I03-Macromolecular Crystallography
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Dylan
Kramer
,
Clarissa
Santos Rocha
,
Christopher A.
Gaulke
,
Marie
Nearing
,
Sumathi
Sankaran-Walters
,
Ikaika
Loque
,
Anugraha
Kidigannappa
,
Eric
Pham
,
Shuang
Hu
,
Patrawin
Wanakumjorn
,
Ramona
Abbattista
,
Abhaya
Dandekar
,
Roland
Faller
,
Satya
Dandekar
Open Access
Abstract: HIV infection disrupts gut epithelial barrier integrity and mucosal immunity, driving chronic inflammation and disease progression which are not fully resolved despite anti-retroviral therapy. Here we identify the microbiota-derived octadecanoid-hydroxy-fatty-acid metabolite 10-hydroxystearic acid (10-HSA), produced by Lactiplantibacillus plantarum, as a key mediator of gut epithelial barrier repair in human intestinal epithelial cells in vitro, ex vivo and in the non-human primate model of HIV/AIDS. X-ray crystallography and transcriptomics combined with functional analyses revealed that 10-HSA directly binds PPARα, inducing lipid metabolism, mitochondrial regeneration and subsequent epigenetic histone crotonylation, thereby promoting gut epithelial renewal. Co-administration of 10-HSA with anti-retroviral therapy in SIV-infected macaques accelerated viral suppression, resolved systemic inflammation, repaired gut epithelial integrity and recovered the gut microbiota. These findings identify a microbiota-derived lipid metabolite–PPARα–histone crotonylation axis that activates gut epithelial regeneration. This study defines a host–microbiome metabolic pathway that restores epithelial–immune homeostasis and enhances the efficacy of anti-retroviral therapy.
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Aug 2026
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Debajit
Dey
,
Naba D.
Salman
,
Charles W. E.
Tomlinson
,
Chunsheng
Jin
,
Grete
Raba
,
Sadie R.
Schaus
,
Marcus
Nilsson
,
Zak
Mciver
,
Reyme
Herman
,
Adam
Simpkin
,
Matthew
Davy
,
Daniel J.
Rigden
,
Mirjam
Czjzek
,
Dominic P.
Byrne
,
Mihai
Oltean
,
Alexander
Case
,
Christoph G.
Baumann
,
Gareth S. A.
Wright
,
Sjoerd
Van Der Post
,
Edwin A.
Yates
,
Eric C.
Martens
,
Lauren
Davey
,
Ana S.
Luis
,
Alan
Cartmell
Diamond Proposal Number(s):
[21970, 3918]
Open Access
Abstract: Excessive foraging of colonic mucin glycans by gut bacteria is associated with diseases such as inflammatory bowel disease. Although Akkermansia muciniphila is an important mucin degrader, the role of carbohydrate sulfatases that facilitate digestion of these heavily sulfated glycans remains unclear. Combining in vitro digestion assays, proteomics and structural biology, we show that A. muciniphila sulfatases, such as Amuc1755 and Amuc0953, have rare adaptations targeted towards known sulfated mucin structures. They show larger degrees of modularity, including a previously unknown mucin-binding domain. When grown on colonic mucin substrates, glycoproteins of reduced size were important for the growth of A. muciniphila. Further mutational analysis and localization studies revealed that desulfation of N-acetyl-d-glucosamine was periplasmic, while desulfation of d-galactose occurred extracellularly and in the periplasm. These data improve our understanding of contexts for the positive health correlations of A. muciniphila while metabolizing colonic mucin as its sole carbon source.
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Jul 2026
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I02-Macromolecular Crystallography
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Open Access
Abstract: The twin-arginine translocation (Tat) system is a mechanistically unique protein transport pathway moving folded proteins across membranes. It is found in all domains of life and is essential for bacterial virulence and plant photosynthesis. The membrane proteins, TatA, TatB and TatC form a core complex to which substrate proteins bind, triggering the recruitment of additional TatA protomers to form the transport site. Here we present cryo-electron microscopy structures of the prototypical TatBC complex from Escherichia coli and the atypical complexes from Nitratifactor salsuginis and Myxococcus xanthus in a resting state, alongside TatAC substrate-bound TatBC and TatABC complexes from E. coli in the early stages of transport. These structures demonstrate that substrate proteins associate with the core complex solely through their N-terminal signal peptides. The Tat targeting sequences of the signal peptides make specific contacts with TatC, and the peptide body is clamped by TatB. The core complex contains highly tilted transmembrane helices that drive extreme local membrane thinning. On the basis of our structures and biochemical and functional analyses, we propose a model for the early steps in Tat transport.
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Jun 2026
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Krios III-Titan Krios III at Diamond
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Patricia
Paracuellos
,
Ambre
Bexter
,
Jonasz B.
Patkowski
,
Steven D.
Kelly
,
Oleksii
Omelchenko
,
Kevin
Mace
,
Aravindan
Ilangovan
,
Sujatha
Subramoni
,
John C.
Whitney
,
Alain
Filloux
,
Tiago R. D.
