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Instruments / Technical Area	Publication Details	Published
Krios I-Titan Krios I at Diamond	CryoEM reveals that ribosomes in microsporidian spores are locked in a dimeric hibernating state Mathew Mclaren , Rebecca Conners , Michail N. Isupov , Patricia Gil-Díez , Lavinia Gambelli , Vicki A. M. Gold , Andreas Walter , Sean R. Connell , Bryony Williams , Bertram Daum Nature Microbiology 400 DOI: 10.1038/s41564-023-01469-w Diamond Proposal Number(s): [25452] Open Access Show Abstract ▼ 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.	Sep 2023
Krios II-Titan Krios II at Diamond	Cryo-electron microscopy of the f1 filamentous phage reveals insights into viral infection and assembly Rebecca Conners , Rayén Ignacia León-Quezada , Mathew Mclaren , Nicholas J. Bennett , Bertram Daum , Jasna Rakonjac , Vicki Gold Nature Communications 14 DOI: 10.1038/s41467-023-37915-w Diamond Proposal Number(s): [25452] Open Access Show Abstract ▼ Abstract: Phages are viruses that infect bacteria and dominate every ecosystem on our planet. As well as impacting microbial ecology, physiology and evolution, phages are exploited as tools in molecular biology and biotechnology. This is particularly true for the Ff (f1, fd or M13) phages, which represent a widely distributed group of filamentous viruses. Over nearly five decades, Ffs have seen an extraordinary range of applications, yet the complete structure of the phage capsid and consequently the mechanisms of infection and assembly remain largely mysterious. In this work, we use cryo-electron microscopy and a highly efficient system for production of short Ff-derived nanorods to determine a structure of a filamentous virus including the tips. We show that structure combined with mutagenesis can identify phage domains that are important in bacterial attack and for release of new progeny, allowing new models to be proposed for the phage lifecycle.	May 2023
Krios I-Titan Krios I at Diamond	Electron cryo-microscopy reveals the structure of the archaeal thread filament Matthew C. Gaines , Michail N. Isupov , Shamphavi Sivabalasarma , Risat U. I. Haque , Mathew Mclaren , Clara L. Mollat , Patrick Tripp , Alexander Neuhaus , Vicki A. M. Gold , Sonja-Verena Albers , Bertram Daum Nature Communications 13 DOI: 10.1038/s41467-022-34652-4 Open Access Show Abstract ▼ Abstract: Pili are filamentous surface extensions that play roles in bacterial and archaeal cellular processes such as adhesion, biofilm formation, motility, cell-cell communication, DNA uptake and horizontal gene transfer. The model archaeaon Sulfolobus acidocaldarius assembles three filaments of the type-IV pilus superfamily (archaella, archaeal adhesion pili and UV-inducible pili), as well as a so-far uncharacterised fourth filament, named “thread”. Here, we report on the cryo-EM structure of the archaeal thread. The filament is highly glycosylated and consists of subunits of the protein Saci_0406, arranged in a head-to-tail manner. Saci_0406 displays structural similarity, but low sequence homology, to bacterial type-I pilins. Thread subunits are interconnected via donor strand complementation, a feature reminiscent of bacterial chaperone-usher pili. However, despite these similarities in overall architecture, archaeal threads appear to have evolved independently and are likely assembled by a distinct mechanism.	Dec 2022
	<em>In situ</em> structure of a dimeric hibernating ribosome from a eukaryotic intracellular pathogen Mathew McLaren , Patricia Gil-Diez , Michail N. Isupov , Rebecca Conners , Lavinia Gambelli , Vicki Gold , Andreas Walter , Sean R. Connell , Bryony Williams , Bertram Daum bioRxiv DOI: 10.1101/2022.04.29.490036 pending processing Show Abstract ▼ 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 cryo-electron tomography (cryoET) and single particle cryoEM. We show that microsporidian spores contain ribosomes primed for host cell invasion and thus shed new light on the infection mechanism of this important pathogen. Prior to host infection, virtually all ribosomes are locked in the 100 S dimeric state, which appears to be formed by a unique dimerization mechanism that is distinct from its bacterial counterparts. Within the dimer, the hibernation factor MDF1 is bound within the E site, locking the L1 stalk in a closed conformation, and thus preventing the translation of mRNAs to polypeptides.Competing Interest StatementThe authors have declared no competing interest.	Jan 2022
Krios I-Titan Krios I at Diamond	CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage Rebecca Conners , Mathew Mclaren , Urszula Łapińska , Kelly Sanders , M. Rhia L. Stone , Mark A. T. Blaskovich , Stefano Pagliara , Bertram Daum , Jasna Rakonjac , Vicki A. M. Gold Nature Communications 12 DOI: 10.1038/s41467-021-26610-3 Diamond Proposal Number(s): [25452] Open Access Show Abstract ▼ Abstract: The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.	Nov 2021
Krios II-Titan Krios II at Diamond	Cryo-electron microscopy reveals two distinct type IV pili assembled by the same bacterium Alexander Neuhaus , Muniyandi Selvaraj , Ralf Salzer , Julian D. Langer , Kerstin Kruse , Lennart Kirchner , Kelly Sanders , Bertram Daum , Beate Averhoff , Vicki A. M. Gold Nature Communications 11 DOI: 10.1038/s41467-020-15650-w Diamond Proposal Number(s): [18258] Open Access Show Abstract ▼ Abstract: Type IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. They are involved in bacterial motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Here, we use cryo-electron microscopy and mass spectrometry to show that the bacterium Thermus thermophilus produces two forms of type IV pilus (‘wide’ and ‘narrow’), differing in structure and protein composition. Wide pili are composed of the major pilin PilA4, while narrow pili are composed of a so-far uncharacterized pilin which we name PilA5. Functional experiments indicate that PilA4 is required for natural transformation, while PilA5 is important for twitching motility.	May 2020

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