I23-Long wavelength MX
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Hannah
Best
,
Lainey J.
Williamson
,
Adam B.
Cutts
,
Marina
Galchenkova
,
Oleksandr
Yefanov
,
Nicole
Bryce-Sharron
,
Emily A.
Heath
,
Raphael
De Wijn
,
Robin
Schubert
,
Anna
Munke
,
Alessandra
Henkel
,
Bjarne
Klopprogge
,
T. Emilie S.
Scheer
,
Viviane
Kremling
,
Salah
Awel
,
Gisel
Pena
,
Juraj
Knoska
,
Anusha
Keloth
,
Julia
Maracke
,
Romain
Letrun
,
Egor
Sobolev
,
Johan
Bielecki
,
Diogo
Melo
,
Sravya
Kantamneni
,
Katerina
Doerner
,
Marco
Kloos
,
Joachim
Schulz
,
P. Lourdu
Xavier
,
Marius
Lauffer
,
Maite
Villanueva
,
Primitivo
Caballero
,
Helen
Waller-Evans
,
Emyr
Lloyd-Evans
,
Charlotte
Uetrecht
,
Richard
Bean
,
Henry N.
Chapman
,
Neil
Crickmore
,
Pierre J.
Rizkallah
,
Colin
Berry
,
Dominik
Oberthuer
Diamond Proposal Number(s):
[36446]
Open Access
Abstract: Bacillus thuringiensis (Bt) strains naturally produce pesticidal proteins as nanocrystalline inclusions that are extraordinarily stable in aqueous environments, but which dissolve selectively at specific pH conditions. These proteins have been used in agriculture for >50 years and are critical to global food security. The majority of previously determined Bt Cry protein structures lack the extended C-terminal “crystallization domain,” which is thought to stabilize crystal packing and control selective solubility in insect targets, often via manipulation of disulfide bridges. It has also recently been shown to influence toxicity and target specificity. Here, we use serial femtosecond crystallography (SFX) to determine high-resolution full-length native structures of Cry1Ca18 (1.65 Å) and Cry8Ba2 (2.27 Å) in their natural nanocrystalline state. Differences in cysteine content (19 versus 4 residues) reveal distinct in vivo crystal-stabilization strategies. Understanding Bt toxin domain architecture and natural crystal formation is essential for improving biopesticide design and advancing agricultural genetic engineering.
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Feb 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[28402]
Open Access
Abstract: Despite the continual emergence of SARS-CoV-2 variants and increasing diversity within the receptor binding domain (RBD), some antibody responses that are directed to conserved regions can display cross-reactivity against variants. We previously isolated an RBD-directed monoclonal antibody (084-7D) from a Beta-infected donor that neutralized Beta and emerging Omicron variants. Here, we solved a high-resolution crystal structure of the 084-7D Fab in complex with the Beta RBD. These data revealed an epitope overlapping both the ACE2 binding site and those of other class 1 antibodies. Furthermore, the epitope includes highly conserved residues, Q409, D420, and Y489, that are present in recent Omicron variants. The N417 residue that emerged with Beta and has since persisted is tolerated within the epitope of 084-7D, explaining the preferential neutralization of contemporaneous N417-containing variants. These structural data defined the mechanism for cross-reactivity of a Beta-elicited neutralizing antibody, potentially informing the design of future broadly reactive SARS-CoV-2 therapeutics.
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Feb 2026
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B21-High Throughput SAXS
Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[24557, 39224]
Open Access
Abstract: Histone variants define distinct chromatin states by modulating the biophysical properties of nucleosomes. Variants play a particularly important role in the parasitic protist Trypanosoma brucei, which has unusual chromatin and lacks a canonical repressive heterochromatin system. Instead, T. brucei utilizes specialized divergent histone variants H3.V and H4.V. However, the biochemical basis of their repressive functions is unknown. Here, we determined the structure of the H3.V-H4.V nucleosome core particle and biochemically characterized variant-containing nucleosomes and nucleosome arrays, probing their unique properties. We discovered that surprisingly for repressive-state nucleosomes, H3.V promotes pronounced DNA splaying, largely via its N-terminal tail region, while retaining overall stability that is comparable to canonical nucleosomes. In contrast, H4.V exhibits near-identical binding to DNA but mediates a slight increase in histone octamer stability. The surface of the H3.V-H4.V nucleosome is altered and provides a differential platform for chromatin-binding proteins, linking the variants to parasite pathogenicity.
