I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[15444]
Abstract: Inorganic/organic hybrids have co-networks of inorganic and organic components, with the aim of obtaining synergy of the properties of those components. Here, a silica-gelatin sol-gel hybrid “ink” was directly 3D printed to produce 3D grid-like scaffolds, using a coupling agent, 3-glycidyloxypropyl)trimethoxysilane (GPTMS), to form covalent bonds between the silicate and gelatin co-networks. Scaffolds were printed with 1 mm strut separation, but the drying method affected the final architecture and properties. Freeze drying produced <40 μm struts and large ~700 μm channels. Critical point drying enabled strut consolidation and optimal mechanical properties, with ~160 μm struts and ~200 μm channels, which improved mechanical properties. This architecture was critical to cellular response: when chondrocytes were seeded on the scaffolds with 200 μm wide pore channels in vitro, collagen Type II matrix was preferentially produced (negligible amount of Type I or X were observed), indicative of hyaline-like cartilaginous matrix formation, but when pore channels were 700 μm wide, Type I collagen was prevalent. This was supported by Sox9 and Aggrecan expression. The scaffolds have potential for regeneration of articular cartilage regeneration, particularly in sports medicine cases.
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Feb 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19800]
Abstract: Cooperative ligand binding is an important phenomenon in biological systems where ligand binding influences the binding of another ligand at an alternative site of the protein via an intramolecular network of interactions. The underlying mechanisms behind cooperative binding remain poorly understood, primarily due to the lack of structural data of these ternary complexes. Using time-resolved fluorescence resonance energy transfer (TR-FRET) studies, we show that cooperative ligand binding occurs for RORγt, a nuclear receptor associated with the pathogenesis of autoimmune diseases. To provide the crucial structural insights, we solved 12 crystal structures of RORγt simultaneously bound to various orthosteric and allosteric ligands. The presence of the orthosteric ligand induces a clamping motion of the allosteric pocket via helices 4 to 5. Additional molecular dynamics simulations revealed the unusual mechanism behind this clamping motion, with Ala355 shifting between helix 4 and 5. The orthosteric RORγt agonists regulate the conformation of Ala355, thereby stabilizing the conformation of the allosteric pocket and cooperatively enhancing the affinity of the allosteric inverse agonists.
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Feb 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[25402]
Open Access
Abstract: For several centuries, microorganisms and enzymes have been used for many different applications. Although many enzymes with industrial applications have already been reported, different screening technologies, methods and approaches are constantly being developed in order to allow the identification of enzymes with even more interesting applications. In our work, we have performed data mining on the Chitinophaga sp. genome, a gram-negative bacterium isolated from a bacterial consortium of sugarcane bagasse isolated from an ethanol plant. The analysis of 8 Mb allowed the identification of the chtcp gene, previously annotated as putative Cht4039. The corresponding codified enzyme, denominated as ChtCP, showed the HEXXH conserved motif of family M32 from thermostable carboxypeptidases. After expression in E. coli, the recombinant enzyme was characterized biochemically. ChtCP showed the highest activity versus benziloxicarbonil Ala-Trp at pH 7.5, suggesting a preference for hydrophobic substrates. Surprisingly, the highest activity of ChtCP observed was between 55 °C and 75 °C, and 62% activity was still displayed at 100 °C. We observed that Ca2+, Ba2+, Mn2+ and Mg2+ ions had a positive effect on the activity of ChtCP, and an increase of 30 °C in the melting temperature was observed in the presence of Co2+. These features together with the structure of ChtCP at 1.2 Å highlight the relevance of ChtCP for further biotechnological applications.
