B21-High Throughput SAXS
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Diamond Proposal Number(s):
[28516]
Abstract: Mutations in the E3 ubiquitin ligase Parkin gene have been linked to early onset Parkinson’s disease. Besides many other roles, Parkin is involved in clearance of damaged mitochondria via mitophagy—a process of particular importance in dopaminergic neurons. Upon mitochondrial damage, Parkin accumulates at the outer mitochondrial membrane and is activated, leading to ubiquitination of many mitochondrial substrates and recruitment of mitophagy effectors. While the activation mechanisms of autoinhibited Parkin have been extensively studied, it remains unknown how Parkin recognizes its substrates for ubiquitination. Here, we characterize a conserved region in the flexible linker between the Ubl and RING0 domains of Parkin, which is indispensable for Parkin interaction with the mitochondrial GTPase Miro1. Our results may explain fast kinetics of Miro1 ubiquitination by Parkin in recombinant assays and provide a biochemical explanation for Miro1-dependent Parkin recruitment to the mitochondrial membrane observed in cells. Our findings are important for understanding mitochondrial homeostasis and may inspire new therapeutic avenues for Parkinson’s disease.
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Aug 2025
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[33542]
Open Access
Abstract: We report on the lyotropic phase behaviour of fully-hydrated mixtures of α-tocopherol (α-toc) with the unsaturated phospholipid dioleoyl phosphatidylcholine (DOPC), as studied by synchrotron small-angle x-ray diffraction. Increasing amounts of α-toc progressively swell the layer spacing of the fluid lamellar Lα phase of DOPC, and then induce a transition to an inverse hexagonal HII phase. Low-resolution electron density profiles show that this increase is largely due to an increased thickness of the bilayer, with little change in the water layer thickness. In the range 30 – 50 mol% α-toc, additional weak low-angle peaks were observed, whose characteristic ratios are in agreement with the presence of swollen inverse bicontinuous cubic phases of spacegroups Im3m / Pn3m. This research has applications both in the biological field and for industrial product development. We show that the effect of α-toc addition in DOPC membranes has some similarities to that of cholesterol by stabilizing inverse curvature structures, which play crucial roles in cell division, membrane trafficking and endocytosis. Concerning industrial applications, the stabilization of inverted hexagonal (HII) and swollen bicontinuous cubic phases offers the opportunity to develop new delivery systems.
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Aug 2025
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B21-High Throughput SAXS
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Beatrice
Mercorelli
,
Alessandro
Bazzacco
,
Michela
Eleuteri
,
Samuele
Di Cristofano
,
Jenny
Desantis
,
Alessandro
Paciaroni
,
Maria Grazia
Ortore
,
Sara
Tuci
,
Francesco
Spinozzi
,
Domenico
Raimondo
,
Laura
Goracci
,
Gabriele
Cruciani
,
Arianna
Loregian
Diamond Proposal Number(s):
[29982]
Open Access
Abstract: SARS-CoV-2 Main protease (Mpro) is the most explored coronavirus antiviral target, being most antivirals approved or under development protease inhibitors. Mpro is active as a dimer and the molecular details of its maturation are poorly understood. Some compounds that crystallize at the dimerization interface rather than at the catalytic pocket have been proposed as allosteric inhibitors. Here, we characterize a series of novel compounds starting from a scaffold identified by an in silico screening for Mpro catalytic pocket. Several compounds showed anti-SARS-CoV-2 activity in infected cells, but they did not inhibit Mpro in vitro. Time-of-addition studies pointed to a stage compatible with Mpro targeting. Molecular modelling studies suggested that compounds 1 and 11 bind Mpro similarly to the allosteric inhibitor AT7519. Small-angle X-ray scattering studies revealed that 1 and 11 strongly shift Mpro equilibrium to the monomeric form, while the allosteric inhibitor pelitinib and the catalytic inhibitors nirmatrelvir and GC376 stabilize the dimer. Compounds 1 and 11 inhibited Mpro proteolytic activity in SARS-CoV-2 infected cells acting as allosteric inhibitors that stabilize the monomeric form. In conclusion, we validated an allosteric site in Mpro that could be exploited for the development of effective anti-SARS-CoV-2 antivirals targeting Mpro with a novel mechanism.
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Aug 2025
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
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Muhammad
Saleem
,
Chan
Li
,
Bubacarr G.
Kaira
,
Alexander K.
Brown
,
Monika
Pathak
,
Shabir
Najmudin
,
Nathan
Cowieson
,
Ingrid
Dreveny
,
Clare
Wilson
,
Aleksandr
Shamanaev
,
David
Gailani
,
Stephanie A.
Smith
,
James H.
