I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Daming
Zhou
,
Abhay
Kotecha
,
James T.
Kelly
,
Peng-Nien
Huang
,
Yi-Yin
Chen
,
Thomas S.
Walter
,
Helen M. E.
Duyvesteyn
,
Raymond J.
Owens
,
Shu-Yuan
Ho
,
Tzou-Yien
Lin
,
Elizabeth E.
Fry
,
Jingshan
Ren
,
Kuan-Ying A.
Huang
,
David I.
Stuart
Diamond Proposal Number(s):
[14744]
Open Access
Abstract: EV-A71 has been responsible for recent severe HFMD outbreaks. We report structures for 12 potently neutralizing human anti–EV-A71 monoclonal antibody Fabs, alone and complexed with virus. Most recognize the native antigenic state with epitopes that span interfaces, together covering 85% of the capsid surface. The majority (8 of 12) bind the canyon, while the others cluster around the icosahedral two- and threefold axes. Blocking SCARB2 receptor binding likely contributes to neutralization for all, and a subset induces empty particles. A predominant gene family (IGHV4-39) does not dictate a common binding pose. Long CDR-H3 loops are frequently key to binding, especially at the canyon, suggesting that antigenicity data based on antibodies with shorter CDR3s (e.g., murine) may be misleading. This dataset reveals neutralization mechanisms for recently circulating EV-A71 genotypes, which will inform immunotherapies. We demonstrate synergy in vitro between canyon binding and both two- and threefold binding antibodies to increase neutralization potency.
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Jun 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
,
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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Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[25452, 32707]
Open Access
Abstract: Antimicrobial resistance is driving the search for new antibiotics and a greater understanding of their mechanism of action. Doxycycline is amongst the most-prescribed antimicrobials. It demonstrates a particularly low minimum inhibitory concentration against the zoonotic pathogen Coxiella burnetii. Doxycycline canonically targets the bacterial ribosome by blocking tRNA binding at the decoding centre (A site) of the small subunit. Using cryo-electron microscopy, we analysed doxycycline binding to C. burnetii and Escherichia coli ribosomes. Both structures reveal doxycycline binding at the exit tunnel in the large subunit. In C. burnetii three doxycycline molecules stack to block the tunnel. In E. coli one doxycycline molecule triggers a major change in the conformation of the ribosome. This rearrangement of the peptidyl transferase centre blocks tRNA binding and nascent chain accommodation, abolishing interactions that are fundamental to ribosome function. We identify a distinct ribosomal protein in the C. burnetii large subunit and characterise an additional member of the prokaryotic ribosome hibernation-promoting factor family. These insights into ribosome function and antibiotic action may aid the development of new ribosome inhibitor antibiotics.
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Jun 2026
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I04-Macromolecular Crystallography
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Jogi
Madhuprakash
,
Amirali
Toghani
,
Hsuan
Pai
,
Madia
Harvey
,
Adam R.
Bentham
,
Benjamin A.
Seager
,
Enoch Lok Him
Yuen
,
Juan Carlos
De La Concepcion
,
David M.
Lawson
,
Clare E. M.
Stevenson
,
Angel
Vergara-Cruces
,
Lida
Derevnina
,
Tolga O.
Bozkurt
,
Mark J.
Banfield
,
Sophien
Kamoun
,
Mauricio P.
Contreras
Diamond Proposal Number(s):
[25108]
Open Access
Abstract: Pathogens counteract central nodes of NLR immune receptor networks to suppress immunity. However, the mechanisms by which pathogens hijack helper NLR pathways are poorly understood. We show that an effector from the late blight pathogen Phytophthora infestans interacts with the host protein NbTOL9a and a helper NLR to suppress immunity. We solved the crystal structure of the RXLR-LWY effector AVRcap1b in complex with the ENTH domain of NbTOL9a. The structure revealed that, unlike other RXLR-LWY effectors, AVRcap1b has a previously unidentified L-shaped fold that defines a distinct structural family of effectors in the genus Phytophthora. We defined the AVRcap1b/NbTOL9a binding interface and designed effector mutants that do not bind NbTOL9a, impairing immune suppression. This suggests that ENTH binding is required for full virulence activity. Last, we show that AVRcap1b associates specifically with activated NbNRC2 independently of NbTOL9a binding. We propose a model in which the effector interconnects NbNRC2 with the NbTOL9a pathway. Our results illustrate a previously uncharacterized pathogen mechanism to hijack NLR pathways and suppress immunity.
