I04-Macromolecular Crystallography
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Stéphane
Bourg
,
Matthieu
Place
,
Chloé
Copin
,
Apirat
Chaikuad
,
Thomas
Robert
,
Hanna
Holzmann
,
Susanne
Müller
,
Stéphane
Bach
,
Sandrine
Ruchaud
,
Stefan
Knapp
,
Frédéric
Buron
,
Sylvain
Routier
,
Pascal
Bonnet
Abstract: CLK1 is one of the four human isoforms of the cdc2-like (CLK) kinases that has been suggested as a therapeutic target in diverse diseases based on its important role regulating mRNA splicing. For example, CLKs and closely related kinases such as DYRK1A have been targeted in Alzheimer’s disease and other diseases in which splice site selection contributes to the disease development. Here we have developed an efficient in silico fragment-based ligand design approach to identify novel CLK1 inhibitors with excellent ligand efficiency based on an imidazo[2,1-b][1,3,4]thiadiazole fragment. More than one million docking poses were generated from 26,225 unique virtual compounds, and after applying several filtering steps, 11 compounds were selected, synthesized and their CLK1 inhibition and cellular potency were evaluated. Gratifyingly, inhibitor potencies were in excellent agreement with predicted values and crystallographic data of an inhibitor bound to CLK1 confirmed the unusual binding mode of the compounds.
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Oct 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[28402, 35088]
Open Access
Abstract: Keratin is an abundant structural fibrous protein and extremely recalcitrant biopolymer. β-Keratin is the major constituent of feathers, which, due to the widespread poultry industry, has become a major waste product. Biotechnological upcycling of feather waste has gained interest as various bacteria and fungi capable of degrading keratin have been isolated. These microorganisms produce proteases, termed keratinases, responsible for the enzymatic hydrolysis of keratin. The structural properties that confer keratinolytic activity to proteases are, however, not well understood. Here, we investigated the structure-function relationship of a subtilisin-like S8 endopeptidase (FerB) from the thermophile Fervidobacterium pennivorans strain T. FerB was crystallized and its structure solved to 1.5 Å resolution, revealing an auto-processed state where the pro-peptide domain is non-covalently attached to the catalytic domain. The carboxyl group of the scissile peptide bond is coordinated in the active site within hydrogen bonding distance of the catalytic triad’s serine residue. Unlike fervidolysin, no β-sandwich domains are present. However, a tyrosine-rich β-hairpin structure is found in the corresponding position within the FerB structure. Deletion of the β-hairpin reduced the protein’s integrity and keratinase activity.
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Aug 2026
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Open Access
Abstract: Radiation damage to macromolecular structures remains a significant challenge for accurate structure solution by X-ray crystallography, leading to incorrect structural and chemical interpretation of the data. Site-specific radiation damage is insidious, typically unidentifiable solely from summary statistics, and is primarily discussed with reference to the predominant forms: disulfide-bond cleavage, metal-centre reduction and decarboxylation of acidic residues. A method is presented for identifying potentially oxidatively damaged cysteines by interrogating the accuracy of the built model within the electron density and the geometry of the difference density peaks surrounding a cysteine. We also highlight that cysteines located within protein active sites or that are in hydrolases are predisposed to this damage.
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Jul 2026
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I24-Microfocus Macromolecular Crystallography
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Jos J. A. G.
Kamps
,
Philip
Hinchliffe
,
Johan
Glerup
,
Emily I.
Freeman
,
Pauline A.
Lang
,
Catherine
Tooke
,
Michael
Beer
,
Laura
Parkinson
,
Do-Heon
Gu
,
Sehan
Park
,
Nicholas
Devenish
,
Tiankun
Zhou
,
Anastasya
Shilova
,
Samanpreet
Kaur
,
Patrick
Rabe
,
Christopher J.
Schofield
,
James
Spencer
,
Jaehyun
Park
,
Robin L.
Owen
,
Allen M.
Orville
,
Pierre
Aller
Diamond Proposal Number(s):
[25260]
Open Access
Abstract: We describe the design and implementation of a drop-on-fixed-target method for time-resolved serial crystallography at both synchrotron and XFEL facilities. A piezoelectric droplet dispensing pipette is employed for addition of picolitre volume aqueous droplets (∼40–90 pl; ∼40–55 µm diameter sphere), containing (co-)substrate(s) or ligand(s), onto enzyme microcrystals previously loaded into the trapezoidal wells of an etched crystalline silicon fixed-target chip containing 25 600 wells in a high-density, square grid with 125 µm centre-to-centre well spacing. These features demand exquisite accuracy and thereby constrain motion controls to enable robust time-resolved crystallographic studies. The system was tested with three enzyme systems, comprising lysozyme and two β-lactamases, CTX-M-15 and AmpCEC. Mitigation strategies for cross-well contamination, including the implementation of interleaved controls, are described; the overall performance of the system at synchrotron and X-ray free-electron laser facilities was evaluated. This drop-on-fixed-target method is a reliable framework for time-resolved crystallography and will improve the consistency of measurements across facilities.