Morrissey
,
Helen
Philippou
,
Jonas
Emsley
Diamond Proposal Number(s):
[37038]
Open Access
Abstract: Factor XIIa (FXIIa) is generated from its zymogen factor XII (FXII) by contact with polyanions such as inorganic polyphosphates. FXIIa cleaves the substrates prekallikrein and factor XI, triggering inflammatory cascades and plasma coagulation. From the N-terminus, FXII has fibronectin type II (FnII), epidermal growth factor-1 (EGF1), fibronectin type I (FnI), EGF2 and kringle domains. The N-terminal domains of FXII mediate polyanion and Zn2+ binding. To understand how ligand binding to polyanions and Zn2+ is coordinated across multiple domains, we determined the crystal structure of recombinant FXII domains 1–5 (FXIIHC5) to 3.4 Å resolution. A separate crystal structure of the isolated FXII FnII domain at 1.2 Å resolution revealed two bound Zn2+ ions. In FXIIHC5 a head-to-tail interaction is formed between the FnII and kringle domains, co-localizing the lysine-binding sites of the kringle domain and the cation-binding site of the FnII domain. Two FXIIHC5 monomers interlock, burying a large surface area of 2067 Å2, such that two kringle domains point outwards separated by a distance of 20 Å. The polyanion-binding site in the EGF1 domain is localized onto a plane together with the FnII and FnI domains. Using native mass spectrometry, we detected a major FXIIHC5 monomer peak and a minor dimer peak. Small-angle X-ray scattering and gel-filtration chromatography revealed the presence of monomers and dimers in solution. These FXII N-terminal domain structures provide a holistic framework to understand how the mosaic domain structure of FXII assembles diverse ligand-binding sites in three dimensions.
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Jul 2025
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I15-1-X-ray Pair Distribution Function (XPDF)
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Janis
Timoshenko
,
David
Kordus
,
Jette K.
Mathiesen
,
Uta
Hejral
,
Patrik
Zeller
,
Gereon
Behrendt
,
Eylül
Öztuna
,
Jihao
Wang
,
Zahra
Gheisari
,
Rene
Eckert
,
Stephan
Reitmeier
,
Andreas
Reitzmann
,
Holger
Ruland
,
Jan
Folke
,
Thomas
Lunkenbein
,
Beatriz
Roldan Cuenya
Diamond Proposal Number(s):
[28439]
Open Access
Abstract: Sample homogeneity on the microscopic scale is critical for the reliable interpretation of x-ray absorption spectra collected in transmission mode. Unfortunately, it is not always easy to ensure it in practice. Especially in operando studies of catalysts and functional materials, the microstructure of the sample can evolve during its operation and even become the key descriptor for understanding structure-property relationships of the material, as exemplified by the transformations taking place in technical iron-based catalysts for ammonia synthesis under operating conditions. Here we present a simple approach for the identification and quantification of the material's microgranular structure effect on its x-ray absorption spectrum. We demonstrate that the quantitative information on the sizes of microscopic sample particles can be extracted from the observed distortions in the x-ray absorption near-edge structure spectra. The obtained insight can also be used to correct for the artifacts in extended x-ray absorption fine structure fitting, associated with the presence of microscopic inhomogeneities in the sample.
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Jul 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[23942]
Open Access
Abstract: Performance plastics, such as poly(methyl methacrylate), underpin the modern economy. Global manufacturing of plastic precursors relies on fossil carbon sources, and the urgently needed shift toward renewable carbon use through biofermentation is hindered by the low tolerance of producer strains to methacrylate esters. The principal mode of butyl methacrylate cellular toxicity is membrane disruption. To understand this process, the conditions for membrane stability, and recovery after solvent shock, we investigate the phase stability of hydrated lipid membranes at high levels of a key intermediate, butyl methacrylate. We assess the role of cis- vs trans-unsaturation in 18-carbon chain phospholipids on butyl methacrylate-induced phase conversion and polymorphism. Using ssNMR, SAXS and cryoEM, we demonstrate the formation of stable lipidic cubic phases in hydrated lipid/solvent (cis-chain phospholipid lipid/butyl methacrylate) systems at a 1:6 molar ratio entirely lacking monoolein. Transient lipidic cubic phases form in trans-chain phospholipid/butyl methacrylate systems, which slowly convert to bilayers through a spontaneous “membrane healing” process during recovery after solvent shock. The observed bicontinuous nanostructures with a cubic phase architecture coexist with a stable, monocontinuous hydrated phase of the same morphology but with simpler topological connectivity, which demonstrates that phase stability in cubic phases does not require topological complementarity. We propose trans-lipid substitutions in membranes of fermentative strains as a key step toward sustainable production of methacrylate esters.
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Jul 2025
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[31849]
Abstract: Contraction of the muscular walls of the heart is driven by an interaction between myosin motors from the thick filaments and actin sites in the thin filaments. Each heartbeat is triggered by calcium binding to troponin in the thin filaments, which unblocks the myosin-binding sites on actin. The strength and speed of contraction is also modulated by the availability of myosin motors, which are sequestered in a helical array on the surface of the thick filaments between heartbeats. The signalling pathway controlling release of the motors from this array during the heartbeat is unknown, but there are three general hypotheses: thick-filament mechano-sensing, thin-to-thick filament signalling, and direct calcium signalling to the thick filament. Here we tested the third hypothesis by replacing the native calcium-binding subunit of troponin (TnC) with a variant which cannot bind calcium. Demembranated trabeculae from rat heart containing this variant generated no active force on addition of calcium. We measured calcium-induced release of myosin motors from the sequestered state by X-ray diffraction and from the orientation of fluorescent probes on the myosin regulatory light chain. Both methods showed the expected calcium-dependent changes in the conformation of the myosin motors in trabeculae containing native TnC, but all these changes were abolished in those containing the TnC variant that cannot bind calcium. We conclude that thick filament activation in rat heart trabeculae is not due to direct binding of calcium to thick filaments, but is mediated by calcium activation of the thin filaments by mechano-sensing or thin-to-thick filament signalling.
