Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[33230]
Open Access
Abstract: Bacterial conjugation, the process of horizontal gene transfer between bacteria, is initiated by mating pair formation (MPF) via a conjugative pilus. Conjugation of the IncP RP4 plasmid is mediated by short mating pili. Here, we report the cryo-EM structure of the RP4 pilus at 2.74 Å resolution. Uniquely, both the structural and quantitative mass spectral analyses revealed that the cyclic TrbC pilin subunit is not lipidated. Consistently, an E. coli pgsA mutant lacking phosphatidylglycerol (PG) can serve as a donor of RP4 but not of F- (pKpQIL), H- (R27) or W- (R388) pili, whose biogenesis and DNA transfer is PG-dependent. RP4 is the first example of a lipid-independent functional mating pilus. This discovery suggests that an amphipathic lipid moiety is not universally essential for the biogenesis of conjugative pili and MPF, providing an alternative model for their assembly and function. These data expand our understanding of the diverse bacterial mechanisms employ to transfer genetic material.
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Jun 2026
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Krios III-Titan Krios III at Diamond
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Ho Fong
Leong
,
Giovanni
Consoli
,
Geoffry A.
Davis
,
Ben
Hancox-Lachman
,
Kenta
Renard
,
Fiazall
Tufail
,
Lauren E.
Lee
,
Lucas
Gautier
,
James W.
Murray
,
Andrea
Fantuzzi
,
A. William
Rutherford
Diamond Proposal Number(s):
[33230]
Open Access
Abstract: Far-red light photoacclimation enables some cyanobacteria to survive in white-light-depleted environments by extending the red limit of photosynthesis. In far-red Photosystem II, paralogous subunits replace their canonical counterparts, allowing the incorporation of some chlorophyll f molecules and one chlorophyll d that are red-shifted and spectrally distinct from the chlorophyll a manifold, and from each other. Here, we present a comparative study of far-red Photosystem II from Chroococcidiopsis thermalis PCC 7203 and Calothrix sp. NIES-3974. In C. thermalis, the cryo-electron microscopy structure reveals the far-red-exclusive subunit, PsbH2’, which forms part of a chlorophyll f binding site. We also assign four chlorophyll f sites using sequence comparisons and electrostatic potential analyses. In Calothrix, psbH2’ is absent, and the same analyses show that only two of these chlorophyll f sites are present. Comparative phylogenetic, structural, and spectroscopic analyses allow the assignment of specific wavelengths to all the red-shifted chlorophylls. This provides the framework needed to model excitation energy transfer in far-red Photosystem II, and to understand the conserved features that allow survival under far-red light.
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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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Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Douglas S. M.
Ferreira
,
Katie
Mclennan
,
Calum
Diamond
,
Melanie
Vollmar
,
Wasim
Kiyani
,
D. Sean
Froese
,
Jola
Kopec
,
Henry J.
Bailey
,
Rod
Chalk
,
Arnaud
Basle
,
Jonathan M.
Elkins
,
Jesse
Coker
,
Wyatt W.
Yue
,
Thomas J.
Mccorvie
Diamond Proposal Number(s):
[34172]
Open Access
Abstract: Human methionine synthase (MTR) is an essential enzyme of one carbon metabolism. Consisting of a catalytic N-half and a cobalamin binding C-half, MTR utilises this intricate organometallic cofactor in the methyl transfer from methyltetrahydrofolate to homocysteine producing methionine. Cobalamin loading into MTR, and its subsequent activation, requires methylmalonic aciduria and homocystinuria Type D (MMADHC) protein and methionine synthase reductase (MTRR), respectively. However, the molecular basis of cobalamin binding and activation of human MTR aided by MMADHC and MTRR remains unknown. Here, using cryo-electron microscopy, we determine structures of human MTR in its apo, and cobalamin bound states. Apo MTR adopts a conformation where the two halves of the enzyme act independently with the C-half posed to bind cobalamin. Binding of cobalamin and its activation causes conformational changes in MTR that result in a flexible catalytically active state. AlphaFold predictions, validated by interaction studies, show that MMADHC interacts with the C-half of apo MTR to facilitate cobalamin loading. Unexpectedly we found that MTRR interacts at two distinct sites within the C-half of MTR which may aid in activation. Collectively these findings lay the groundwork to uncover the mechanisms through how MMADHC and MTRR coordinate cobalamin loading and activation of human MTR.