Costa
Diamond Proposal Number(s):
[25127]
Open Access
Abstract: Type VI secretion systems (T6SSs) are widespread bacterial nanomachines that deliver effectors into prokaryotic and eukaryotic cells. How an effector cargo is recruited and loaded into the Hcp ring assemblies that form the tube injected by the T6SS remains poorly understood. Pseudomonas aeruginosa has four T6SSs, each associated with a different Hcp protein. Here we use cryo-electron microscopy to resolve the structure of the Tce1 cargo loaded into a Hcp3 ring from the P. aeruginosa H3-T6SS. We show that a single Tce1 monomer interacts asymmetrically with, and is enclosed by, two hexameric Hcp3 rings, engaging key residues lining the inner surface of the Hcp3 disc. Our data indicate a stepwise loading mechanism, where an initial heterodimeric Hcp–cargo complex forms before ring encapsulation around the effector. Structural modelling suggests similar effector–Hcp3 interactions for a second T6SS effector, Tce2, which has antifungal activity. We propose that this mechanism enables coordinated delivery of a broad payload into target cells.
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May 2026
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I24-Microfocus Macromolecular Crystallography
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Ying
Xu
,
Reed M.
Stubbendieck
,
Raghuvir
Viswanatha
,
Ajda
Krc
,
Lisa S.
Baik
,
Won Se
Suh
,
Yanhui
Hu
,
Huan
Wang
,
Linxiang
Yin
,
Enzo
Mameli
,
Anne
Van Der Meij
,
John R.
Carlson
,
Andrew C.
Doxey
,
Pal
Stenmark
,
Norbert
Perrimon
,
Cameron R.
Currie
,
Min
Dong
Diamond Proposal Number(s):
[29948]
Open Access
Abstract: Streptomyces and insects engage in complex interactions shaped by millions of years of evolution. While many beneficial relationships are well recognized, it remains unknown whether Streptomyces produce virulence factors targeting insects specifically. Here, through bioinformatic analysis, we identified diphtheria toxin (DT) homologues, which we named Streptomyces antiquus insecticidal proteins (SAIP), within a monophyletic lineage of Streptomyces that emerged more than 100 million years ago. SAIP is cytotoxic to insect cells and lethal to Drosophila melanogaster, suppressing neuronal activity and immune responses in vivo. Structural and functional studies validated that SAIP is homologous to DT and acts by ADP ribosylation of eukaryotic elongation factor 2. CRISPR–Cas9 screening identified the insect protein Flower as the SAIP receptor across a range of insects. Toxigenic Streptomyces can consume dead insects and produce bioactive secondary metabolites while growing on insect carcasses. These findings establish an insecticidal toxin in Streptomyces and demonstrate that Streptomyces have evolved highly specific virulence factors against insects.
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Apr 2026
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Aquilos-CryoFIB at Diamond
Krios III-Titan Krios III at Diamond
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Zhen
Hou
,
Yao
Shen
,
Stanley
Fronik
,
Juan
Shen
,
Jiong
Shi
,
Jialu
Xu
,
Long
Chen
,
Nathan
Hardenbrook
,
Alan N.
Engelman
,
Christopher
Aiken
,
Peijun
Zhang
Diamond Proposal Number(s):
[29812]
Open Access
Abstract: Lentiviruses, such as HIV-1, infect non-dividing cells by traversing the nuclear pore complex (NPC); however, the detailed molecular processes remain unclear. Here we reconstituted functional HIV-1 nuclear import using permeabilized T cells and isolated HIV-1 cores, which significantly increases import events, and developed an integrated three-dimensional cryo-correlative workflow to specifically target and image 1,489 native HIV-1 cores at 4 distinct nuclear import stages using cryo-electron tomography. We found HIV-1 nuclear import depends on both capsid elasticity and nuclear pore adaptability. The NPC acts as a selective filter, preferentially importing smaller cores, while expanding and deforming to accommodate their passage. Brittle mutant cores fail to enter the NPC, while CPSF6-binding-deficient cores enter but stall within the NPC, leading to impaired nuclear import. This study uncovers the interplay between the HIV-1 core and the NPC and provides a framework to dissect HIV-1 nuclear import and downstream events, such as uncoating and integration.
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Jul 2025
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I04-Macromolecular Crystallography
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Hugo
Belda
,
David
Bradley
,
Evangelos
Christodoulou
,
Stephanie D.
Nofal
,
Malgorzata
Broncel
,
David
Jones
,
Heledd
Davies
,
M. Teresa
Bertran
,
Andrew G.
Purkiss
,
Roksana W.
Ogrodowicz
,
Dhira
Joshi
,
Nicola
O’reilly
,
Louise
Walport
,
Andrew
Powell
,
David
House
,
Svend
Kjaer
,
Antoine
Claessens
,
Christian R.