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Jun 2026
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I03-Macromolecular Crystallography
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Abstract: The widespread emergence of bacterial resistance to β-lactam antibiotics, driven primarily by serine β-lactamases (SBLs), continues to threaten the clinical utility of this cornerstone class of antibacterial agents. Although β-lactamase inhibitors such as sulbactam and tazobactam have historically restored the activity of partner antibiotics, their effectiveness has been eroded by the proliferation of inhibitor-resistant enzymes, particularly class C (AmpC) and class D (OXA) β-lactamases. Consequently, there remains a pressing need for new inhibitor scaffolds that combine potent enzyme inhibition with improved chemical stability and tunable physicochemical properties.
This thesis explores sulfoximine substitution on the penam scaffold as a strategy to expand the chemical and biological properties of β-lactamase inhibitors. Sulfoximines offer distinct advantages over classical sulfones, including stereochemical control, dual hydrogen-bond donor/acceptor capability, and enhanced opportunities for structural diversification. Leveraging recent advances in nitrene-transfer chemistry, robust and stereocontrolled synthetic routes to penam-sulfoximines were developed from sulbactam-derived sulfoxides. Both (S)- and (R)-configured penam sulfoximines were prepared, including synthetically challenging NH-sulfoximines that closely mimic the parent sulbactam framework. Single-crystal X-ray diffraction confirmed stereochemical integrity and structural assignments.
The scope and limitations of sulfoximine N-functionalization were systematically investigated, enabling access to amide, urea, and N-aryl derivatives while revealing intrinsic stability constraints of the penam core under basic or reductive conditions. These studies establish a versatile synthetic platform for the generation of structurally diverse penam-sulfoximines suitable for biological evaluation.
Biochemical and microbiological studies demonstrated that penam-sulfoximines are potent inhibitors of clinically relevant SBLs, including AmpC and OXA-type enzymes. A free NH penam-sulfoximine displayed particularly strong enzyme inhibition and measurable antibacterial activity against Acinetobacter baumannii, supported by enzyme inhibition assays, protein-binding studies, antimicrobial susceptibility testing, and structural characterization of enzyme–inhibitor complexes. However, many substituted sulfoximines exhibited reduced stability under physiological conditions, highlighting a trade-off between potency and chemical robustness.
Overall, this work establishes penam-sulfoximines as a chemically accessible and biologically active class of β-lactamase inhibitors, delineates key structure–stability–activity relationships, and provides a foundation for the future design of sulfoximine-based agents targeting resistant Gram-negative pathogens.
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Jun 2026
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I03-Macromolecular Crystallography
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Monisha
Singha
,
Liam A.
Wilson
,
Elisabete C.
C. M. Moura
,
Maria M.
Trush
,
Karina
Calvopina
,
Gurleen
Kaur
,
Greta
Zaborskytė
,
Toms
Kalniņš
,
Tharindi
Panduwawala
,
Matthew J.
Bowen
,
Matthew J.
Beech
,
Jurgen
Brem
,
Peter J.
Mchugh
,
Edgars
Suna
,
Timothy R.
Walsh
,
Christopher J.
Schofield
,
Alistair J. M.
Farley
Diamond Proposal Number(s):
[31353]
Open Access
Abstract: Use of the clinically vital β-lactam antibiotics is increasingly compromised by resistance, commonly mediated by β-lactamases. While clinically used serine-β-lactamase (SBL) inhibitors have long been available, metallo-β-lactamase (MBL) inhibitors are not yet approved for clinical use. We report the structure-guided development of pyrrole-2-carboxylic acid derivatives as potent inhibitors of the clinically important di-Zn(II) ion containing B1 MBLs (NDM-1, VIM-1, VIM-2, IMP-1). Crystallographic studies reveal the pyrrole-2-carboxylic acids inhibit B1 MBLs via active site Zn(II)-coordination of the inhibitor carboxylate and trapping of the di-Zn(II) ion bridging hydroxide, the latter of which reacts with the substrate β-lactam ring during hydrolysis. Appropriately derivatized pyrrole-2-carboxylic acids enhance the activity of carbapenems against MBL producing Gram-negative clinical isolates. The results support further development of metalloenzyme inhibitors that exploit binding to structural or catalytically important water molecules, an approach which may help in achieving selectivity over other metalloenzymes compared to metal-chelation based approaches.
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May 2026
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I03-Macromolecular Crystallography
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Huazhang
Shu
,
Julian M.
