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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Open Access
Abstract: A comparison is provided between the use of electrons and X-rays for collecting diffraction data from small protein crystals and imaging data from cells and tissues. The paper contains a review element written to enable an understanding of the relevant properties of electrons by those (including one of the authors) more used to X-ray imaging and diffraction. Radiation-damage mechanisms, sample thickness-dependent dose efficiency and energy-dependent scattering cross sections are discussed, together with contrast mechanisms in electron and X-ray imaging. Crossover points are calculated for diffraction from crystals where electrons and X-rays could yield equivalent data quality in the presence of radiation damage. Increasing the electron energy from 300 to 1000 keV results in a ∼43% rise in the electron/X-ray crossover point. However, the maximum information coefficient (useful signal/absorbed dose) for electrons alone occurs for a 250 nm crystal examined at around 800 keV. The impact of inelastic scattering on electron imaging and diffraction is examined, including its role in coherence loss, Bragg spot broadening and background elevation. The possibilities are investigated for locating regions of interest using X-rays for subsequent higher resolution imaging using electrons. For locating a 30 nm diameter protein or virus, the required X-ray dose would be much less than the tolerable dose for electron imaging at 0.5 or 0.25 nm. Overall, these findings are relevant for imaging at different length scales while minimizing dose-induced structural damage.
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May 2026
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Open Access
Abstract: Analytical expressions for the damage-limited resolution (DLR) are developed and applied to X-ray and electron imaging of beam-sensitive specimens, allowing for variation of the characteristic radiation dose with spatial resolution. The dependence of DLR on specimen thickness is illustrated for the common modes of X-ray and transmission electron-microscope imaging. Similarities and differences between the radiolysis damage caused by electrons and X-rays are discussed. The meaning of a `Bragg boost' in diffracted intensity is discussed.
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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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Holger
Greschik
,
Florian
Friedrich
,
Ludwig
Seifert
,
Farnoush
Mousavizadeh
,
Francesco
Fiorentino
,
Johannes
Walz
,
Lin
Zhang
,
Jianyu
Li
,
Emanuele
Fabbrizi
,
Stefano
Tomassi
,
Farhad
Panahi
,
Niklas
Papenkordt
,
Silas L.
Wurnig
,
Johannes
Osterroth
,
Anna M.
Strasser
,
Jan
Ruprecht
,
Aurélien F. A.
Moumbock
,
Martin
Hügle
,
Manuela
Sum
,
Ling
Peng
,
Sheng
Wang
,
Adina A.
Baniahmad
,
Laura
Pulido-Cortés
,
H. Th. Marc
Timmers
,
Ralf
Flaig
,
Eric
Metzger
,
Bernhard
Breit
,
Oliver
Einsle
,
Stefan
Günther
,
Dante
Rotili
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Antonello
Mai
,
Roland
Schüle
,
Manfred
Jung
Open Access
Abstract: The chromatin remodeler CHD1, a regulator of gene activity and potential drug target in prostate cancer (PCa), contains a tandem chromodomain (tCD) binding histone H3 trimethylated at lysine 4 (H3K4me3). We developed the first submicromolar inhibitors (2n and 2s) that target the H3K4me3 binding site of the CHD1 tCD with Kd values of 0.15 μM and 0.14 μM, respectively. Co-crystal structures of these quinoline-based compounds revealed aromatic cage interactions and extended ligand contacts in other parts of the H3K4me3 peptide pocket as the main determinants of high-affinity ligand binding. 2n and 2s engage endogenous CHD1 in cell lysates or the exogenous CHD1 tCD in cells. Furthermore, we provide evidence for selectivity against a panel of methyl-lysine readers and epigenetic enzymes as well as impairment of PCa cell viability. Due to their high potency and defined binding mode, our ligands offer new directions for further optimization.
