I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[17221, 23620]
Open Access
Abstract: Type 4 filaments (T4F)—of which type 4 pili (T4P) are the archetype—are a superfamily of nanomachines nearly ubiquitous in prokaryotes. T4F are polymers of one major pilin, which also contain minor pilins whose roles are often poorly understood. Here, we complete the structure/function analysis of the full set of T4P pilins in the opportunistic bacterial pathogen Streptococcus sanguinis. We determined the structure of the minor pilin PilA, which is unexpectedly similar to one of the subunits of a tip-located complex of four minor pilins, widely conserved in T4F. We found that PilA interacts and dramatically stabilizes the minor pilin PilC. We determined the structure of PilC, showing that it is a modular pilin with a lectin module binding a subset of glycans prevalent in the human glycome, the host of S. sanguinis. Altogether, our findings support a model whereby the minor pilins in S. sanguinis T4P form a tip-located complex promoting adhesion to various host receptors. This has general implications for T4F.
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Jan 2023
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Abstract: Crystal engineering has, so far, exclusively focused on the development of advanced materials based on small organic molecules. We demonstrate how the cocrystallization of a polymer significantly enhances its thermal stability without compromising its mechanical flexibility, while isomorphous replacement of one of the cocrystal components enables the formation of solid solutions with melting points that can be readily fine-tuned over a practically wide temperature range. The results of this study credibly extend the scope of crystal engineering and cocrystallization from small molecules to polymers.
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Jan 2023
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I15-1-X-ray Pair Distribution Function (XPDF)
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Open Access
Abstract: Noncovalent interactions are essential in the formation and properties of a diverse range of materials. However, reliably identifying the noncovalent interactions remains challenging using conventional methods such as X-ray diffraction, especially in nanocrystalline, poorly crystalline or amorphous materials which lack long-range lattice periodicity. Here, we demonstrate the accurate determinations of deviations in the local structure and tilting of aromatic rings during the temperature-induced first order structural transition in the 1:1 adduct of 4,4’-bipyridinium squarate from low temperature form HAZFAP01 to high temperature HAZFAP07. This work demonstrates how Pair Distribution Function (PDF) analyses can improve our understanding of local structural deviations resulting from noncovalent bonds and guide the development of novel functional materials.
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Jan 2023
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Open Access
Abstract: X-ray photoemission and X-ray absorption spectroscopy are important techniques to characterize chemical bonding at surfaces and are often used to identify the strength and nature of adsorbate–substrate interactions. In this study, we judge the ability of X-ray spectroscopic techniques to identify different regimes of chemical bonding at metal–organic interfaces. To achieve this, we sample different interaction strength regimes in a comprehensive and systematic way by comparing two topological isomers, azulene and naphthalene, adsorbed on three metal substrates with varying reactivity, namely the (111) facets of Ag, Cu, and Pt. Using density functional theory, we simulate core-level binding energies and X-ray absorption spectra of the molecular carbon species. The simulated spectra reveal three distinct characteristics based on the molecule-specific spectral features which we attribute to types of surface chemical bonding with varying strength. We find that weak physisorption only leads to minor changes compared to the gas-phase spectra, weak chemisorption leads to charge transfer and significant spectral changes, and strong chemisorption leads to a loss of the molecule-specific features in the spectra. The classification we provide is aimed at assisting interpretation of experimental X-ray spectra for complex metal–organic interfaces.
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Jan 2023
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I03-Macromolecular Crystallography
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Open Access
Abstract: The biosynthetic enzyme, ForT, catalyses the formation of a C-C bond between 4-amino-1H-pyrazoledicarboxylic acid and MgPRPP to produce a C-nucleoside precursor of formycin A. The transformation catalysed by ForT is of chemical interest because it is one of only a few examples in which C-C bond formation takes place via an electrophilic substitution of a small, aromatic heterocycle. In addition, ForT is capable of discriminating between the aminopyrazoledicarboxylic acid and an analogue in which the amine is replaced by a hydroxyl group; a remarkable feat given the steric and electronic similarities of the two molecules. Here we report biophysical measurements, structural biology and quantum chemical calculations that provide a detailed molecular picture of ForT-catalysed C-C bond formation and the conformational changes that are coupled to catalysis. Our findings set the scene for employing engineered ForT variants in the biocatalytic production of novel, anti-viral C-nucleoside and C-nucleotide analogues.