Landry
,
Moritz
Treeck
Diamond Proposal Number(s):
[25587]
Open Access
Abstract: Of 250 Plasmodium species, 6 infect humans, with P. falciparum causing over 95% of 600,000 annual malaria-related deaths. Its pathology arises from host cell remodelling driven by over 400 exported parasite proteins, including the FIKK kinase family. About one million years ago, a bird-infecting Plasmodium species crossed into great apes and a single non-exported FIKK kinase gained an export element. This led to a rapid expansion into 15–21 atypical, exported Ser/Thr effector kinases. Here, using genomic and proteomic analyses, we demonstrate FIKK differentiation via changes in subcellular localization, expression timing and substrate motifs, which supports an individual important role in host–pathogen interactions. Structural data and AlphaFold2 predictions reveal fast-evolving loops in the kinase domain that probably enabled rapid functional diversification for substrate preferences. One FIKK evolved exclusive tyrosine phosphorylation, previously thought absent in Plasmodium. Despite divergence of substrate preferences, the atypical ATP binding pocket is conserved and we identified a single compound that inhibits all FIKKs. A pan-specific inhibitor could reduce resistance development and improve malaria control strategies.
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Jun 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Thomas C.
Mclean
,
Francisco
Balaguer-Pérez
,
Joshua
Chandanani
,
Christopher M.
Thomas
,
Clara
Aicart-Ramos
,
Sophia
Burick
,
Paul Dominic B.
Olinares
,
Giulia
Gobbato
,
Julia E. A.
Mundy
,
Brian T.
Chait
,
David M.
Lawson
,
Seth A.
Darst
,
Elizabeth A.
Campbell
,
Fernando
Moreno-Herrero
,
Tung
Le
Diamond Proposal Number(s):
[25108]
Open Access
Abstract: Examples of long-range gene regulation in bacteria are rare and generally thought to involve DNA looping. Here, using a combination of biophysical approaches including X-ray crystallography and single-molecule analysis for the KorB–KorA system in Escherichia coli, we show that long-range gene silencing on the plasmid RK2, a source of multi-drug resistance across diverse Gram-negative bacteria, is achieved cooperatively by a DNA-sliding clamp, KorB, and a clamp-locking protein, KorA. We show that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters up to 1.5 kb away. We resolved the tripartite crystal structure of a KorB–KorA–DNA co-complex, revealing that KorA latches KorB into a closed clamp state. DNA-bound KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes. Together, our findings explain the mechanistic basis for KorB role switching from a DNA-sliding clamp to a co-repressor and provide an alternative mechanism for long-range regulation of gene expression in bacteria.
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Jan 2025
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Scios-Scios at Diamond
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Vojtěch
Pražák
,
Yuliia
Mironova
,
Daven
Vasishtan
,
Christoph
Hagen
,
Ulrike
Laugks
,
Yannick
Jensen
,
Saskia
Sanders
,
John M.
Heumann
,
Barbara G.
Klupp
,
Thomas C.
Mettenleiter
,
Michael
Grange
,
Kay
Grunewald
,
Jens B.
Bosse
Abstract: The viral nuclear egress complex (NEC) allows herpesvirus capsids to escape from the nucleus without compromising the nuclear envelope integrity. The NEC lattice assembles on the inner nuclear membrane and mediates the budding of nascent nucleocapsids into the perinuclear space and their subsequent release into the cytosol. Its essential role makes it a potent antiviral target, necessitating structural information in the context of a cellular infection. Here we determined structures of NEC–capsid interfaces in situ using electron cryo-tomography, showing a substantial structural heterogeneity. In addition, while the capsid is associated with budding initiation, it is not required for curvature formation. By determining the NEC structure in several conformations, we show that curvature arises from an asymmetric assembly of disordered and hexagonally ordered lattice domains independent of pUL25 or other viral capsid vertex components. Our results advance our understanding of the mechanism of nuclear egress in the context of a living cell.
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Jul 2024
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I24-Microfocus Macromolecular Crystallography
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Phillip
Nußbaum
,
Danguole
Kureisaite-Ciziene
,
Dom
Bellini
,
Chris
Van Der Does
,
Marko
Kojic
,
Najwa
Taib
,
Anna
Yeates
,
Maxime
Tourte
,
Simonetta
Gribaldo
,
Martin
Loose
,
Jan
Lowe
,
Sonja-Verena
Albers
Diamond Proposal Number(s):
[28677]
Abstract: Cell division in all domains of life requires the orchestration of many proteins, but in Archaea most of the machinery remains poorly characterized. Here we investigate the FtsZ-based cell division mechanism in Haloferax volcanii and find proteins containing photosynthetic reaction centre (PRC) barrel domains that play an essential role in archaeal cell division. We rename these proteins cell division protein B 1 (CdpB1) and CdpB2. Depletions and deletions in their respective genes cause severe cell division defects, generating drastically enlarged cells. Fluorescence microscopy of tagged FtsZ1, FtsZ2 and SepF in CdpB1 and CdpB2 mutant strains revealed an unusually disordered divisome that is not organized into a distinct ring-like structure. Biochemical analysis shows that SepF forms a tripartite complex with CdpB1/2 and crystal structures suggest that these two proteins might form filaments, possibly aligning SepF and the FtsZ2 ring during cell division. Overall our results indicate that PRC-domain proteins play essential roles in FtsZ-based cell division in Archaea.
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Mar 2024
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