays. Collectively, our results suggest that SALM3 dimerization is a pre-requisite for the SALM3-PTPσ complex to exert synaptogenic activity.
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Jul 2020
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