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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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[2452]
Open Access
Abstract: Phages can use a small-molecule communication arbitrium system to coordinate lysis–lysogeny decisions, but the underlying mechanism remains unknown. Here we determined that the arbitrium system in Bacillus subtilis phage phi3T modulates the bacterial toxin–antitoxin system MazE–MazF to regulate the phage life cycle. We show that phi3T expresses AimX and YosL, which bind to and inactivate MazF. AimX also inhibits the function of phi3T_93, a protein that promotes lysogeny by binding to MazE and releasing MazF. Overall, these mutually exclusive interactions promote the lytic cycle of the phage. After several rounds of infection, the phage-encoded AimP peptide accumulates intracellularly and inactivates the phage antiterminator AimR, a process that eliminates aimX expression from the aimP promoter. Therefore, when AimP increases, MazF activity promotes reversion back to lysogeny, since AimX is absent. Altogether, our study reveals the evolutionary strategy used by arbitrium to control lysis–lysogeny by domesticating and fine-tuning a phage-defence mechanism.
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Jan 2024
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Simon G.
Caulton
,
Carey
Lambert
,
Jess
Tyson
,
Paul
Radford
,
Asmaa
Al-Bayati
,
Samuel
Greenwood
,
Emma J.
Banks
,
Callum
Clark
,
Rob
Till
,
Elisabete
Pires
,
R. Elizabeth
Sockett
,
Andrew L.
Lovering
Diamond Proposal Number(s):
[26803, 19880]
Open Access
Abstract: Predatory bacteria, like the model endoperiplasmic bacterium Bdellovibrio bacteriovorus, show several adaptations relevant to their requirements for locating, entering and killing other bacteria. The mechanisms underlying prey recognition and handling remain obscure. Here we use complementary genetic, microscopic and structural methods to address this deficit. During invasion, the B. bacteriovorus protein CpoB concentrates into a vesicular compartment that is deposited into the prey periplasm. Proteomic and structural analyses of vesicle contents reveal several fibre-like proteins, which we name the mosaic adhesive trimer (MAT) superfamily, and show localization on the predator surface before prey encounter. These dynamic proteins indicate a variety of binding capabilities, and we confirm that one MAT member shows specificity for surface glycans from a particular prey. Our study shows that the B. bacteriovorus MAT protein repertoire enables a broad means for the recognition and handling of diverse prey epitopes encountered during bacterial predation and invasion.
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Jan 2024
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[25452]
Open Access
Abstract: Translational control is an essential process for the cell to adapt to varying physiological or environmental conditions. To survive adverse conditions such as low nutrient levels, translation can be shut down almost entirely by inhibiting ribosomal function. Here we investigated eukaryotic hibernating ribosomes from the microsporidian parasite Spraguea lophii in situ by a combination of electron cryo-tomography and single-particle electron cryo-microscopy. We show that microsporidian spores contain hibernating ribosomes that are locked in a dimeric (100S) state, which is formed by a unique dimerization mechanism involving the beak region. The ribosomes within the dimer are fully assembled, suggesting that they are ready to be activated once the host cell is invaded. This study provides structural evidence for dimerization acting as a mechanism for ribosomal hibernation in microsporidia, and therefore demonstrates that eukaryotes utilize this mechanism in translational control.
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Sep 2023
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