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Feb 2021
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I19-Small Molecule Single Crystal Diffraction
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Diamond Proposal Number(s):
[15833]
Open Access
Abstract: The solid state supramolecular interactions of diketopyrrolopyrrole derivatives (DPPs) and their correlation with thin film optical properties are of particular interest because of the applications of these materials in organic electronics. In this study, we report the single crystal X-ray structures of several phenyl DPP derivatives, containing 4-methoxyphenyl, 4-hydroxyphenyl and 4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl aryl units, and show how subtle changes in the substituent chains at side or end positions of the chromophore can lead to very different packing. They are compared to their phenyl counterpart to explore how the nature of both the alkyl chain and the aryl unit influence the optical properties that have been measured in solid and solution states. Importantly, for the three families of N-substituted compounds studied, the structures are changed by the conformation of the molecules and are apparently dominated by crystal packing effects where edge-to-face interactions are favoured rather than π stacking, with only one of the compounds showing a flat form, promoted by intermolecular contacts between the aromatic regions. It is therefore possible that the twist between DPP and phenyl units in crystals of DPPs results from edge-to-face interactions (rather than steric interactions between the N-substituent and the protons attached to the aromatic ring) that might be overcome in more extended structures. Hydrogen bonding dominates the packing to generate chains of DPP units for phenol derivatives. Remote bulky groups do affect the core conformation. The emission of the materials as thin films is dominated by local effects in the packing of the materials that are unique for each case as the structures are distinct from one another. Charge mobility (as calculated from the crystal structures) is not favoured because of twisted conformations and large displacement, but the sometimes high emission and large Stokes shift could make the materials interesting for other purposes, such as light emitters.
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Feb 2021
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I19-Small Molecule Single Crystal Diffraction
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Estefanía
Tiburcio
,
Rossella
Greco
,
Marta
Mon
,
Jordi
Ballesteros-soberanas
,
Jesus
Ferrando-soria
,
Juan-carlos
Hernandez-garrido
,
Judit
Oliver-meseguer
,
Carlo
Marini
,
Mercedes
Boronat
,
Donatella
Armentano
,
Antonio
Leyva-perez
,
Emilio
Pardo
,
Miguel
López-haro
Diamond Proposal Number(s):
[18768, 22411]
Abstract: Metal single-atom catalysts (SACs) promise great rewards in terms of
metal atom efficiency. However, the requirement of particular conditions and
supports for their synthesis, together with the need of solvents and additives for
catalytic implementation, often precludes their use under industrially viable
conditions. Here, we show that palladium single atoms are spontaneously formed
after dissolving tiny amounts of palladium salts in neat benzyl alcohols, to catalyze
their direct aerobic oxidation to benzoic acids without ligands, additives, or solvents.
With this result in hand, the gram-scale preparation and stabilization of Pd SACs
within the functional channels of a novel methyl-cysteine-based metal−organic
framework (MOF) was accomplished, to give a robust and crystalline solid catalyst
fully characterized with the help of single-crystal X-ray diffraction (SCXRD). These
results illustrate the advantages of metal speciation in ligand-free homogeneous organic reactions and the translation into solid
catalysts for potential industrial implementation.
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Feb 2021
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[27906]
Open Access
Abstract: Multicomponent hydrogels offer a tremendous opportunity to prepare useful and exciting materials that cannot be accessed using a single component. Here, we describe an unusual multi‐component low molecular weight gelling system that exhibits pH‐responsive behavior involving cooperative hydrogen bonding between the components, allowing it to maintain a gel phase across a wide pH range. Unlike traditional acid‐triggered gels, our system undergoes a change in the underlying molecular packing and maintains the β‐sheet structure both at acidic and basic pH. We further establish that autonomous programming between these two gel states is possible by an enzymatic reaction which allows us to prepare gels with improved mechanical properties.
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Feb 2021
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I12-JEEP: Joint Engineering, Environmental and Processing
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Diamond Proposal Number(s):
[27011]
Abstract: A two-phase near-β titanium alloy (Ti–10V–2Fe–3Al, or Ti-1023) in its as-forged state is employed to illustrate the feasibility of a Bayesian framework to identify single-crystal elastic constants (SEC). High Energy X-ray diffraction (HE-XRD) obtained at the Diamond synchrotron source are used to characterize the evolution of lattice strains for various grain orientations during in situ specimen loading in the elastic regime. On the other hand, specimen behavior and grain deformation are estimated using the elastic self-consistent (ELSC) homogenization scheme. The XRD data and micromechanical modelling are revisited with a Bayesian framework. The effect of different material parameters (crystallographic and morphological textures, phase volume fraction) of the micromechanical model and the biases introduced by the XRD data on the identification of the SEC of the β phase are systematically investigated. In this respect, all the three cubic elastic constants of the β phase (
) in the Ti-1023 alloy have been derived with their uncertainties. The grain aspect ratio in the ELSC model, which is often not considered in the literature, is found to be an important parameter in affecting the identified SEC. The Bayesian inference suggests a high probability for non-spherical grains (aspect ratio of
):
. The uncertainty obtained by Bayesian approach lies in the range of ∼1-3 GPa for the shear modulus
, and ∼7 GPa for the shear modulus
, while it is significantly larger in the case of the bulk modulus
(∼17-24 GPa).