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Jul 2026
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I03-Macromolecular Crystallography
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Barbara
Forte
,
Fiona
Bellany
,
Peter S.
Campbell
,
Giulia
Chemi
,
Alice
Dawson
,
Mark
Anderson
,
Yaw
Aniweh
,
Anna Y.
Burkhard
,
Anna Caroline Campos
Aguiar
,
Alisje
Churchyard
,
Caitlin A.
Cooper
,
Amália
Dos Santos Ferreira
,
Mufuliat Toyin
Famodimu
,
Francis G.
Fang
,
Xiao
Hu
,
Tonnie
Huijs
,
Delphine
Baud
,
Chimed
Jansen
,
María Belén
Jiménez Díaz
,
Roger
Bonnert
,
Susan
Boyd
,
Benigno
Crespo-Fernández
,
Branko
Mitasev
,
Simone
Montagna
,
Sachel
Mok
,
Dinakaran
Murugesan
,
Sunil K.
Narwal
,
Neil R.
Norcross
,
John
Okombo
,
Heekuk
Park
,
Caroline
Peet
,
Dhelio B.
Pereira
,
John M.
Post
,
Janette
Reader
,
Jennifer
Riley
,
David A.
Robinson
,
Raku
Shinkyo
,
Frederick R. C.
Simeons
,
Laura
Simpson
,
Alasdair
Smith
,
Dennis
Smith
,
Josefine
Striepen
,
Carolina B. G.
Teles
,
Rianne
Van Der Laak
,
Anne-Catrin
Uhlemann
,
Amélie
Vantaux
,
Caroline
Wilson
,
Benoît
Witkowski
,
Gavin
Wood
,
Tomas
Yeo
,
Fabio
Zuccotto
,
Iñigo
Angulo-Barturen
,
Jake
Baum
,
Judith M.
Bolscher
,
Rafael Victorio Carvalho
Guido
,
Lyn-Marié
Birkholtz
,
Michael J.
Delves
,
Laurent
Dembele
,
David A.
Fidock
,
Francisco Javier
Gamo
,
Dennis E.
Kyle
,
Steven P.
Maher
,
Jean
Popovici
,
Chris
Walpole
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Fabian
Gusovsky
,
Paul A.
Willis
,
Kevin D.
Read
,
Ian H.
Gilbert
,
Beatriz
Baragaña
Diamond Proposal Number(s):
[10071, 26793]
Open Access
Abstract: A fused dihydropyrrolidino-pyrimidine hit with low lipophilicity and excellent ligand efficiency was identified in a biochemical screen of the Global Health Chemical Diversity Library (GHCDL) against Plasmodium lysyl-tRNA synthetase (KRS). Structure-guided lead optimization delivered analogues with potent parasite growth inhibition, excellent biochemical and cellular selectivity (>1000-fold), and oral efficacy in the malaria NOD-scid-IL2Rγnull (SCID) mouse model. Structural information and computational methods were deployed to identify a potent and selective basic KRS inhibitor (30) with an extended half-life to reduce the dose regimen to a single-dose cure. Compound 30 displayed a long half-life across preclinical species, favorable safety, and activity across Plasmodium species as well as against drug-resistant and sensitive P. falciparum strains and field isolates. Unfortunately, 30 lacked oral bioavailability, which could not be mitigated with a prodrug approach. Nevertheless, learnings from this series will assist future KRS programs in delivering a clinical candidate with this novel mode of action.
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Jun 2026
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Julian M.
Ludäscher
,
Emma
Scaletti Hutchinson
,
Guillem
Vila-Julià
,
Ann-Sofie
Jemth
,
Saher
Shahid
,
Elisee
Wiita
,
Israel
Cabeza De Vaca
,
Szymon
Pach
,
Lukas
Gajdos
,
Swati
Aggarwal
,
Ellen
Walse
,
Oliver
Mortusewicz
,
Thomas
Helleday
,
Jens
Carlsson
,
Pal
Stenmark
Diamond Proposal Number(s):
[29948]
Open Access
Abstract: Human single-strand-selective monofunctional uracil DNA glycosylase 1 (hSMUG1) removes uracil, 5-hydroxymethyluracil (5hmU) and 5-fluorouracil (5FU) from DNA, thereby initiating the base excision repair (BER) process. hSMUG1 is important for maintaining genomic integrity and plays a significant role in cancer biology. Here, we present the structures of hSMUG1, including complexes with products (uracil and 5FU) and an enzyme-product complex of hSMUG1 with double-stranded DNA (dsDNA). Analysis of our hSMUG1-dsDNA complex reveals how uracil is flipped out of the dsDNA for excision and identifies key residues that we confirm to be critical for both DNA binding and enzymatic activity. Furthermore, our hSMUG1 substrate complexes, molecular dynamics simulations and neutron diffraction data suggest a mechanism by which the substrate uracil rotates following base excision. The structural and functional information presented here will be highly useful for the future development of inhibitors and/or activators targeting hSMUG1.
