B21-High Throughput SAXS
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Diamond Proposal Number(s):
[38885]
Open Access
Abstract: Cholesterol is an essential sterol in cell membranes that regulates organization and fluidity. This biomolecule has been identified as one of the critical factors in the internalization process of various viruses in human cells. Therefore, understanding these mechanisms is crucial for a deeper comprehension of viral pathogenicity in the search for practical therapeutic approaches against viral diseases. The biochemical and biophysical processes related to these diseases are highly complex. For this reason, studying model systems capable of mimicking the interaction of lipid membranes with cholesterol and proteins is fundamental. In this work, we propose to study the structural and elastic changes in mono-, bi-, and tridimensional lipid systems composed of dipalmitoylphosphatidylcholine (PC) with varying amounts of cholesterol in the presence and absence of the S protein (Spike) and its receptor-binding domain (RBD) from SARS-CoV-2. To characterize these systems, we used both experimental and theoretical approaches such as Langmuir trough, atomic force microscopy (AFM), small-angle X-ray scattering (SAXS), electrochemical methods, and molecular dynamics (MD) simulations. With the interpretation of all results obtained in this work, it was possible to propose a structural model of the membrane in the presence of cholesterol and the interaction with the Spike protein and RBD. The behavior of the adsorption isotherm and SAXS data, together with the results provided by MD simulations, led us to conclude that cholesterol in PC monolayers promotes local alterations, inducing the formation of more rigid membrane regions. More importantly, cholesterol plays a crucial role in facilitating the allocation of SARS-CoV-2 proteins in lipid systems. This is especially true for the Spike protein, which displayed a non-ACE2 mediated stable binding to the lipid membrane with high internalization.
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Jun 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[38885]
Open Access
Abstract: This study reports the preparation and characterization of zeolitic imidazolate framework-67 (ZIF-67) through the recycling of spent synthesis liquor containing unreacted 2-methylimidazole. The recycling enhances the yield of ZIF-67 while maintaining the dodecahedral rhombic morphology of the particles. In the first synthesis, a molar ratio of 1:26 (Co2:2-methylimidazole) results in particles with an average diameter of 230 nm and a surface area of 1374 m2 g−1. The first recycling step produces ZIF-67 particles that doubl in diameter and surface area, reaching 520 nm and 1690 m2 g−1, respectively. In the second recycling step, the particles further increase to 1040 nm in diameter and 1806 m2 g−1 in surface area. This increase in diameter is attributed to changes in the metal-to-ligand ratio, which affects the nucleation and growth rates. Increased surface area is linked to a reduction in the average micropore diameter, which decreases from 1.42 nm (first synthesis) to 1.37 nm (second recycling step). There is a 6 m2 g−1 increase in surface area for every 0.001 cm3 g−1 increase in the volume of micropores. This indicates that the spent liquor can be utilized in consecutive batches to produce ZIF-67, minimizing reagent waste.
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Jun 2025
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B21-High Throughput SAXS
I22-Small angle scattering & Diffraction
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Ester
Serrano
,
Tianxiao
Zhao
,
David R.
Mark
,
Mostafa
Soroor
,
Iris
Floria
,
Nicholas J.
Terrill
,
Nikil
Kapur
,
Arwen I. I.
Tyler
,
Mathew H.
Horrocks
,
Andrew J.
Roe
,
Olwyn
Byron
Diamond Proposal Number(s):
[28516]
Open Access
Abstract: Enterohaemorrhagic Escherichia coli causes sporadic, and sometimes large-scale, food poisoning outbreaks, for which antibiotic treatment in humans is contraindicated. As an alternative form of therapy, previous studies developed the family of salicylidene acylhydrazide (SA) anti-virulence compounds. One target of the SA compounds is AdhE, an enzyme that converts acetyl-CoA to ethanol and vice versa. AdhE oligomerizes, forming helicoidal filaments, heterogeneous in length, called spirosomes. We show it is possible to only partially fractionate AdhE spirosomes because in vitro they oligomerize in the absence of stimuli, and that spirosome formation is necessary to regulate the direction of AdhE enzymatic reactions. We also show that the SA compound ME0054 binds and perturbs AdhE spirosomes, enhancing the conversion of ethanol to acetyl-CoA. This mechanistic understanding of how ME0054 impacts AdhE function will help in the development of SA compounds as novel anti-virulence inhibitors.
