I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[25402, 33658]
Open Access
Abstract: Environment-sensitive fluorescent probes are indispensable tools for studying biological systems and advancing drug discovery. This study reports the development of 4-sulfamoyl-7-aminobenzoxadiazole (SBD)-based fluorescent probes for the allosteric site of the liver isoform of pyruvate kinase (PKL). By integrating SBD moieties into known activator scaffolds, such as mitapivat and diarylsulfonamide (DASA) ligands, probes for indicator displacement assays were designed to quantify ligand interactions in the allosteric site. Compound 4a displayed dose-dependent fluorescence enhancement in response to PKL binding and was used in a competitive binding assay with unlabelled ligands: mitapivat, TEPP-46, DASA-58 and reported activator 21. Structure–activity relationship (SAR) analysis revealed key structural features influencing activity and fluorescence sensitivity. The probes report selectively on the allosteric site ligands as the binding was not affected by natural ligands, such as ADP, fructose-1,6-bisphosphate (FBP), phosphoenolpyruvate (PEP), and phenylalanine. These findings provide a practical framework for detecting allosteric ligand engagement in PKL and expand the repertoire of molecular tools for advancing PKL-targeted therapies.
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Nov 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[31850]
Open Access
Abstract: To ensure safety, pharmaceuticals are rigorously tested for lipopolysaccharide (LPS) contamination, as this can trigger severe immune reactions in patients. Low Endotoxin Recovery (LER), describing the masking of spiked LPS controls in Limulus Amebocyte Lysate (LAL) assays, has been associated with the presence of chelating agents and surfactants in pharmaceutical formulations. The addition of excipients, such as Mg2+, have shown the ability to mitigate the effects of LER, however, inconsistencies in various studies regarding the influence of the excipients on LPS aggregate characteristics and LER occurrence hinder a clear understanding of the mechanisms underlying LER. In this study, dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) were employed to systematically assess the impact of chelating agents, surfactants, and divalent cations on the size and shape of LPS aggregates across various formulations. Our results indicate that surfactant-only formulations generally reduce LPS aggregate size, whereas chelating agent-only formulations do not. Notably, the smallest aggregates were observed when both chelating agents and surfactants were present, with the extent of size reduction being specific to the particular excipients used. Additionally, Mg2+ generally inhibited the excipients’ capacity to decrease aggregate size, most effectively in phosphate-containing samples. Despite these variations in size, the overall aggregate shape remained largely unchanged in all formulations. These findings suggest that LPS aggregate size or shape does not distinguish formulations causing LER; instead, factors such as the characteristics of the LPS aggregate surface in different formulations should be explored in the future.
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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):
[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):
[31378]
Open Access
Abstract: Amphiphilic compounds, such as phospholipids or surface-active substances, are present in biological systems and can be part of pharmaceutical formulations. As a consequence, all pharmaceutically active ingredients will encounter amphiphilic compounds, either in the formulation or after administration. With the growing interest in peptide-based pharmaceuticals, there is a need to enhance the understanding of the interactions between peptides and amphiphilic compounds.
In this particular study, we have chosen to study mixtures of the comparatively small cyclical octapeptide lanreotide and the conventional anionic surfactant sodium dodecylsulfate (SDS). This was done by examining the self-assembly structures formed in lanreotide-SDS mixtures using light scattering and small-angle X-ray scattering (SAXS).
Above the critical micelle concentration (cmc) of SDS, the large excess of SDS could solubilize all lanreotide and form small micelles with lanreotide attached to the interface. Upon dilution to concentrations below the cmc of SDS, a suspension with dispersed solid nanoparticles is formed. The solid nanoparticles grow in size with decreasing concentration and, eventually, precipitate. The precipitated material is arranged in a liquid crystalline micellar phase, consisting of small close-packed SDS micelles with peptide adsorbed at the interface.
We were able to conclude that lanreotide does not form mixed micelles with SDS, indicating that it lacks the amphiphilic properties required to integrate fully with SDS behaving as a cosurfactant. In contrast, lanreotide attaches to the interface of SDS micelles, resembling the interactions of polymers, proteins, and nucleic acids with surfactants.
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Nov 2025
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I24-Microfocus Macromolecular Crystallography
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Natalia
Venetz-Arenas
,
Tim
Schulte
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Sandra
Müller
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Karin
Wallden
,
Stefanie
Fischer
,
Tom
Resink
,
Nadir
Kadri
,
Maria
Paladino
,
Nicole
Pina
,
Filip
Radom
,
Denis
Villemagne
,
Sandra
Bruckmaier
,
Andreas
Cornelius
,
Tanja
Hospodarsch
,
Evren
Alici
,
Hans-Gustaf
Ljunggren
,
Benedict J.