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Feb 2026
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[38021]
Open Access
Abstract: Prenylated flavin mononucleotide (prFMN) is a modified flavin cofactor required by the UbiD family of (de)carboxylase enzymes. While the reduced prFMNH2 form is produced by the flavin prenyltransferase UbiX, the corresponding two-electron oxidized prFMNiminium form is required to support UbiD catalysis. Thus, oxidative maturation of prFMNH2 is required, which can be catalyzed by UbiD. However, heterologous (over)expression of UbiDs frequently leads to the accumulation of the stable but non-active one-electron oxidized purple prFMNradical species. A dedicated prFMN maturase enzyme (PhdC) from Mycolicibacterium fortuitum was recently identified as capable of catalyzing the oxidative maturation of prFMNradical to prFMNiminium, thereby enabling an effective supply of active cofactor to the associated phenazine-1-carboxylate (de)carboxylase PhdA. We report the crystal structure of PhdC in complex with flavin, revealing it is a distant member of the class I HpaC-like family of short-chain dimeric flavin reductases and demonstrate catalytic conversion of the prFMNradical species to prFMNiminium in the presence of oxygen or ferricyanide. Co-expression of PhdC or a distant homologue from Priestia megaterium (YclD) with the canonical UbiD from Escherichia coli leads to activation of the latter, similar in effect to co-expression with the prFMNH2-binding chaperone LpdD. Conserved Glu residues in the PhdC active site suggest catalysis occurs through C1′ proton-abstraction coupled oxidation. This study thus provides both structural and mechanistic insight into the function of PhdC, adding to the expanding repertoire of prFMN-binding proteins associated with the widespread UbiDX system.
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Dec 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Sites of protein-protein interaction (PPI) are potentially more selective binding sites for therapeutics than protein substrate-binding sites. PPIs include distinct regions frequently called “hotspots,” sites of key amino acid interactions. Prospective identification of these hotspots through X-ray crystallographic screening could assist in the identification of separation of function mutants for experimental validation, enhance confidence in AI-generated multiprotein complex predictions, and accelerate development of selective chemical probes. To explore these applications, we utilize the FragLite library to examine the binding surfaces of CDK2-cyclin A. The many protein- and peptide-CDK2-cyclin A complexes that have been structurally characterized make this complex an appropriate test case. We show that FragLites comprehensively map both known sites of protein-protein interaction on CDK2-cyclin A and identify a possible uncharacterized site, providing a structural method toward directing mechanistic studies and starting points for chemical probe design.
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Aug 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Open Access
Abstract: We describe the generation and characterization of camelid single-domain antibodies (nanobodies) raised against tumor suppressor protein p16INK4a (p16). p16 is a cell cycle regulator that inhibits cyclin-dependent kinases CDK4 and CDK6 and is inactivated in sporadic and familial cancers. The majority of p16 missense mutations cause loss of function by destabilizing the protein’s structure. We identify nanobodies that bind p16 with nanomolar affinities and restore the stability of many different cancer-associated p16 mutations located at sites throughout the protein. The crystal structure of a nanobody-p16 complex reveals that the nanobody binds to the opposite face of p16 to the CDK-binding interface permitting formation of a ternary complex. We confirm that nanobodies bind to p16 in a cellular setting and do not preclude p16 binding to CDK6 and its ability to induce cell-cycle arrest. These findings indicate that nanobodies merit testing as pharmacological chaperones for p16 reactivation in the cell.
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Aug 2025
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I12-JEEP: Joint Engineering, Environmental and Processing
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Ilaria
Quaratesi
,
Ioan
Călinescu
,
Petre
Chipurici
,
Elisa-Gabriela
Dumbravă
,
Andrei
Cucos
,
Mohamed Yassine
Zaki
,
Pellegrino
La Manna
,
Adrian
Bercea
,
Miruna Silvia
Stan
,
Stefan
Michalik
,
Chloe
Pearce
,
Marianne
Odlyha
,
Genoveva
Burca
,
Elena
Badea
Diamond Proposal Number(s):
[35634]
Open Access
Abstract: This study presents an ultrasound-assisted synthesis of β-cyclodextrin/hydroxyapatite composites to be used as green and safe auxiliaries in the tanning process. A combination of spectroscopic and non-spectroscopic techniques such as DLS (dynamic light scattering), ZP (zeta potential), XRD (X-ray diffraction), SEM (scanning electron microscopy) and ATR-FTIR (attenuated total reflectance-Fourier transform infrared spectroscopy) were used to thoroughly characterize the eight composites obtained by varying the ultrasound process parameters. While not cytotoxic, all composites had strong antibacterial action against Brevibacterium lines, Staphylococcus aureus, Escherichia coli, and Staphylococcus epidermis. All composites underwent lab-scale tanning tests, but only those exhibiting the most suitable set of tanning abilities underwent pilot-scale testing. The composites' interaction with the collagen matrix was assessed by micro-DSC (micro-differential scanning calorimetry), TG/DTG/DTA (thermal analysis), 1H unilateral NMR (proton nuclear magnetic resonance), ATR-FTIR, in-situ temperature synchrotron-based XRD and standard tests (UNI EN ISO 3380: 2015, UNI EN ISO 2589: 2016, UNI EN ISO 105- B02:2014). Thermal stability, dye penetration, thickness, colour fastness, surface appearance and microbiological protection were all improved for the leather treated with a small amount of composite added to the wet finish float. These findings demonstrate the benefits of β-cyclodextrin/hydroxyapatite composites as safe and sustainable tanning additives.
