I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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María
Conde-Giménez
,
Sandra
Salillas
,
María
Galiana-Cameo
,
Juan E.
Martínez-Oliván
,
Alejandro
Mahía
,
Manuel
Ledesma
,
Juan José
Galano-Frutos
,
Ritwik
Maity
,
Adrián
Velázquez-Campoy
,
María D.
Díaz-De-Villegas
,
Ramon
Hurtado-Guerrero
,
Javier
Sancho
Diamond Proposal Number(s):
[14739]
Open Access
Abstract: henylketonuria (PKU) is an inherited metabolic disorder caused by pathogenic variants in phenylalanine hydroxylase (PAH), leading to toxic phenylalanine accumulation and severe neurological complications if untreated. Current pharmacological treatment relies on tetrahydrobiopterin (BH4), which benefits only a subset of patients, highlighting a major unmet need for alternative therapies. Here, we combined high-throughput screening, computational modelling, and drug repurposing to identify pharmacological chaperones capable of rescuing PAH function. We evaluated 26 structurally diverse small molecules in HEK293T cells expressing wild-type PAH or one of eight PKU-associated variants spanning phenotypes from mild to classical disease. Chaperoning efficacy was strongly variant-dependent, and for every variant tested at least one compound produced a greater activity increase than BH4 under identical assay conditions. Notably, belinostat, a clinically approved histone deacetylase inhibitor, emerged as the most effective compound for several clinically severe variants. Mechanistically, functional rescue consistently correlated with an increased population of tetrameric, catalytically competent PAH, as quantified by mass photometry. The crystal structure of the PAH–belinostat complex (PDB ID: 9T1O), together with structural models for all compounds, provide a framework for rational optimization. These results establish a preclinical proof-of-concept for genotype-guided pharmacological chaperone therapy in PKU and support the feasibility of personalized, variant-specific treatment strategies.
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Jun 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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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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I04-Macromolecular Crystallography
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Philipp
Münick
,
Dimitrios-Ilias
Balourdas
,
Julianne S.
Funk
,
Büşra
Yüksel
,
Danai
Mavridi
,
Justin
Heftel
,
Birgit
Dreier
,
Jonas V.
Schaefer
,
Birgit
Schäfer
,
Stefan
Knapp
,
Tümay
Telatar
,
Baki
Akgül
,
Andreas
Plückthun
,
Thorsten
Stiewe
,
Andreas C.
Joerger
,
Volker
Dötsch
Abstract: The tumor suppressor p53 is the most frequently mutated protein in tumors and a target for drug development. More than 2000 cancer-associated p53 missense mutations have been reported, most of them located in the DNA-binding domain (DBD). Due to the low intrinsic thermostability of the latter, they often lead to unfolding at physiological temperature. Stabilizing the DBD with small molecules has been shown to be effective in reactivating the cavity-creating cancer mutant Y220C. Unfortunately, the majority of p53 mutants seem to lack druggable binding pockets for small molecules. Here we show that a designed ankyrin repeat protein (DARPin) that binds to the p53 DBD stabilizes temperature-sensitive (TS) p53 cancer mutants, thereby compensating for mutation-induced loss of stability. We determined high-resolution crystal structures of multiple DARPin–mutant p53 complexes, providing mechanistic insights into this mode of stabilization. Reporter gene assays across a comprehensive panel of cancer-associated mutants revealed reactivation of the majority of TS mutants, whereas DNA-contact mutants and those with local misfolding of the DNA-binding surface remained inactive, as expected. We demonstrate that this reactivation induces the transcription of canonical p53 target genes and elicits antiproliferative effects in cancer cell lines. A combination of this DARPin with an mRNA/lipid nanoparticle-based transfection approach may have the potential to reactivate most TS p53 mutants and resensitize cancer cells to chemotherapy.
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May 2026
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[31353, 38144]
Open Access
Abstract: Iron–sulfur (Fe-S) clusters are ubiquitous as redox-active protein cofactors, but it is often difficult to collect protein structures in which redox centres are in uniform and well-defined oxidation states. Using spinach ferredoxin I (Fdx) as a model redox protein, we demonstrate an integrated methodological pathway for electrochemical modulation of redox state in protein crystals coupled with in crystallo EPR and online-UV-visible spectroscopy to verify oxidation state. We show that Fdx crystals can be electrochemically reduced, reversibly, without compromising lattice integrity or X-ray diffraction quality. We show that redox levels can be precisely ascertained in crystallo via EPR and UV-visible spectroscopy, enabling a direct correlation between protein structure and electronic state of the metal cluster. In this way, we generate and compare ’oxidised’, ‘reduced’ and ‘re-oxidised’ structures of Fdx. Overall, our approach demonstrates a pipeline which will be applicable to structure-function studies of a wide range of electron-transfer proteins and redox enzymes.
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May 2026
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B23-Circular Dichroism
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[10442, 11638, 13119]
Open Access
Abstract: Achiral bivalent ligands capable of simultaneous occupation of both hormone binding sites of transthyretin generate a substantial and complex induced near ultraviolet circular dichroism spectrum during protein binding, revealing the dynamics of this process. Reduced temperature and pH slow the interaction and reveal two phases consistent with formation of an encounter complex and progression of the interaction through the core of the transthyretin tetramer. The x-ray structure of the protein-ligand complex confirms the endpoint of the binding trajectory and shows evidence of plasticity in the structure, with substantial disturbances of some mainchain and sidechains within and adjacent to the binding channel. This study highlights the effective complementarity of CD and x-ray investigations.
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May 2026
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I03-Macromolecular Crystallography
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Open Access
Abstract: The bacteriophages with single-stranded RNA (ssRNA) genomes (class Leviviricetes) are among the simplest known viruses that encode only three core proteins: a receptor-binding protein, a capsid protein, and an RNA-dependent RNA polymerase. The number of isolated ssRNA phages has remained very low, but the accumulating RNA metagenome data have uncovered a large variety of these viruses in many environments. Besides the core proteins, many of these genomes putatively encode additional proteins, which up to now have remained uncharacterized. We looked for non-conserved open reading frames (ORFs) in Leviviricetes sequences from the IMG/VR virus metagenome database and used sequence- and structure-based clustering to organize them into similarity groups. Potential ORFs were found throughout the ssRNA phage genomes but almost exclusively on the positive-sense RNA strand, suggestive of their protein-coding potential. The prevalence of the non-conserved ORFs varied in various phage lineages, and their distribution among different genome positions was markedly uneven. Most of the identified ORFs encode all-α proteins, a portion of which contain transmembrane segments that resemble a group of known ssRNA phage lysis proteins, while many others represent previously uncharacterized families of globular or semi-globular α-helical proteins. We additionally uncovered a major class of globular α/β proteins and experimentally determined the structure of a representative protein of this group. These results pave the way for further functional studies of novel ssRNA phage proteins for a better understanding of this diverse virus group.
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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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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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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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