Ludäscher
,
Sushma
Sharma
,
Seher
Alam
,
Lilian
Frank
,
Emma
Scaletti Hutchinson
,
Marianna
Tampere
,
Chloé
Lévêque
,
André B. P.
Van Kuilenburg
,
Nicholas C. K.
Valerie
,
Mikael
Altun
,
Andrei
Chabes
,
Pal
Stenmark
,
Sean G.
Rudd
,
Si Min
Zhang
Diamond Proposal Number(s):
[36026]
Open Access
Abstract: Molnupiravir is a nucleoside analogue antiviral drug against RNA viruses, including its clinical indication SARS-CoV-2. Whilst its mechanism-of-action is well defined, host factors that regulate its therapeutic responses have not been thoroughly deciphered and characterized. Here we show that uridine cytidine kinases (UCKs), key enzymes in pyrimidine salvage, effectively phosphorylate and thereby bioactivate N4-hydroxycytidine (NHC) – the active compound of molnupiravir, thus dictating its anti-SARS-CoV-2 efficacy and furthermore selectivity. In vitro, both isoforms of UCKs (UCK1 and UCK2) effectively phosphorylated NHC, where the structural basis of the catalysis was further deciphered via the first complete substrate bound co-crystal structure of UCK, i.e., UCK1-NHC-AMPPNP. In SARS-CoV-2-infected cells, UCK2 knockdown via siRNA hampered the intracellular accumulation of the tri-phosphorylated antiviral metabolite of NHC, resulting in a 10-fold reduction of the antiviral efficacy, and surprisingly, 2-fold reduction of its selectivity, which were critically recapitulated in a dose-dependent manner using a pan-UCK inhibitor. Altogether, this work underscores UCKs as pivotal players in upholding molnupiravir efficacy and therapeutic window of molnupiravir, and furthermore as pharmacologically tractable targets for tailoring the drug response.
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May 2026
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I03-Macromolecular Crystallography
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Sarah
Hijazi
,
Francesco
Marchesani
,
Marialaura
Marchetti
,
Valeria
Buoli Comani
,
Paul
Brear
,
Barbara
Campanini
,
Luca
Ronda
,
Serena
Faggiano
,
Eleonora
Gianquinto
,
Somayeh
Asgharpour Hassankiade
,
Barbara
Rolando
,
Francesca
Spyrakis
,
Carlotta
Compari
,
Loretta
Lazzarato
,
Omar
De Bei
,
Emanuela
Frangipani
,
Stefano
Bettati
Diamond Proposal Number(s):
[25402]
Open Access
Abstract: Infections caused by Staphylococcus aureus depend on its ability to access essential nutrients, including acquiring iron from human hemoglobin (Hb) through the iron-regulated surface determinant (Isd) system. The compound 4-[(2-{[5-(1H-indol-3-yl)-1,3,4-oxadiazol-2-yl]sulfanyl}acetyl)amino]benzoic acid (C35) was recently identified as a promising antimicrobial agent for its ability to bind Hb and hamper its interaction with the staphylococcal hemophore IsdB in vitro. Here, we show that C35 inhibits S. aureus growth by targeting the hemophore-driven iron-acquisition system, highlighting its potential as an inhibitor and validating hemophores as antibacterial targets. Furthermore, for drug design purposes, we solved the X-ray structure of Hb:C35 complex. In contrast to the predicted binding pose, C35 binds tetrameric Hb in a cleft between the α subunits, stabilizing a relaxed conformation (R2) and increasing Hb oxygen affinity. This serendipitous result hints to C35 as a promising scaffold for developing compounds with diverse, or even dual, therapeutic aims, with antimicrobial and Hb-modulating activity.
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May 2026
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Open Access
Abstract: Respiratory syncytial virus (RSV) remains a leading cause of severe lower respiratory tract disease in infants, older adults, and immunocompromised individuals. Over the past decade, advances in structural biology, particularly cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), have transformed our understanding of RSV architecture, dynamics, and the mechanisms of entry and replication. High-resolution structures of the prefusion F glycoprotein (pre-F) and its complexes with neutralizing antibodies established the rationale for structure-guided antigen stabilization and directly enabled the development of the first licensed RSV vaccines. Complementary structures of the ribonucleoprotein, polymerase complex, and matrix lattice have broadened therapeutic targets beyond F. Here, we summarize these structural advances; review current structure-guided vaccine, antibody, and antiviral development efforts; and highlight priorities for next-generation vaccines and therapeutics.
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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
,
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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