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May 2026
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B21-High Throughput SAXS
Krios III-Titan Krios III at Diamond
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Abstract: In conclusion, this work provides a comprehensive and detailed view of the
functioning of Tse5, establishing it as a unique paradigm within the Rhs toxins of the T6SS
system. Tse5 integrates a highly specialized delivery machinery, capable of recognizing
and anchoring to the target cell membrane, with a potent effector domain that
executes cell death through the formation of depolarizing pores. These findings not only
significantly expand our understanding of the mechanistic diversity of bacterial
weapons used in intercellular competition, but they also offer a solid molecular basis
for the future development of new biotechnological and antimicrobial strategies inspired
by sophisticated bacterial secretion systems.
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May 2026
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Krios II-Titan Krios II at Diamond
Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[19435, 18258]
Open Access
Abstract: Type IV pili are long, filamentous structures that extend from bacterial cell surfaces, enabling cells to respond to changing environments and facilitating genome plasticity. Thermus thermophilus HB27 produces two different type IV pili, each exhibiting distinct structural and functional properties. Here, we combine cryo-electron tomography, mutagenesis, and AlphaFold predictions to generate hypothetical in situ models of the T. thermophilus type IV pilus assembly machinery. Using single-particle cryo-electron microscopy, we determine structures of both filament types, enabling modelling of their surface glycans. Molecular dynamics simulations further reveal the flexibility of these glycans on extrusion. Integration of the filament structures with our hypothetical model of the assembly machinery offers a framework for further dissecting T4P architecture and biogenesis.
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Mar 2026
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[33230, 39228]
Open Access
Abstract: Adeno-associated viruses (AAVs) are parvoviruses utilized as gene therapy vectors. However, the AAV packaging mechanism is unresolved at the molecular level, creating a bottleneck for vector manufacturing, safety, and efficacy. Here, cryo-EM structures of the Rep helicase packaging motor in complex with the packaging marker DNA (ITR) and the Rep-AAV8 capsid complex are presented. Rep-ITR complexes reveal dynamic oligomeric states on the DNA, elucidating the strand separation mechanism coupled to its ATPase cycle. We observe Rep preferentially bound to empty capsids, with a binding interface likely conserved across the virus family. This complex also unveils a cryptic capsid ATP-binding site which, alongside Rep binding, triggers structural rearrangements priming the capsid for packaging. Collectively, these findings advance the understanding of Rep-mediated packaging, with significant implications for parvovirus virology and viral vector design.
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Mar 2026
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Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Donghwi
Ko
,
Raili
Ruonala
,
Alexandre
Faille
,
Eva
Hellmann
,
Hanna
Help
,
Huili
Liu
,
Ronni
Nielsen
,
Anders
Haakonsson
,
Nuria
De Diego
,
Anja
Paatero
,
Mariia V.
Shcherbii
,
Karolina
Stefanowicz
,
Sanja
Ćavar Zeljković
,
Tine
Drud Lundager Rasmussen
,
Ondrej
Novak
,
Zsuzsanna
Bodi
,
Gugan
Eswaran
,
Brecht
Wybouw
,
Matthieu
Bourdon
,
Cristina
Urbez
,
Xiaonan
Liu
,
Kari
Salokas
,
Tiina
Öhman
,
Tanya
Waldie
,
Petri
Törönen
,
Sedeer
El-Showk
,
Martin
Balcerowicz
,
Fabrice
Besnard
,
Xiaomin
Liu
,
Patrick
Perkins
,
Serina
Mazzoni-Putman
,
Julia P.
Vainonen
,
Maija
Sierla
,
Mikko J.
Frilander
,
Susanne
Mandrup
,
Teva
Vernoux
,
Karin
Ljung
,
Alejandro
Ferrando
,
Miguel A.
Blazquez
,
Liisa
Holm
,
Rupert
Fray
,
Markku
Varjosalo
,
Ottoline
Leyser
,
Ville O.
