I04-1-Macromolecular Crystallography (fixed wavelength)
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Michael
Berlin
,
Jennifer
Cantley
,
Fabio
Broccatelli
,
Lin
Cao
,
Huifen
Chen
,
Tommy K.
Cheung
,
Andrew P.
Crew
,
Dean
Dinicola
,
Hanqing
Dong
,
Matthew
Grimmer
,
Brian D.
Hamman
,
Alicia
Harbin
,
Mingtao
He
,
Xiuxian
Hu
,
Alison J.
Hole
,
Thomas
Januario
,
Philip S.
Kerry
,
Xiangjia
Liu
,
Connor
Quinn
,
Christopher M.
Rose
,
Emma
Rousseau
,
Lawrence B.
Snyder
,
Leanna R.
Staben
,
Gan
Wang
,
Jing
Wang
,
Xiaofen
Ye
,
Robert L.
Yauch
,
Peter S.
Dragovich
Diamond Proposal Number(s):
[23279]
Open Access
Abstract: Modification of the VHL-binding fragments contained in proteolysis targeting chimeras (PROTACs) that potently degrade the BRM protein (also known as SMARCA2) improved degradation selectivity over the closely-related paralog protein BRG1 (SMARCA4). In particular, replacement of the phenyl-thiazole entity commonly employed in the generation of VHL-dependent PROTACs with pyridyl-thiazole, phenyl imidazole, and phenyl-nitrile moieties consistently improved the BRG1/BRM degradation selectivity ratios of multiple, structurally-diverse degrader compounds. Crystal structures of these new VHL-binding fragments in complex with the VHL protein were obtained to better understand their interactions. Some of these VHL alterations, the phenyl-nitrile substitution in particular, afforded molecules that displayed strong antiproliferative activities against BRM-dependent (BRG1-mutant) cancers but minimal potency toward wild-type cell lines. One such compound (21, G-9293) was profiled in detailed broad proteomics and chromatin accessibility experiments, and its biological properties were clearly differentiated from a less-selective BRM-degrader (5, A947) in the latter assessment. The highly selective molecule (21, G-9293) was also extensively profiled in vitro using a panel of lung cancer cell lines (defined by BRG1 or BRM status) along with several prostate cancer lines. It exhibited similar antiproliferation activity relative to the less-selective BRM-degrader (5, A947) against the lung lines but significantly diminished potency toward the prostate cancer cells.
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May 2026
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VMXi-Versatile Macromolecular Crystallography in situ
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Open Access
Abstract: Macromolecular crystallography provides mechanistic understanding of biological processes and can be applied in drug design. Nowadays, the use of robotic systems for crystal growth and diffraction analysis is widespread and high-throughput protein-to-structure pipelines for ligand and fragment screening are revolutionizing the field. However, the identification of crystals is still largely carried out through manual inspection, sometimes involving tens of thousands of images, which represents a bottleneck in an otherwise highly automated process. Here we describe AXIS, an AI-based Crystal Identification System combining the DINOv2 computer vision model, state-of-the-art transfer learning and MARCO, the largest crystallization dataset available to date, for automated crystal detection. AXIS can operate with both visible and UV light images and integrates a Lab-in-the-Loop approach combining ML and expert inputs for iterative learning and specialization. AXIS enables automated annotation of large crystallization image datasets with performance and accuracy comparable to that of human experts, and the Lab-in-the-Loop approach introduced here enables efficient adaptation to local conditions, facilitating widespread application, which has been a major limitation to date. AXIS can help to correct human errors in image annotation and removes critical bottlenecks, particularly in the context of extensive crystallization screens or high-throughput applications like fragment and ligand screening, unlocking the potential for higher levels of automation that are key in both fundamental and translational research.
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May 2026
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I24-Microfocus Macromolecular Crystallography
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Marina
Lucic
,
Johan
Glerup
,
Pierre
Aller
,
Danny
Axford
,
Nicholas
Devenish
,
Jaehyun
Park
,
Anastasiia
Shilova
,
Arturo
Landeros De La Isla
,
Richard W.
Strange
,
Tiankun
Zhou
,
Robin L.
Owen
,
Jonathan A. R.
Worrall
,
Michael A.
Hough
Diamond Proposal Number(s):
[19458, 28583]
Open Access
Abstract: Metalloenzymes containing a heme cofactor catalyse a wide range of oxidative reactions critical to life. Understanding the structure and electronic states of the heme across the catalytic cycle is essential in understanding the oxidative chemistry performed on the substrate. This work demonstrates in crystallo manipulation of the heme-iron oxidation state in a B-type dye-decolourizing peroxidase from Streptomyces lividans (DtpB) using multiple, complementary, serial crystallography approaches. Fixed-target drop-on-chip serial femtosecond crystallography (SFX) together with dose-resolved serial synchrotron crystallography (SSX) allowed DtpB to be driven between multiple iron oxidation states. Drop-on-chip addition of hydrogen peroxide with fixed-target SFX is used to generate a ferryl [Fe(IV)=O] species, while the X-ray-driven approach modulates the iron oxidation state, with an apparent two-electron reduction leading to a return to a ferric state. The formation and dose response of the Fe(IV)—O state is highly variable between the chemically identical heme groups of the DtpB hexamer, highlighting the importance of understanding the effect of the crystalline lattice on observed changes in time- and dose-resolved crystallography.