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Jan 2023
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Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[22238, 17057]
Abstract: The molecular mode of action of biguanides, including the drug metformin, which is widely used in the treatment of diabetes, is incompletely characterized. Here, we define the inhibitory drug-target interaction(s) of a model biguanide with mammalian respiratory complex I by combining cryo–electron microscopy and enzyme kinetics. We interpret these data to explain the selectivity of biguanide binding to different enzyme states. The primary inhibitory site is in an amphipathic region of the quinone-binding channel, and an additional binding site is in a pocket on the intermembrane-space side of the enzyme. An independent local chaotropic interaction, not previously described for any drug, displaces a portion of a key helix in the membrane domain. Our data provide a structural basis for biguanide action and enable the rational design of medicinal biguanides.
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Jan 2023
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B18-Core EXAFS
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Diamond Proposal Number(s):
[30958]
Open Access
Abstract: The heterogeneous solid–gas reactions of crystals of [Rh(L2)(propene)][BArF4] (1, L2 = tBu2PCH2CH2PtBu2) with H2 and propene, 1-butene, propyne, or 1-butyne are explored by gas-phase nuclear magnetic resonance (NMR) spectroscopy under batch conditions at 25 °C. The temporal evolution of the resulting parahydrogen-induced polarization (PHIP) effects measures catalytic flux and thus interrogates the efficiency of catalytic pairwise para-H2 transfer, speciation changes in the crystalline catalyst at the molecular level, and allows for high-quality single-scan 1H, 13C NMR gas-phase spectra for the products to be obtained, as well as 2D-measurements. Complex 1 reacts with H2 to form dimeric [Rh(L2)(H)(μ-H)]2[BArF4]2 (4), as probed using EXAFS; meanwhile, a single-crystal of 1 equilibrates NMR silent para-H2 with its NMR active ortho isomer, contemporaneously converting into 4, and 1 and 4 each convert para-H2 into ortho-H2 at different rates. Hydrogenation of propene using 1 and para-H2 results in very high initial polarization levels in propane (>85%). Strong PHIP was also detected in the hydrogenation products of 1-butene, propyne, and 1-butyne. With propyne, a competing cyclotrimerization deactivation process occurs to afford [Rh(tBu2PCH2CH2PtBu2)(1,3,4-Me3C6H3)][BArF4], while with 1-butyne, rapid isomerization of 1-butyne occurs to give a butadiene complex, which then reacts with H2 more slowly to form catalytically active 4. Surprisingly, the high PHIP hydrogenation efficiencies allow hyperpolarization effects to be seen when H2 is taken directly from a regular cylinder at 25 °C. Finally, changing the chelating phosphine to Cy2PCH2CH2PCy2 results in initial high polarization efficiencies for propene hydrogenation, but rapid quenching of the catalyst competes to form the zwitterion [Rh(Cy2PCH2CH2PCy2){η6-(CF3)2(C6H3)}BArF3].
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Jan 2023
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19880]
Abstract: Rieske monooxygenases undertake complex catalysis integral to marine, terrestrial and human gut-ecosystems. Group-I to IV Rieske monooxygenases accept aromatic substrates and have well characterised catalytic mechanisms. Nascent to our understanding are Group-V members catalysing the oxidation/breakdown of quaternary ammonium substrates. Phylogenetic analysis of Group V highlights a cysteine residue-pair adjacent to the mononuclear Fe active site with no established role. Following our elucidation of the carnitine monooxygenase CntA structure, we probed the function of the cysteine pair Cys206/Cys209. Utilising biochemical and biophysical techniques, we found the cysteine residues do not play a structural role nor influence the electron transfer pathway, but rather are used in a non-stoichiometric role to ensure the catalytic iron centre remains in an Fe(II) state.
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Jan 2023
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I08-Scanning X-ray Microscopy beamline (SXM)
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Christina L.
Davis
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Ryan A.
Venturelli
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Alexander B.
Michaud
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Jon R.
Hawkings
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Amanda M.
Achberger
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Trista J.
Vick-Majors
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Brad E.
Rosenheim
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John E.
Dore
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August
Steigmeyer
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Joel D.
Barker
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Liane G.
Benning
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Matthew R.
Siegfried
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John C.