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Jun 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[30763]
Open Access
Abstract: Metal nanocrystals (M-NCs) and their supramolecular assemblies have attracted significant interest from the scientific community due to their wide range of applications arising from the possibility of accurately tuning the M-NCs properties through self-assembly into supramolecular aggregates. In this study, we investigate the complex interplay between capping agent surface coverage and solvent-capping agent interactions in the self-assembly process of M-NCs into supramolecular structures. Specifically, we explore the self-assembly behavior of gold (Au-NCs), silver (Ag-NCs), and platinum (Pt-NCs) nanocrystals upon functionalization with oleic acid (OA) in water using a microemulsion approach. Through a multi-technique analysis, we demonstrate the critical role of ligand density and solvent choice in driving the formation of highly ordered supramolecular structures. By increasing the surface coverage of the M-NC ligands, we observed a transition to more organised assemblies, with the interaction between the oleylamine alkyl chain and the functionalization medium further modulating the type of supramolecular arrangement. Moreover, we highlight the profound influence of both the external environment and supramolecular aggregation on the optical properties of the M-NCs. This work provides crucial insights into the factors that govern nanocrystals’ self-assembly and optical behavior, with broad implications for the design and application of nanomaterials in nanotechnology and materials science.
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Jun 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[20161, 21035, 25270]
Open Access
Abstract: The SARS-CoV-2 nucleocapsid (N) protein is essential for the viral life cycle, facilitating RNA packaging, replication, and host-cell interactions. Its ability to self-assemble and undergo phase separation is critical for these functions but remains poorly understood. Using an integrated approach combining small-angle X-ray scattering (SAXS), nuclear magnetic resonance spectroscopy, computational modeling, and biophysical assays, we uncover key mechanisms underpinning N-protein’s dynamic self-assembly. We show that the N-protein’s interdomain linker (IDL) contains a conserved coiled-coil (CC) motif that drives transient interactions between protein subunits, enabling the formation of progressively larger complexes at higher concentrations. SAXS analysis and ensemble modeling reveal that the IDL exists in a concentration-dependent equilibrium between monomeric, dimeric, and trimeric states. The CC motif facilitates parallel, head-to-head oligomerization of N-protein dimers, transitioning between compact (closed) and extended (open) configurations depending on the interaction network within the IDL. This linker-driven assembly modulates phase separation, impacting the size, stability, and dynamics of biomolecular condensates. Here, we present the most comprehensive conformational landscape analysis of the N-protein to date, providing a detailed model of its self-assembly and phase separation. Our findings highlight how the structural plasticity of the IDL and CC-mediated interactions are pivotal to its roles in the SARS-CoV-2 life cycle.
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Jun 2025
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B21-High Throughput SAXS
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Alexander
Leithner
,
Oskar
Staufer
,
Tanmay
Mitra
,
Falk
Liberta
,
Salvatore
Valvo
,
Mikhail
Kutuzov
,
Hannah
Dada
,
Jacob
Spaeth
,
Weijie
Zhou
,
Felix
Schiele
,
Sophia
Reindl
,
Herbert
Nar
,
Stefan
Hoerer
,
Maureen
Crames
,
Stephen
Comeau
,
David
Young
,
Sarah
Low
,
Edward
Jenkins
,
Simon J.
Davis
,
David
Klenerman
,
Andrew
Nixon
,
Noah
Pefaur
,
David
Wyatt
,
Omer
Dushek
,
Srinath
Kasturirangan
,
Michael L.
Dustin
Open Access
Abstract: Bispecific T cell engagers (TcEs) link T cell receptors to tumor-associated antigens on cancer cells, forming cytotoxic immunological synapses (IS). Close membrane-to-membrane contact (≤13 nm) has been proposed as a key mechanism of TcE function. To investigate this and identify potential additional mechanisms, we compared four immunoglobulin G1-based (IgG1) TcE Formats (A–D) targeting CD3ε and Her2, designed to create varying intermembrane distances (A < B < C < D). Small-angle X-ray scattering (SAXS) and modeling of the conformational states of isolated TcEs and TcE–antigen complexes predicted close contacts (≤13 nm) for Formats A and B and far contacts (≥18 nm) for Formats C and D. In supported lipid bilayer (SLB) model interfaces, Formats A and B recruited, whereas Formats C and D repelled, CD2–CD58 interactions. Formats A and B also excluded bulky Quantum dots more effectively. SAXS also revealed that TcE–antigen complexes formed by Formats A and C were less flexible than complexes formed by Formats B and D. Functional data with Her2-expressing tumor cells showed cytotoxicity, surface marker expression, and cytokine release following the order A > B = C > D. In a minimal system for IS formation on SLBs, TcE performance followed the trend A = B = C > D. Addition of close contact requiring CD58 costimulation revealed phospholipase C-γ activation matching cytotoxicity with A > B = C > D. Our findings suggest that when adhesion is equivalent, TcE potency is determined by two parameters: contact distance and flexibility. Both the close/far-contact formation axis and the low/high flexibility axis significantly impact TcE potency, explaining the similar potency of Format B (close contact/high flexibility) and C (far contact/low flexibility).