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May 2026
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I03-Macromolecular Crystallography
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Olesia
Werbowy
,
Maria
Håkansson
,
Sebastian
Dorawa
,
Aleksandra
Stefańska-Kaźmierczak
,
L. Anders
Svensson
,
Salam
Al-Karadaghi
,
Agata
Jurczak-Kurek
,
Karolina
Kwiatkowska-Semrau
,
Magdalena
Plotka
,
Olafur H.
Fridjonsson
,
Gudmundur O.
Hreggvidsson
,
Arnthór
Aevarsson
,
Sławomir
Dąbrowski
,
Anna‐karina
Kaczorowska
,
Tadeusz
Kaczorowski
Diamond Proposal Number(s):
[23282]
Abstract: We present the structural and functional characterization of a single-stranded DNA-binding protein (SSB-M5) identified from a hot spring metagenome in Vatnajökull National Park, Iceland. This small protein (136 aa; 15,695 Da) shares 100% amino acid sequence identity with two previously uncharacterized SSBs from hyperthermophilic Fervidobacterium species. Functional complementation assay demonstrated that SSB-M5 can substitute for Escherichia coli SSB in an ssb− mutant strain, confirming its biological activity. A recombinant C-terminally His-tagged SSB-M5 was overproduced, purified to homogeneity, and subjected to structural, biochemical, and biophysical analysis. The crystal structure revealed that SSB-M5 forms a dimer through a crystallographic twofold axis, with each monomer contributing to a large antiparallel β-sheet. The flat surfaces of the β-sheets from the two dimers are packed together via a second crystallographic twofold axis, forming a tetramer that serves as the functional unit of the SSB-M5. Electrophoretic mobility shift assays showed that SSB-M5, after heat treatment up to 100°C, forms stable DNA-protein complexes with the (dT)40 oligo. Quantitative analyses revealed that SSB-M5 binds (dT)70 oligonucleotide with very high affinity (KD = 72 ± 6 pM). Hill analysis indicated cooperative binding, yielding an EC50 of 141 pM and a Hill coefficient of 2. Moreover, inclusion of SSB-M5 in PCR reactions significantly enhanced amplification by eliminating non-specific products. Together, these findings identify SSB-M5 as a hyperthermostable, high-affinity single-stranded DNA-binding protein with potential applications in molecular biology and biotechnology.
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Apr 2026
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[32728]
Open Access
Abstract: Ferritins play a key role in iron management in organisms from all kingdoms of life. Excess iron is sequestered in mineral form within the hollow protein shell and can be liberated when supply becomes restricted. The protein consists of 24 isostructural monomeric units that pack with 4-, 3-, and 2-fold symmetry. Channels through the protein coat at the 3-fold axes of ferritins localised in the cytosol of animal cells contain a strictly conserved LCDFXEX ‘twin carboxylate’ motif, and have been shown to be the major iron entry route to animal ferritins, facilitating access to the H-chain intra-subunit catalytic ferroxidase centre. In the ferritin localised to the mitochondria of animals, there is natural variation within the residues lining this channel, such that the Asp residue of the twin carboxylate motif (Asp131) is not strictly conserved. Here we report X-ray crystallographic and solution kinetic studies of the properties of D131N variants of H-chain and mitochondrial ferritins. X-ray structures revealed significant perturbation of metal binding at the three-fold channels and ferroxidase centres of H-chain ferritin, but a relatively minor effect on mitochondrial ferritin. Likewise, kinetic data showed that rapid Fe2+ uptake was abolished in the D131N variant of H-chain ferritin, but less severely impacted in the equivalent variant of mitochondrial ferritin. Differences were also observed in rates of mineralisation and extent of iron release in the D131N variants of the two ferritins. The implications for the physiological role of mitochondrial versus cytosolic ferritin are discussed.
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Apr 2026
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Stefania
Patti
,
Simone A.
De Rose
,
Michail N.
Isupov
,
Ilya V.
Kublanov
,
Ilaria
Magrini Alunno
,
Sergio
Riva
,
Ivan
Bassanini
,
Eleonora
Dore
,
Christina
Stracke
,
Bettina
Siebers
,
Erica Elisa
Ferrandi
,
Jennifer A.