Chambers
,
Xiao
Han
,
Renhua
Sun
,
Marta
Carroni
,
Victor
Levitsky
,
Tatyana
Sandalova
,
Marcel
Walser
,
Adnane
Achour
Diamond Proposal Number(s):
[21625]
Open Access
Abstract: The balance between affinity and specificity in T cell receptor (TCR)-dependent targeting of HLA-restricted tumor-associated antigens presents a significant challenge for immunotherapy development. T cell engagers that circumvent these limitations are therefore of particular interest. We established a process to generate bispecific Designed Ankyrin Repeat Proteins (DARPins) that simultaneously target HLA-I/peptide complexes and CD3e. These high-affinity T cell engagers elicited CD8+ T cell activation against tumor targets with strong peptide specificity, as confirmed by X-scanning mutagenesis and functional killing assays. A cryo-EM structure of the ternary DARPin/HLA-A*0201/NY-ESO1157-165 complex revealed a rigid, concave DARPin surface spanning the full length of the peptide-binding cleft, contacting both α-helices and the peptide. The present findings reveal promising immuno-oncotherapeutic approaches and demonstrate the feasibility of rapidly developing DARPins with high affinity and specificity for HLA/peptide targets, which can be readily combined with a new generation of anti-CD3e-specific DARPins.
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Nov 2025
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I03-Macromolecular Crystallography
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Cholpon
Tilegenova
,
Tun
Liu
,
Qian
Zhao
,
Mahati
Are
,
Yu
Zhao
,
Woo Suk
Choi
,
Anusarka
Bhaumik
,
Ruth
Steele
,
Nicholas A.
Manieri
,
Bengi
Turegun
,
Alex
Ni
,
Rosa M. F.
Cardoso
,
Paul
Shaffer
,
Desmond
Clark
,
Robin
Ernst
,
Wenyu
Li
,
Tracy
Taylor
,
Suresh Kumar
Swaminathan
,
Bhargavi
Ramaraju
,
Kevin
Liaw
,
Steven A.
Jacobs
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Sujata
Sharma
,
Wan Cheung
Cheung
,
Adam
Zwolak
Diamond Proposal Number(s):
[34711]
Abstract: Bispecific antibodies (bsAbs) can enable therapeutic mechanisms, such as dual antigen targeting or receptor agonism, that are impossible using monoclonal antibodies. BsAbs with IgG-like format (bsIgG) are comprised of two unique heavy chains, each having a cognate light chain. Co-expression of these four unique polypeptides often leads to several mispaired species that are difficult to separate from the target bsIgG due to their similar biophysical properties. Here we describe a set of mutations called ProAla that exploit a the unfolded protein response pathway of cells. ProAla heavy chains are engineered with higher folding energy barriers such that only the cognate light and heavy chains can induce folding, chaperone release and secretion. The structures of the ProAla Fab and Fc regions are identical in structure to normal antibodies, enabling maintenance of half-life and function. Mispaired polypeptides fail to secrete from the cell due to enhanced interaction with the endoplasmic reticulum chaperone BiP, resulting in increased purity of secreted bsIgGs.
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Oct 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Anaïs F. M.
Noisier
,
Jenny
Sandmark
,
Fredrik
Edfeldt
,
Anna
Backmark
,
Johan
Broddefalk
,
Joanna
Wandzik
,
Ulrik
Jurva
,
Margareta
Ek
,
Carina A.
Johansson
,
Louise
Barlind
,
Jenny
Gunnarsson
,
Janna M.
Bigalke
,
Yafeng
Xue
,
Andrey I.
Frolov
,
Cecilia
Kankkonen
,
Robert G.
Roth
,
Maria
Fritsch
,
Sophie
Watcham
,
Katerine
Van Rietschoten
,
Gemma E.
Mudd
,
Helen
Harrison
,
Liuhong
Chen
,
Michael J.
Skynner
,
David J.
Craik
,
Sunay V.
Chankeshwara
,
Malin
Lemurell
Abstract: The GDF15–GFRaL–RET signaling complex is involved in a broad range of disease states, with agonistic action of GDF15 affecting metabolism and body weight control, while inhibition is indicated in cancer and wasting disorders like cachexia. Here, we describe the discovery of the peptide inhibitors of the GDF15–GFRaL protein–protein interaction to prevent RET-induced signaling using both a structure-guided design and a phage display approach. Phage display provided bicyclic peptide hits with high affinity for GFRaL, and these were dimerized to mimic the bidentate interaction of homodimeric GDF15. Guided by structural data, the monomeric peptides were converted into tandem Bicycle molecules with picomolar affinities, similar to that of the endogenous GDF15 ligand. These dimerized protein mimetics inhibited cell signaling in a functional assay and showed improved pharmacokinetic properties compared with their monomeric counterparts. This is the first example of a homodimeric Bicycle molecule inhibiting receptor complex formation, thereby antagonizing the intracellular signaling response.