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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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I24-Microfocus Macromolecular Crystallography
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Katleen
Van Nerom
,
Andres
Ainelo
,
Kyo
Coppieters ‘t Wallant
,
Ariel
Talavera-Perez
,
Dannele
Echemendia-Blanco
,
Sarah
Peeters
,
Brahim
El Khalfaoui Oulali
,
Hedvig
Tamman
,
Tatsuaki
Kurata
,
Mohammad
Roghanian
,
Chloé
Martens
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Els
Pardon
,
Jan
Steyaert
,
Vasili
Hauryliuk
,
Abel
Garcia-Pino
Open Access
Abstract: Long RSH enzymes, Rel and RelA, are master regulators of bacterial (p)ppGpp alarmones levels. Bifunctional Rel transitions between a compact hydrolysis-competent (HDON) state, a relaxed catalytically inactive (HDOFF/SYNTHOFF) state, and an elongated synthesis-competent (SYNTHON) state, whereas RelA samples only the latter two. The distribution of these states is controlled by starved ribosomes and regulatory proteins, including DarB, EIIANtr, ACP, NirD and YtfK. Here, we identify and characterize camelid nanobodies that act as selective allosteric modulators by stabilizing Rel and RelA in defined conformational states. Nanobodies that sequester the TGS domain of RelA prevent activation by deacylated tRNA on starved ribosomes, strongly inhibiting (p)ppGpp synthesis and suppressing Escherichia coli virulence in an animal model. Nb898 stabilizes Rel in the open SYNTHON state, enhancing synthesis while suppressing hydrolysis, whereas Nb585 traps Rel in a hydrolysis-competent HDON/SYNTHOFF conformation. Structural and biochemical analyses show that nanobodies, like endogenous allosteric regulators, restrict the conformational landscape of long RSH enzymes, establishing them as powerful tools for dissecting RSH function and as frameworks for developing protein-based RSH modulators.
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May 2026
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: The organic cation choline is essential for eukaryotic metabolism. Recently, the feline leukemia virus subgroup C receptor–related (FLVCR, SLC49) family was demonstrated as central for basal choline transport, questioning the role of the choline transporter-like (CTL, SLC44) family in this capacity. Here, we use Xenopus laevis oocytes to confirm that FLVCR1 (SLC49A1) and FLVCR2 (SLC49A2) proteins are choline transporters. CTL1 (SLC44A1) does not transport choline under the same conditions, supported by other CTL proteins, Arabidopsis thaliana CherI and Saccharomyces cerevisiae PNS1, which also display no choline transport activity. We present the atomic structures of FLVCR2, CTL1, and PNS1. The 3.4 Å cryo-EM structure of FLVCR2 has choline in the binding pocket. The 3.3 Å cryo-EM structure of CTL1 and the 2.7 Å crystal structure of PNS1 reveal an unusual protein fold, weakly related to the mitochondrial carrier family (SLC25). The unusual fold appears incompatible with transmembrane transport and implies a different and, so far, unknown function for CTL proteins. Our results support FLVCR proteins as choline transporters and suggest a nontransport role for CTL proteins.
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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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Michael J.
Lambrecht
,
Jun
Liang
,
Peter Man-Un
Ung
,
Malcolm P.
Huestis
,
Bing-Yan
Zhu
,
Lisa M.
Barton
,
Georgette M.
Castanedo
,
Jason R.
Zbieg
,
Robin
Larouche-Gauthier
,
Araz
Jakalian
,
Jean-Philippe
Leclerc
,
Arun
Yadav
,
Pouyan
Haghshenas
,
Samuel
Aubert-Nicol
,
Hossein
Ismaili
,
Liang
Zhao
,
Mélissa
Leblanc
,
Jian
Wang
,
Shouliang
Wang
,
Qiuyue
Wang
,
Thomas
Garner
,
Sophia
Tan
,
Madeleine
Prangley
,
Fabio
Broccatelli
,
Jodie
Pang
,
Jeremy
Murray
,
Christine
Yu
,
Peter
Hsu
,
Sascha
Rutz
,
Satoko
Kakiuchi-Kiyota
,
Isabel
Ishizuka
,
Dennis H.
Leung
,
Ponien
Kou
,
Linda
Bao
,
Xiaojing
Wang
Abstract: Casitas B-lineage lymphoma-b (Cbl-b), an E3 ubiquitin ligase, is a key negative regulator of immune function, and its inhibition is a promising strategy for cancer immunotherapy. Here, we show the optimization of a series of inactive-state Cbl-b inhibitors to improve their potency and pharmacokinetic properties. Through systematic modification of a benzylic amine and a linker region, compound 16 was identified, which demonstrates a favorable balance of biochemical potency, cellular activity, and in vitro ADME properties. Despite exhibiting high IV clearance in vivo, compound 16 achieved oral exposures sufficient to demonstrate significant tumor growth inhibition in a murine CT26 colon-cancer model.
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May 2026
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