Priscu
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Brent C.
Christner
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Carlo
Barbante
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Mark
Bowling
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Justin
Burnett
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Timothy
Campbell
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Billy
Collins
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Cindy
Dean
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Dennis
Duling
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Helen A.
Fricker
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Alan
Gagnon
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Christopher
Gardner
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Dar
Gibson
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Chloe
Gustafson
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David
Harwood
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Jonas
Kalin
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Kathy
Kasic
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Ok-Sun
Kim
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Edwin
Krula
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Amy
Leventer
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Wei
Li
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W. Berry
Lyons
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Patrick
Mcgill
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James
Mcmanis
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David
Mcpike
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Anatoly
Mironov
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Molly
Patterson
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Graham
Roberts
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James
Rot
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Cathy
Trainor
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Martyn
Tranter
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John
Winans
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Bob
Zook
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Mark L.
Skidmore
Diamond Proposal Number(s):
[25828]
Open Access
Abstract: Ice streams that flow into Ross Ice Shelf are underlain by water-saturated sediments, a dynamic hydrological system, and subglacial lakes that intermittently discharge water downstream across grounding zones of West Antarctic Ice Sheet (WAIS). A 2.06 m composite sediment profile was recently recovered from Mercer Subglacial Lake, a 15 m deep water cavity beneath a 1087 m thick portion of the Mercer Ice Stream. We examined microbial abundances, used 16S rRNA gene amplicon sequencing to assess community structures, and characterized extracellular polymeric substances (EPS) associated with distinct lithologic units in the sediments. Bacterial and archaeal communities in the surficial sediments are more abundant and diverse, with significantly different compositions from those found deeper in the sediment column. The most abundant taxa are related to chemolithoautotrophs capable of oxidizing reduced nitrogen, sulfur, and iron compounds with oxygen, nitrate, or iron. Concentrations of dissolved methane and total organic carbon together with water content in the sediments are the strongest predictors of taxon and community composition. δ¹³C values for EPS (−25 to −30‰) are consistent with the primary source of carbon for biosynthesis originating from legacy marine organic matter. Comparison of communities to those in lake sediments under an adjacent ice stream (Whillans Subglacial Lake) and near its grounding zone provide seminal evidence for a subglacial metacommunity that is biogeochemically and evolutionarily linked through ice sheet dynamics and the transport of microbes, water, and sediments beneath WAIS.
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Jan 2023
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B21-High Throughput SAXS
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Laura C.
Clark
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Kate E.
Atkin
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Fiona
Whelan
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Andrew S.
Brentnall
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Gemma
Harris
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Aisling M.
Towell
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Johan P.
Turkenburg
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Yan
Liu
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Ten
Feizi
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Samuel C.
Griffiths
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Joan A.
Geoghegan
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Jennifer R.
Potts
Diamond Proposal Number(s):
[7864, 18598]
Open Access
Abstract: Staphylococcus aureus and Staphylococcus epidermidis are frequently associated with medical device infections that involve establishment of a bacterial biofilm on the device surface. Staphylococcal surface proteins Aap, SasG and Pls are members of the Periscope Protein class and have been implicated in biofilm formation and host colonisation; they comprise a repetitive region (“B region”) and an N-terminal host colonisation domain within the “A region”, predicted to be a lectin domain. Repetitive E-G5 domains (as found in Aap, SasG and Pls) form elongated ‘stalks’ that would vary in length with repeat number, resulting in projection of the N-terminal A domain variable distances from the bacterial cell surface. Here, we present the structures of the lectin domains within A regions of SasG, Aap and Pls and a structure of the Aap lectin domain attached to contiguous E-G5 repeats, suggesting the lectin domains will sit at the tip of the variable length rod. We demonstrate that these isolated domains (Aap, SasG) are sufficient to bind to human host desquamated nasal epithelial cells. Previously, proteolytic cleavage or a deletion within the A domain have been reported to induce biofilm formation; the structures suggest a potential link between these observations. Intriguingly, whilst the Aap, SasG and Pls lectin domains bind a metal ion, they lack the non-proline cis peptide bond thought to be key for carbohydrate binding by the lectin fold. This suggestion of non-canonical ligand binding should be a key consideration when investigating the host cell interactions of these bacterial surface proteins.
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Jan 2023
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