I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[36690]
Abstract: The online structure development during tenter-frame biaxial stretching of linear low-density (LLDPE), high-density polyethylene (HDPE), and their 50/50 blend was investigated using synchrotron Wide-Angle X-ray Scattering (WAXS). During stretching, crystallite reorientation, crystallographic slip phenomena activated at yielding, and lamellar fragmentation at higher strains are responsible for a decrease in crystallinity. These processes induce a morphological transformation from the initial semisolid spherulitic morphology into an oriented fibrillar network with lamellae in edge-on orientation, and the b- and c-axes constrained within the film plane, as confirmed by ex-situ WAXS and SAXS. These phenomena are more important for HDPE, which exhibits higher susceptibility to biaxial stretching. Upon cooling, the morphology developed during stretching is preserved, as newly crystallized material grows epitaxially on the preexisting structure. These findings highlight the higher structural sensitivity of HDPE during semisolid biaxial stretching compared with LLDPE, providing unique information to understand and control the biaxial processing of polyethylene.
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Mar 2026
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labSAXS-Offline SAXS and Sample Environment Development
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Diamond Proposal Number(s):
[39236]
Open Access
Abstract: Designing polymers that combine performance with sustainability remains a critical challenge. Here, we report high-performance elastomers derived from CO2 and biobased monomers that integrate both mechanical toughness and closed-loop chemical recyclability through a single material feature: dynamic metal–ionomer cross-links. These ABA block polymers, synthesized from ε-decalactone, δ-jasmolactone, CO2, and bicyclic epoxides, incorporate abundant and inexpensive metal carboxylates (Na(I), Zn(II), and Al(III)) into the midblock, forming reversible networks that enhance tensile strength by 150% while maintaining high strain at break (>1500%) and elastic recovery (>85%). The same cross-links act as built-in catalysts, enabling energy-efficient depolymerization of both polyester and polycarbonate domains at 200 °C, recovering the original monomers. This dual-function approach advances circular polymer design by combining enhanced performance with efficient, low-energy, closed-loop recycling.
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Mar 2026
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[38168]
Open Access
Abstract: Cyclophilins (Cyps) are ubiquitous cytosolic proteins with peptidyl-prolyl cis-trans isomerase (PPIase) activity and the ability to bind the immunosuppressant cyclosporin A (CsA). The genome of Toxoplasma gondii, the parasite responsible for toxoplasmosis, encodes multiple putative Cyps, whose specific functions remain largely unexplored.
Here, we characterize TgCyp21, a predicted Cyp from T. gondii. TgCyp21 displays PPIase activity and is inhibited by CsA in vitro. Importantly, its activity decreases markedly under oxidizing conditions but is partially restored by reducing agents, including dithiothreitol (DTT) and the parasite endogenous thioredoxin (TgTrx). TgCyp21 contains four cysteines, with Cys87 and Cys141 predicted to be spatially close based on structural modeling. Substitution of both residues significantly reduced PPIase activity, with Cys87 emerging as the main contributor to this loss. Structural modeling further indicates that Cys87 and Cys141 are suitably oriented to interact with the conserved active-site cysteines of TgTrx. This interaction is supported experimentally by mixed disulfide trapping, which identifies a stable disulfide-linked intermediate between TgCyp21 and TgTrx, consistent with a thiol-disulfide exchange mechanism. Small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy further confirm the formation of the complex.
Taken together, our data indicate that TgCyp21 behaves in vitro as a redox-responsive Cyp and a substrate for Trx, suggesting a potential involvement in Trx-mediated redox processes in T. gondii.