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Jun 2026
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B22-Multimode InfraRed imaging And Microspectroscopy
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Open Access
Abstract: Measuring live cells by FTIR spectroscopy is challenging due to their small size and the absorbance of water in the mid-IR region. However, measuring cells in their live state is important to observe changes in the biological processes of cells that are unaffected by fixation and drying processes. Recently, ZnS hemispheres were used to sandwich live cells in a 6 µm layer of cell medium, which simultaneously limit the absorbance of water and increase the spatial resolution by x2.25, thereby enabling high quality spectra to be acquired from living cells. So far, this method has been used as an imaging technique to showcase the distribution of biomolecules within a single cell. In this work, we present an alternative use of these ZnS hemispheres as a high throughput screening tool. We obtained high quality spectra of a single cell at a measurement rate of ∼1 min/cell. We’ve applied this technique to observe the biochemical effects of various polystyrene microplastics on two mammalian cell lines (J774A.1 and A549), however the method can easily be expanded to other cell lines, microplastics, and alternative xenobiotics.
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Jun 2026
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[37221]
Open Access
Abstract: Biocatalytic cascades offer a promising route for CO2-fixation into valuable chemicals, addressing the urgent need for efficient, sustainable technologies to reduce CO2 emissions. This paper describes an enzymatic route converting gaseous CO2 and acetaldehyde into enantiopure lactic acid, widely used in diverse industries. A newly characterized pyruvate decarboxylase from Neoasia chiangmaiensis (NcPDC) enabled acetaldehyde carboxylation to pyruvate. To suppress the competing carboligation to acetoin, acetaldehyde was reversibly trapped with Tris. Pyruvate was reduced to lactate by lactate dehydrogenase, coupled with glucose dehydrogenase for NADH regeneration via D-glucose oxidation to D-gluconic acid. Up to 65% lactate yield was achieved. Repeated acetaldehyde dosing resulted in a 27 mM titer, representing a >100-fold improvement over previous reports. At 0.5 L scale, using a gas mixture mimicking industrial-grade CO2, we obtained 21 mM D-(–)-lactic acid, 42% yield and >98% e.e., demonstrating scalability and robustness. Finally, replacing the D-(–)-selective lactate dehydrogenase with an L-(+)-selective variant at small scale enabled production of L-(+)-lactic acid at 41% yield and >93% e.e, allowing switchable access to either enantiomer. A volumetric productivity of 1.1 × 10−2 g L−1 h−1 ranks among the most efficient minimal enzymatic routes developed to date for CO2-to-lactate conversion.
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Jun 2026
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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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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26793]
Open Access
Abstract: Bacteria coexist in polymicrobial communities where they engage in complex interactions, including interbacterial antagonism. The environmental bacterial pathogen Chromobacterium violaceum possesses an active type VI secretion system (T6SS), which relies mainly on VgrG3 for its activity and role in interbacterial competition. However, the arsenal of toxic effectors delivered by this T6SS remains unknown. Here, we identify the repertoire of C. violaceum T6SS effectors and characterize a novel antibacterial Rhs-family effector, RhsF (Rhs with a FIX domain), and its cognate immunity protein, RhsFi. Using mass spectrometry analyses of secreted proteins and proteins co-immunoprecipitated with VgrG3, we identified six novel effector candidates, namely four phospholipases, a protein of unknown function, and the previously-uncharacterized Rhs protein, RhsF (CV_1431). RhsF contains an N-terminal FIX domain and can intoxicate susceptible bacteria in a T6SS-dependent manner. The action of the C-terminal toxin domain of RhsF (RhsF-CT) is prevented by RhsFi (CV_1430), confirming that RhsF-RhsFi comprises an effector-immunity pair. The structure of the RhsF-CT/RhsFi complex determined by X-ray crystallography (1.85 Å resolution) revealed that RhsF-CT shares structural similarity with ADP-ribosyltransferase toxins and that RhsFi inhibits toxicity via direct occlusion of the RhsF-CT catalytic site. Functional assays indicated that RhsF-CT ADP-ribosylates RNA in vitro and that RhsF toxicity requires a catalytic triad composed of R1403, Y1456, and E1497 residues. Overall, our findings reveal effectors secreted by the T6SS of C. violaceum, establish RhsF as a potent antibacterial toxin, and confirm T6SS-dependent delivery of a FIX-containing Rhs protein, expanding the known repertoire of bacterial arms involved in microbial competition.
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Jun 2026
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