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Apr 2025
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[26876]
Abstract: The R2TP complex is a specialized HSP90 cochaperone essential for the maturation of macromolecular complexes such as RNAPII and TORC1. R2TP is formed by a hetero-hexameric ring of AAA-ATPases RuvBL1 and RuvBL2, which interact with RPAP3 and PIH1D1. Several R2TP-like complexes have been described, but these are less well characterized. Here, we identified, characterized and determined the cryo-electron microscopy (cryo-EM) structure of R2T from Arabidopsis thaliana, which lacks PIH1D1 and is probably the only form of the complex in seed plants. In contrast to R2TP, R2T is organized as two rings of AtRuvBL1-AtRuvBL2a interacting back-to-back, with one AtRPAP3 anchored per ring. AtRPAP3 has no effect on the ATPase activity of AtRuvBL1-AtRuvBL2a and binds with a different stoichiometry than in human R2TP. We show that the interaction of AtRPAP3 with AtRuvBL2a and AtHSP90 occurs via a conserved mechanism. However, the distinct architectures of R2T and R2TP suggest differences in their functions and mechanisms.
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Feb 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[17201]
Open Access
Abstract: N-utilization substance A (NusA) is a regulatory factor with pleiotropic functions in gene expression in bacteria. Archaea encode two conserved small proteins, NusA1 and NusA2, with domains orthologous to the two RNA binding K Homology (KH) domains of NusA. Here, we report the crystal structures of NusA2 from Sulfolobus acidocaldarius and Saccharolobus solfataricus obtained at 3.1 Å and 1.68 Å, respectively. NusA2 comprises an N-terminal zinc finger followed by two KH-like domains lacking the GXXG signature. Despite the loss of the GXXG motif, NusA2 binds single-stranded RNA. Mutations in the zinc finger domain compromise the structural integrity of NusA2 at high temperatures and molecular dynamics simulations indicate that zinc binding provides an energy barrier preventing the domain from reaching unfolded states. A structure-guided phylogenetic analysis of the KH-like domains supports the notion that the NusA2 clade is ancestral to the ribosomal protein eS7 in eukaryotes, implying a potential role of NusA2 in translation.
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Nov 2024
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Zhihan
Bo
,
Thomas
Rowntree
,
Steven
Johnson
,
Hilman
Nurmahdi
,
Richard J.
Suckling
,
Johan
Hill
,
Boguslawa
Korona
,
Philip C.
Weisshuhn
,
Devon
Sheppard
,
Yao
Meng
,
Shaoyan
Liang
,
Edward D.
Lowe
,
Susan M.
Lea
,
Christina
Redfield
,
Penny A.
Handford
Diamond Proposal Number(s):
[31353, 7495, 12346]
Open Access
Abstract: The Notch receptor is activated by the Delta/Serrate/Lag-2 (DSL) family of ligands. The organization of the extracellular signaling complex is unknown, although structures of Notch/ligand complexes comprising the ligand-binding region (LBR), and negative regulatory region (NRR) region, have been solved. Here, we investigate the human Notch-1 epidermal growth factor-like (EGF) 20-27 region, located between the LBR and NRR, and incorporating the Abruptex (Ax) region, associated with distinctive Drosophila phenotypes. Our analyses, using crystallography, NMR and small angle X-ray scattering (SAXS), support a rigid, elongated organization for EGF20-27 with the EGF20-21 linkage showing Ca2+-dependent flexibility. In functional assays, Notch-1 variants containing Ax substitutions result in reduced ligand-dependent trans-activation. When cis-JAG1 was expressed, Notch activity differences between WT and Ca2+-binding Ax variants were less marked than seen in the trans-activation assays alone, consistent with disruption of cis-inhibition. These data indicate the importance of Ca2+-stabilized structure and suggest the balance of cis- and trans-interactions explains the effects of Drosophila Ax mutations.
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Nov 2024
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