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Nov 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[36130]
Open Access
Abstract: The initiation of allergic responses critically depends on the recognition of an allergenic epitope by the paratope of IgE antibodies. While previous structural studies have focused on recombinant fragments or engineered forms of IgE, the structure of full-length IgE in its native state remains poorly understood. In this study, we investigate the conformational changes of a native murine IgE (2F5), both in its free form and upon binding to the Hevea brasiliensis allergen profilin (Hev b 8). Small-angle X-ray scattering (SAXS) data reveal that unbound IgE adopts an extended conformation with open Fab arms. However, when it binds to profilin, it transitions to a more compact arrangement characterized by closer proximity of the arms. Molecular dynamics (MD) simulations of the Fab region further identified conformational rearrangements upon allergen binding, including a twisting motion and partial disruption of interactions between the naturally paired heavy and light chains. These findings indicate that there may be allosteric communication between Fab and Fc regions, even in the absence of a hinge region, which is not present in IgE. Overall, this study provides valuable insights into the dynamic structural properties of native IgE and enhances our understanding of the molecular mechanisms underlying allergen recognition.
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Nov 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[33176, 35926]
Open Access
Abstract: Mycobacterial infections, including tuberculosis, remain a major global health challenge, causing millions of deaths annually. Their treatment is increasingly hindered by limited therapeutic options and rising antimicrobial resistance, highlighting the urgent need for alternative strategies. Mycobacteriophage LysA endolysins are complex multi-domain peptidoglycan hydrolases emerging as potential tools to treat mycobacterial infections. However, despite the therapeutic prospects of LysAs, our understanding of their mechanism of action remains limited. This study provides a comprehensive structural-functional analysis of the catalytic domains of D29LysA and DS6ALysA endolysins (D29N4/D29GH19 and DS6AGH19/DS6AAmi2B), characterised alone and in complex with PG analogues, using protein engineering, X-ray crystallography, small-angle X-ray scattering, and in silico tools. Our results reveal precise details of the substrate-binding site and the catalytic platforms at each domain, including information about substrate-binding mode and conformational changes associated with peptidoglycan recognition and hydrolysis. Moreover, these findings also suggest a coordinated mechanism of action of both catalytic domains in DS6ALysA lysin. These insights represent a significant advance in understanding the structural basis of mycobacterial cell-wall degradation by mycobacteriophage endolysins. Information that may aid in further exploring these endolysins as therapeutic antimicrobial tools in the future.
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Nov 2025
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[23087]
Open Access
Abstract: The utilization of CO2 as a sustainable feedstock for oxygenated polymers offers a promising route to high-performance materials while addressing environmental challenges. This study investigates the synthesis of high-molar-mass, nonlinear polymer architectures using switchable catalysis, focusing on multiarm star block polymers derived from vinyl-cyclohexene oxide (vCHO), CO2, and ε-decalactone (ε-DL). A [Zn(II)Mg(II)] organometallic catalyst and multifunctional chain-transfer agents (CTAs) are employed in a “core-first” approach to produce tri-, tetra-, and hexafunctional star block polymers. Thermomechanical and morphological properties were evaluated as a function of molar mass, number of arms, and architecture, indicating the differences between star and linear structures. Postpolymerization modification of the polycarbonate block, via thiol–ene chemistry, introduced pendant hydroxyl groups, enhancing hydrogen bonding and microphase separation, significantly impacting thermal and mechanical performance. This work highlights the versatility of switchable catalysis in accessing star polymers while underscoring the potential of integrating architectural control and functionalization to enhance the performance and applicability of CO2-derived poly(ester-b-carbonate)s.
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Oct 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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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[37593]
Abstract: 4-Hydroxyphenylacetate-3-monooxygenase from the thermophilic bacterium Geobacillus mahadii Geo-05 catalyzes the hydroxylation of 4-hydroxyphenylacetate (4-HPA) to 3,4-dihydroxyphenylacetate (3,4-DHPA), marking the initial step of the 4-HPA degradation pathway. This enzyme comprises of two components: an oxygenase and reductase. In this study, the gene encoding the oxygenase component, GMHpaB was successfully cloned, overexpressed in Escherichia coli BL21 (DE3) and purified to homogeneity. Purified GMHpaB was shown to bind reduced chromogenic cofactors, evidenced by maximal absorbance peaks at 375 nm. GMHpaB is optimally active at 55 °C and demonstrates thermostability, retaining 96 % of its catalytic activity after 30 min of incubation at its optimum temperature. Furthermore, GMHpaB displays versatility, showing high enzymatic activity with both FMN and FAD as cofactors, with relative activity increases by 250 % and 383 %, respectively, compared to the cofactor-free control. The overall fold classifies GMHpaB as group D flavin-dependent monooxygenase, but distinct loop conformations set it apart from homologs within the group. Notably, residue Glu212, positioned on the substrate binding loop of GMHpaB plays a critical role in anchoring and stabilizing the flavin binding loop, potentially contributing to the enzymes dual cofactor compatibility. These biochemical and structural insights lay the groundwork for future applications, particularly in high-temperature biocatalysis.