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Oct 2025
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I14-Hard X-ray Nanoprobe
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Gaewyn
Ellison
,
Rhiannon E.
Boseley
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Meg
Willans
,
Sarah
Williams
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Evelyn S.
Innes
,
Paige
Barnard
,
Julia
Koehn
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Somayra S. A.
Mamsa
,
Paul
Quinn
,
Daryl L.
Howard
,
Simon A.
James
,
Mark J.
Hackett
Diamond Proposal Number(s):
[34101]
Open Access
Abstract: Understanding the role of metal ions in normal and abnormal cell function continues to emerge as a critical research area in the biological and biochemical sciences. This is especially true in the context of brain health and neurodegenerative diseases, as the brain is especially enriched in metal ions. A range of microscopy and bioanalytical techniques are available to assist in characterizing and observing changes to the brain metallome. As is the case in many other scientific fields, the integration of multiple analytical methods often yields a more complete chemical picture and deeper biological understanding. Herein, we present a case study applying 4 different analytical methods to provide spatially resolved characterization of chemical and biochemical parameters relating to the iron (Fe) metallome within a specific brain region, cornu ammonis sector 1 (CA1) of the hippocampus. The CA1 hippocampal sector was chosen for investigation due to its known endogenous enrichment in Fe and its selective vulnerability to neurodegeneration. The 4 analytical techniques applied were X-ray fluorescence microscopy (to quantify Fe distribution); X-ray absorption near-edge structure (XANES) spectroscopy to reveal information on Fe oxidation state and coordination environment; immuno-fluorescence to reveal relative abundance of Fe storage proteins (heavy chain ferritin and mitochondrial ferritin); and spatial transcriptomics to reveal gene expression pathways relevant to Fe homeostasis. Collectively, the results highlight that although pyramidal neurons in lateral and medial regions of the hippocampal CA1 sector are morphologically similar, key differences in the Fe metallome are evident. The observed differences within the hippocampal CA1 sector potentially indicate a higher oxidative environment and higher metabolic turnover in medial CA1 neurons relative to lateral CA1 neurons, which may account for the heightened vulnerability to neurodegeneration that is observed in the medial CA1 sector.
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Oct 2025
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Krios I-Titan Krios I at Diamond
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Andreas
Schedlbauer
,
Xu
Han
,
Wouter
Van Bakel
,
Tatsuya
Kaminishi
,
Borja
Ochoa-Lizarralde
,
Idoia
Iturrioz
,
Retina
Çapuni
,
Ransford
Parry
,
Ronny
Zegarra
,
David
Gil-Carton
,
Jorge P.
López-Alonso
,
Kristina
Barragan Sanz
,
Letizia
Brandi
,
Claudio O.
Gualerzi
,
Paola
Fucini
,
Sean R.
Connell
Diamond Proposal Number(s):
[17171, 31586]
Open Access
Abstract: The initiation phase is the rate-limiting step of protein synthesis (translation) and is finely regulated, making it an important drug target. In bacteria, initiation is guided by three initiation factors and involves positioning the start site on the messenger RNA within the P-site on the small ribosomal subunit (30S), where it is decoded by the initiator fMet–tRNA. This process can be efficiently inhibited by GE81112, a natural hydrophilic, noncyclic, nonribosomal tetrapeptide. It is found in nature in three structural variants (A, B, and B1 with molecular masses of 643–658 Da). Previous biochemical and structural characterization of GE81112 indicates that the primary mechanism of action of this antibiotic is to (i) prevent the initiator fMet–tRNA from binding correctly to the P-site and (ii) block conformational rearrangements in initiation factor IF3, resulting in an unlocked 30S preIC state. In this study, using cryo-EM, we have determined the binding site of GE81112 in initiation complexes (3.2–3.7 Å) and on empty ribosomes (2.09 Å). This binding site is within the mRNA channel but remote from the binding site of the initiation factors and initiator fMet–tRNA. This suggests that it acts allosterically to prevent the initiator fMet–tRNA from being locked into place. The binding mode is consistent with previous biochemical studies and recent work identifying the key pharmacophores of GE81112.
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Oct 2025
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