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Feb 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Shanshan
Zhou
,
Hussain
Bhukya
,
Nicolas
Malet
,
Peter J.
Harrison
,
Dean
Rea
,
Matthew J.
Belousoff
,
Hariprasad
Venugopal
,
Paulina K.
Sydor
,
Kathryn M.
Styles
,
Lijiang
Song
,
Max J.
Cryle
,
Lona M.
Alkhalaf
,
Vilmos
Fulop
,
Gregory L.
Challis
,
Christophe
Corre
Diamond Proposal Number(s):
[8359, 8388]
Abstract: Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture1. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear2. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by the binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR3. Here we report the X-ray crystal structure of an MmfR–AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding through the single-particle cryo-electron microscopy structure of an MmfR–operator complex. DNA binding and release assays with MmfR mutants and synthetic AHFCA analogues define the role of individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of actinobacterial hormones and their associated TFTRs in synthetic biology and in the discovery of new antibiotics.
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Feb 2021
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Krios II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[22238]
Open Access
Abstract: The ATP synthase complexes in mitochondria make the ATP required to sustain life by a rotary mechanism. Their membrane domains are embedded in the inner membranes of the organelle, and they dimerize via interactions between their membrane domains. The dimers form extensive chains along the tips of the cristae with the two rows of monomeric catalytic domains extending into the mitochondrial matrix at an angle to each other. Disruption of the interface between dimers by mutation affects the morphology of the cristae severely. By analysis of particles of purified dimeric bovine ATP synthase by cryo-electron microscopy, we have shown that the angle between the central rotatory axes of the monomeric complexes varies between ca. 76 and 95°. These particles represent active dimeric ATP synthase. Some angular variations arise directly from the catalytic mechanism of the enzyme, and others are independent of catalysis. The monomer–monomer interaction is mediated mainly by j subunits attached to the surface of wedge-shaped protein-lipid structures in the membrane domain of the complex, and the angular variation arises from rotational and translational changes in this interaction, and combinations of both. The structures also suggest how the dimeric ATP synthases might be interacting with each other to form the characteristic rows along the tips of the cristae via other interwedge contacts, molding themselves to the range of oligomeric arrangements observed by tomography of mitochondrial membranes, and at the same time allowing the ATP synthase to operate under the range of physiological conditions that influence the structure of the cristae.
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Feb 2021
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B21-High Throughput SAXS
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Yoshifumi
Itoh
,
Michael
Ng
,
Akira
Wiberg
,
Katsuaki
Inoue
,
Narumi
Hirata
,
Katiucia Batista Silva
Paiva
,
Noriko
Ito
,
Kim
Dzobo
,
Nanami
Sato
,
Valentina
Gifford
,
Yasuyuki
Fujita
,
Masaki
Inada
,
Dominic
Furniss
Abstract: Dupuytren's Disease (DD) is a common fibroproliferative disease of the palmar fascia. We previously identified a causal association with a non-synonymous variant (rs1042704, p.D273N) in MMP14 (encoding MT1-MMP). In this study, we investigated the functional consequences of this variant, and demonstrated that the variant MT1-MMP (MT1-N273) exhibits only 17% of cell surface collagenolytic activity compared to the ancestral enzyme (MT1-D273). Cells expressing both MT1-D273 and MT1-N273 in a 1:1 ratio, mimicking the heterozygous state, possess 38% of the collagenolytic activity compared to the cells expressing MT1-D273, suggesting that MT1-N273 acts in a dominant negative manner. Consistent with the above observation, patient-derived DD myofibroblasts with the alternate allele demonstrated around 30% of full collagenolytic activity detected in ancestral G/G genotype cells, regardless of the heterozygous (G/A) or homozygous (A/A) state. Small angle X-ray scattering analysis of purified soluble Fc-fusion enzymes allowed us to construct a 3D-molecular envelope of MT1-D273 and MT1-N273, and demonstrate altered flexibility and conformation of the ectodomains due to D273 to N substitution. Taking together, rs1042704 significantly reduces collagen catabolism in tissue, which tips the balance of homeostasis of collagen in tissue, contributing to the fibrotic phenotype of DD. Since around 30% of the worldwide population have at least one copy of the low collagenolytic alternate allele, further investigation of rs1042704 across multiple pathologies is needed.
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Feb 2021
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