Littlechild
,
Daniela
Monti
Diamond Proposal Number(s):
[22563]
Open Access
Abstract: 2-Phosphoglycerate kinase (2PGK) and cyclic 2,3-diphosphoglycerate synthase (cDPGS) are key enzymes involved in the biosynthesis of cyclic 2,3-diphosphoglycerate (cDPG), an extremolyte known to stabilize proteins in hyperthermophilic Archaea. Using bioinformatics approaches, two candidate genes for each enzyme were identified from a range of thermophilic bacterial and archaeal genomes and metagenomes. Significantly, one gene pair derived from the Taman mud volcano metagenome represents the first indication of a bacterial cDPG biosynthesis pathway. The recombinant expression and purification of these enzymes paved the way to their biochemical and structural characterization. One 2PGK candidate displayed predominant ATPase activity, while the newly identified cDPGS variants demonstrated cDPG synthase activity. Moreover, one of the latter biocatalysts, Ts-cDPGS from the hyperthermophilic archaeon Thermococcus sibiricus, demonstrated a notable thermostability and its 3D structure was resolved at a resolution of 2.2 Å. These findings broaden our understanding of extremophilic enzyme systems and lay the foundation for biotechnological applications involving extremolyte production.
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Apr 2026
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[39203]
Abstract: Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a critical regulator of extracellular matrix remodelling and an important mediator of remyelination in demyelinating disorders such as multiple sclerosis. In addition, TIMP-1 has emerged as a promising therapeutic target in cancer due to its interaction with CD63, which promotes tumorigenic signalling and carcinogenesis. Although several structures of TIMP-1 bound to matrix metalloproteinases have been reported, no unbound structure with all druggable sites available has previously been reported. Here, we present the first unbound crystal structure of human TIMP-1, resolved at 1.95 Å resolution. Comparison with the MMP-bound complex reveals localized conformational changes and altered intramolecular hydrogen bonding in the unbound structure, indicating increased structural plasticity in the absence of the protease. Crystals were obtained in multiple conditions, but only two diffracted to high resolution. Although optimization and seeding did not significantly improve the morphology, the additive screen enhanced both the morphology and reproducibility and provided intrinsic cryoprotection. The resulting crystal form proved compatible with soaking-based screening campaigns, providing a robust structural basis for the discovery of TIMP-1 ligands with clinical potential.
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Apr 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[38144]
Open Access
Abstract: The use of monoclonal fragments antigen binding (Fabs) is a prevalent methodology facilitating protein structure determination via both crystallography and cryo-EM. The development of a synthetic Fab against the BRIL domain improved the accessibility of this approach, providing a general fiducial applicable to any protein of interest via the simple curation of a BRIL fusion protein. Here, we document the generation of a T7 Express ΔcybC strain allowing contaminant-free bacterial expression of the synthetic anti-BRIL Fab BAG2. We also report the crystal structure of BAG2 in complex with native cytochrome b562, a complex arising from expression in canonical Escherichia coli strains.
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Apr 2026
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I12-JEEP: Joint Engineering, Environmental and Processing
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Diamond Proposal Number(s):
[30778]
Open Access
Abstract: The thermo-mechanical response of reduced activation ferritic martensitic steel EUROFER97 is governed by dislocation-mediated deformation processes, which are critical for designing and predicting structural behavior under fusion-relevant loading conditions. In this study, the deformation behavior of batch-2 EUROFER97 is investigated using in-situ high energy synchrotron X-ray diffraction during monotonic tensile testing from room temperature to elevated temperature (nominal 500°C, corresponding to a measured value of 460°C) and asymmetric cyclic tensile loading at 460°C. Monotonic tensile tests are used to establish baseline temperature-dependent evolution of dislocation character and density. The primary focus is on asymmetric cyclic tensile loading, representative of ratcheting conditions, during which cycle-resolved analysis of dislocation recovery is realized. Diffraction line profile analysis is employed to quantify total dislocation density and the relative contributions of edge and screw components. The experimental data are interpreted using physically based recovery models, allowing extraction of activation energies and volumes associated with character-specific dislocation recovery. The results show that, while the overall deformation behavior is consistent with previously reported trends in EUROFER97 and related steels, the present work provides quantitative characterization of recovery parameters under non-fully reversed cyclic loading. These parameters offer physically meaningful input for dislocation-based constitutive models describing cyclic plasticity and ratcheting. The findings support the transferability of micromechanical deformation behavior across industrial batches, while emphasizing the importance of dislocation kinetics in designing and modelling fusion structural materials under thermo-mechanical loading.
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Apr 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[34349]
Open Access
Abstract: Recently, there has been growing interest in the function and physiological importance of human TANGO2 (transport and Golgi organization 2 homolog), particularly whether it acts as a heme-trafficking protein. To address this question, we experimentally determined the three-dimensional structure of TANGO2. Our crystallographic analysis indicates that interactions between heme and TANGO2 are nonspecific. Structural comparison of the TANGO2 crystal structure with known cysteine Ntn-hydrolases allowed us to identify a putative active site, catalytic residues and a substrate-binding cavity that correspond to residues that are mutated in pathogenic TANGO2 variants. Based on these features, we propose that TANGO2 may utilize fatty-acid derivatives as substrates, suggesting a potential role in lipid metabolism. Mutations in the human TANGO2 gene cause TANGO2 deficiency disorder, a multisystem, life-threatening disease with onset in early childhood. Together, our results provide new insights into the molecular function of TANGO2 and help to resolve the ongoing debate regarding whether it functions as a heme-trafficking protein.
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Apr 2026
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