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Jul 2025
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Elena
Romagnoli
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Emiliano
Laudadio
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Giovanna
Mobbili
,
Leonardo
Sorci
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Giovanni
Birarda
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Federica
Piccirilli
,
Lisa
Vaccari
,
Hendrik
Vondracek
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Brenda
Romaldi
,
Massimo
Marcaccio
,
Paola
Storici
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Marta
Semrau
,
Roberta
Galeazzi
,
Andrea
Toma
,
Vincenzo
Aglieri
,
Pierluigi
Stipa
,
Tatiana
Armeni
,
Cristina
Minnelli
Open Access
Abstract: Tyrosine kinase inhibitors (TKIs) targeting the oncogene Epidermal Growth Factor Receptor (EGFR) are widely used in the treatment of non-small cell lung cancer (NSCLC). In this context, the introduction of fourth-generation TKIs has significantly advanced targeted therapy for T790M and C797S EGFR mutations. Current therapeutic strategies are increasingly focusing on the design of orthoallosteric TKIs, which have shown promise in stabilizing the inactive conformation of mutated EGFR. In this context, we report the discovery of FL30, a small molecule with a flavone core that exhibits nanomolar potency against the EGFR-L858R/T790M mutation, even in the presence of the C797S mutation. The IC50 comparable to the Osimertinib - one of the most renowned EGFR-TKIs - emphasizes the remarkable success of the design approach. In NSCLC models, FL30 effectively inhibits cancer growth and EGFR phosphorylation selectively in cells with the EGFR mutations. Kinetic studies, molecular modeling, and Plasmon Internal Reflection Surface-Enhanced Infrared Absorption (PIR-SEIRA) microscopy suggests that FL30 binds to the orthosteric site while inducing the transition of the mutant EGFR toward an inactive-like state. These findings highlight FL30's potential for further optimization and propose a novel approach for developing targeted therapies that combine orthosteric binding with allosteric modulation.
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Jun 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[36130]
Open Access
Abstract: CD93 is a receptor predominantly expressed on the surface of endothelial cells, where it plays a pivotal role in angiogenesis through its interaction with the extracellular matrix. In our previous studies, we identified the monoclonal antibody 4E1 as a potent inhibitor of angiogenesis by targeting the CD93-Multimerin-2 axis. Here, we report the development of 4E1 as a recombinant whole immunoglobulin and a single-chain variable fragment, designated sc-4E. Both formats retained the binding properties of the parental monoclonal antibody and exhibited comparable inhibitory effects on endothelial cell migration and differentiation. To elucidate the molecular basis of the 4E1-CD93 interaction, we initially employed machine learning-based modeling and docking analyses of the variable heavy and light domains of 4E1. Subsequent crystallographic analysis of sc-4E provided high-resolution structural insights, confirming and validating the predicted model. Further docking experiments and molecular dynamics simulations using the crystallographic structures of CD93 and sc-4E revealed that the interaction is primarily mediated by the CDR-H3 and CDR-L2 loops. Notably, these regions engage with the sushi-like domain of CD93, which is critical for its interaction with Multimerin-2. This comprehensive structural and functional characterization of 4E1 and sc-4E underscores their potential as anti-angiogenic agents. By effectively inhibiting endothelial cell migration and differentiation, 4E1 derivatives represent promising therapeutic candidates for the treatment of ocular vascular diseases driven by pathological angiogenesis.
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Apr 2025
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