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Jun 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[34289]
Open Access
Abstract: Pseudomonas aeruginosa employs the Type VI secretion system (T6SS) to outcompete other bacteria in its environment. Among the effectors secreted by the T6SS of P. aeruginosa PAO1, Tse4 is known for its potent antibacterial activity. This study elucidates the molecular function of Tse4, which promotes cell depolarization in competing bacteria. Our results show that Tse4 spontaneously incorporates into lipid monolayers and forms multiionic channels in planar bilayers, with either ohmic conduction or diode-like rectifying currents and a preference for cations over anions. These observations allow us to propose a model of action whereby Tse4 channels couple cell depolarization with K+ efflux. These insights into Tse4’s pore-forming activity enhance our understanding of bacterial competition and exemplify a finely tuned antibacterial strategy, coupling its ability to cause membrane depolarization with potassium efflux that synergises with other T6SS effectors. These results highlight the sophistication of Pseudomonas aeruginosa’s competitive arsenal.
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Jun 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[39167]
Open Access
Abstract: We report the first reversible addition–fragmentation chain transfer polymerisation-induced self-assembly (RAFT-PISA) in ionic liquid (IL) that proceeds under emulsion conditions. Moreover, this formulation exploits refractive index contrast matching to generate highly transparent nanoparticle dispersions. Specifically, 1-ethyl-3-methyl-imidazolium ethylsulfate, [EMIM][EtOSO3], was used as the solvent for the chain extension of poly(2-hydroxyethyl methacrylate) (PHEMA) macromolecular chain transfer agents (macro-CTAs) using n-butyl methacrylate (BuMA) via RAFT emulsion polymerisation. Two series of PHEMAx-b-PBuMAy diblock copolymers with target PBuMA degrees of polymerisation (DPs) varying from 50 to 1000 were synthesised using either a PHEMA21 or PHEMA77 macro-CTA. All resulting nanoparticle dispersions yielded highly transparent dispersions, even when nanoparticle diameters exceeded 100 nm, due to the closely matched refractive index values of the [EMIM][EtOSO3] solvent and PBuMA nanoparticle core. Detailed analysis using small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM) confirmed the presence of spherical nanoparticles. Furthermore, the synthesis of PHEMA-b-PBuMA via this new PISA formulation was directly compared to equivalent block copolymer syntheses conducted in N,N-dimethylformamide (DMF) or ethanol/water mixtures. It was found that syntheses conducted in [EMIM][EtOSO3] resulted in the highest monomer conversions (up to >99%) and lowest dispersity (ĐM) values (as low as 1.16) in the shortest reaction times (2 hours) compared to the other solvent systems. This work demonstrates the use of ILs as a more sustainable and effective solvent for RAFT–PISA via the development of the first emulsion PISA formulation in IL.
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May 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[31952]
Abstract: Chirality and non-covalent chemistry are essential features in the construction of complex molecular systems that constitute living organisms. The specific interactions between molecular components with defined geometric orientations enable nature’s dynamism, responsiveness, specificity, and selection. The precision with which nature controls its homochirality and functional structures inspired the design of synthetic supramolecular systems. These systems have aimed at understanding the origins of these properties and mimicking their complexity. Research in the last decades has led to many insights into synthetic and natural supramolecular polymerization processes and principles of amplification of asymmetry. However, the complexity observed in nature remains far from being understood. In particular, detailed studies of assembly mechanisms and amplification of asymmetry principles in water-compatible supramolecular systems are scarce due to the challenging contribution of hydrophobic effects leading to non-equilibrium states of supramolecular assemblies. Valuable contributions to this field have been made in recent years, including the introduction of in situ chemical modifications and dissipative non-equilibrium assembly states, leading to evolutionary features such as self-replication. This chapter introduces the concepts of supramolecular polymerization and shows the analogies between natural and synthetic systems. In particular, the influence of homochirality in natural macroscopic structures is emphasized, showing the importance of this property in water-compatible supramolecular systems. Therefore, key developments in research on the origin of homochirality and challenges for future research to understand this essential feature of life are outlined.
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May 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[26855]
Abstract: There are over 1 million Alu elements in the human genome which can be transcribed into discrete, RNA polymerase III transcribed non-coding Alu RNAs. These Alu RNAs often interact with and are regulated by the protein heterodimer SRP9/SRP14. This interaction is dependent on a 5’ pseudoknot domain in the Alu RNA that is thought to be held together by a canonical nucleotide triad within a U-turn motif. Herein, we discover a significant reduction in BC200 expression after mutation of a critical guanosine in the U-turn motif within its pseudoknot domain. We studied a recently discovered short human Alu RNA, EB120 that lacked the canonical Alu RNA U-turn nucleotide triad. We tested the expression of EB120 in 18 different human cell lines and tissues. EB120 was found to lack association with SRP9/SRP14 in a cellular context. Small angle X-ray scattering followed by atomistic computation structure prediction suggests the BC200 Alu domain and its U-turn mutant both possess a canonical Alu RNA fold, while EB120 lacks one. Our results highlight the structural diversity of Alu RNA, and the impact mutations may have on Alu RNA function.
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May 2025
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