Paavilainen
,
Ari Pekka
Mähönen
,
Anna
Stepanova
,
Jose
Alonso
,
Steffen
Heber
,
Robert
Malinowski
,
Finn
Kirpekar
,
Alan J.
Warren
,
Ykä
Helariutta
Diamond Proposal Number(s):
[37678]
Abstract: Polyamines are often associated with ribosomes and are thought to stabilize their integrity. In Arabidopsis, the polyamine thermospermine (tSpm) affects xylem cell fate. tSpm induces translation of SUPPRESSOR-OF-ACAULIS51 (SAC51) and SAC51-LIKEs (SACLs), which inhibit heterodimerization of the xylem development proteins LONESOME-HIGHWAY (LHW) and TARGET-OF-MONOPTEROS5. Here, we report a methyltransferase, OVERACHIEVER, that methylates the peptidyl transferase center of the 25S ribosomal RNA (rRNA). Residue m3U2952 promotes functional tSpm binding to a specific site connecting the P-site transfer RNA (tRNA) with rRNA residues in the peptidyl transferase center. This interaction enhances the translation of SACLs but inhibits that of LHW. Our study uncovers the dependency between a conserved rRNA base methylation and a polyamine in orchestrating cell fate decisions, highlighting a role for the ribosome chemical landscape in translational regulation.
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Feb 2026
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[21404]
Abstract: Background: IgE is the central driver of allergic responses. Prior studies have defined the conformation of the IgE Fc fragment bound to the FcεRIα ectodomain and the dynamic properties of the IgE Fc. It remains unknown, how these prior studies translate to the complex of a full antibody including the Fab arms with the receptor. Methods: For structural analysis, crystallography, cryo-EM and negative stain EM (ns-EM) were combined. IgE variants were analyzed by mediator release and CD23 binding assays. Results: An ensemble of 10 cryo-EM structures of the full-size IgE FcεRIα complex was obtained revealing that the receptor bound IgE adopts a pronounced T-like conformation. Either Fab arm may rotate up to 40°. Two additional conformations with different arrangements of the Fab arms were captured in ns-EM. The introduction of additional flexibility into the Fab-Fc hinge does not compromise the biological activity of IgE, suggesting that the observed conformations of the IgE Fab-Fc hinge exhibit equivalent biological function. Comparison of the full IgE receptor complex with recent cryo-EM structures of the intact receptor reveals that FcεRI conformations differ markedly by the orientation of the ectodomain. Hence, our ensemble of IgE FcεRIα structures including the Fab arms enabled critical evaluation of FcεRI conformations. Conclusion: Our data reveal the architecture of a full-size IgE antibody bound to its receptor and a new layer of dynamics in FcεRIα bound IgE on top of the well-established IgE Fc conformations. Development of novel anti-IgE therapeutics may take into account these properties of FcεRIα bound IgE.
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Nov 2025
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Krios III-Titan Krios III at Diamond
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Giovanni
Consoli
,
Fiazall
Tufail
,
Ho Fong
Leong
,
Stefania
Viola
,
Geoffry A.
Davis
,
Nicholas
Rew
,
Daniel
Medranda
,
Michael
Hofer
,
Paul
Simpson
,
Marco
Sandrin
,
Benoit
Chachuat
,
Jenny
Nelson
,
Thomas
Renger
,
James W.
Murray
,
Andrea
Fantuzzi
,
A. William
Rutherford
Diamond Proposal Number(s):
[25127]
Abstract: The discovery of chlorophyll f-containing photosystems, with their long-wavelength photochemistry, represented a distinct, low-energy paradigm for oxygenic photosynthesis. Structural studies on chlorophyll f-containing photosystem I could identify some chlorophylls f sites, but none among the photochemically active pigments and concluded that chlorophyll f plays no photochemical role. Here, we report two cryo-EM structures of far-red PSI from Chroococcidiopsis thermalis PCC 7203, allowing the assignment of eight chlorophylls f molecules, including the redox active A-1B. Simulations of absorption difference spectra induced by charge separation indicate that the experimental spectra can be reproduced only by considering the presence of a chlorophyll f at the A-1B site. The chlorophyll f locations, wavelength assignments, and conserved far-red-specific residues provide functional insights for efficient use of long wavelength photons.
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Oct 2025
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