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Jul 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[26334]
Open Access
Abstract: Fas-activated serine/threonine kinase (FASTK) is the founding member of the FASTKD protein family, which was shown to regulate the fate of mRNA molecules on multiple levels. The mitochondrial variant of FASTK co-localizes with mitochondrial RNA granules and regulates the degradation of mitochondrial mRNAs, whereas the cytoplasmic and nuclear forms of FASTK are involved in the regulation of alternative splicing, cytoplasmic RNA granule formation, and mRNA translation. Despite these multiple roles of FASTK in mRNA biology, the exact rules of RNA recognition by this protein remained undetermined. Here, we demonstrate direct RNA binding by purified human FASTK and show its preference for single-stranded G-rich oligonucleotides, including those with a tendency to form RNA G-quadruplexes. Addition of FASTK alone was sufficient to achieve protection of mitochondrial mRNAs from degradation by the degradosome. Structural characterization by SAXS (Small-Angle X-ray Scattering) showed that FASTK in solution is a monomer with an extended conformation. Point mutagenesis studies supported the structural predictions of an exposed RNA-binding interface in the central helical region, preceded by a smaller, flexibly attached helical N-terminal domain. We provide the first such extensive in vitro characterization of the RNA binding properties for a representative of the FASTKD protein family and suggest how these intrinsic properties may underlie FASTK function in mRNA metabolism.
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Jul 2025
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B21-High Throughput SAXS
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Diego E.
Sastre
,
Stylianos
Bournazos
,
Maros
Huliciak
,
Barbara Ann C.
Grace
,
E. Josephine
Boder
,
Jonathan
Du
,
Nazneen
Sultana
,
Tala
Azzam
,
Trenton J.
Brown
,
Maria W.
Flowers
,
Pete
Lollar
,
Ting
Xu
,
Tatiana A.
Chernova
,
Alasdair D.
Keith
,
Meredith
Keen
,
Abigail
Saltzman
,
Ana
Martinez Gascueña
,
Beatriz
Trastoy
,
Marcelo
Guerin
,
Filipp
Frank
,
Eric A.
Ortlund
,
Jeffrey V.
Ravetch
,
Eric J.
Sundberg
Diamond Proposal Number(s):
[34289]
Open Access
Abstract: Corynebacterium diphtheriae clade species secrete single-domain endo-β-N-acetylglucosaminidases (ENGases) that specifically bind to human IgG antibodies and hydrolyze their N297-linked glycans. Here, we define the molecular mechanisms of IgG-specific deglycosylation for the entire family of corynebacterial IgG-specific ENGases, including but not limited to CU43 and CM49. By solving the crystal structure of CU43 in a 1:1 complex with the IgG1 Fc region, combined with targeted and saturation mutagenesis analysis and activity measurements using engineered antibodies, we establish an inter-protomeric mechanism of recognition and deglycosylation of IgG antibodies. Using in silico modeling, small-angle X-ray scattering and saturation mutagenesis we determine that CM49 uses a unique binding site on the Fc region, to process N297-linked glycans. Moreover, we demonstrate that CU43 treatment is highly effective in abrogating Fc effector functions in humanized mouse models, while preserving the neutralizing capacity of anti-influenza IgG antibodies, thereby conferring protection against lethal influenza challenge.
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Jul 2025
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I15-1-X-ray Pair Distribution Function (XPDF)
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Diamond Proposal Number(s):
[34358]
Open Access
Abstract: Heterogeneous catalysts are often undergoing subtle structural changes in both the nanoparticle and the support phase during operation conditions. Catalytic cycling in in-situ experiments between catalysis and dropout conditions allows for exploiting the power of modulation excitation (ME) experiments. While phase sensitive detection (PSD) of ME experiments has been showcased for infrared and X-ray absorption spectroscopy in the past, here we present the first application to pair distribution function (PDF) analysis. We have performed in-situ X-ray total scattering studies on two alumina supported nickel catalysts (Ni@alumina) under methanation condition and hydrogen dropouts, and showcase how PSD analysis of the modulation excitation pair distribution function (ME-PDF) data improves the signal-to-noise ratio in the phase-resolved PDF data, enabling us to detect otherwise hidden structural changes. We identify a metal-support interaction of the Ni nanoparticles with the γ-Al2O3 support when choosing the deposition-precipitation method for catalyst preparation. By way of contrast, an industrial catalyst with comparable catalytic performance and nanoparticle diameters showed a dynamic surface oxidation of Ni nanoparticles during unstable H2 supply.
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Jul 2025
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