In Situ Imaging and Structure Determination of Biomolecular Complexes Using Electron Cryo-Tomography

Kaplan, Mohammed; Nicolas, William J; Wei, Zhao; Carter, Stephen D.; Metskas, Lauren Ann; Chreifi, Georges; Ghosal, Debnath; Jensen, Grant J.

doi:10.1007/978-1-0716-0966-8_4

Cited by 12 publications

(7 citation statements)

References 54 publications

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“…Most of the previous work relied on low-resolution imaging methods or conventional electron microscopy preparations, in which dehydration and fixation disrupt cell membranes and obscure macromolecular details. Cryogenic electron tomography (cryo-ET) allows the investigation of cellular processes in a fully-hydrated frozen state with macromolecular resolution (Ghosal et al, 2019a;Kaplan et al, 2021a;Oikonomou and Jensen, 2017), but so far it has only been applied to study the ultrastructure of individual B. bacteriovorus cells in the attack phase. While these studies revealed important structural features of this stage (Borgnia et al, 2008;Butan et al, 2011;Fenton et al, 2010b), much remains to be learned about the full invasion cycle of B. bacteriovorus.…”

Section: Introductionmentioning

confidence: 99%

Dynamic structural adaptations enable the endobiotic predation ofbdellovibrio bacteriovorus

Kaplan

Chang

Oikonomou

et al. 2022

Preprint

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Bdellovibrio bacteriovorusis an endobiotic microbial predator that offers promise as a living antibiotic for its ability to kill Gram-negative bacteria, including human pathogens. Even after six decades of study, fundamental details of its predation cycle remain mysterious. Here, we used cryo-electron tomography to comprehensively image the lifecycle ofB. bacteriovorusat nanometer-scale resolution. In addition to providing the first high-resolution images of predation in a native (hydrated, unstained) state, we also discover several surprising features of the process, including novel macromolecular complexes involved in prey attachment/invasion and a flexible portal structure lining a hole in the prey peptidoglycan that tightly seals the prey outer membrane around the predator during entry. Unexpectedly, we find thatB. bacteriovorusdoes not shed its flagellum during invasion, but rather resorbs it into its periplasm for degradation. Finally, following replication and division in the bdelloplast, we observe a transient and extensive ribosomal lattice on the condensedB. bacteriovorusnucleoid.Graphical abstract

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Section: Introductionmentioning

confidence: 99%

Dynamic structural adaptations enable the endobiotic predation ofbdellovibrio bacteriovorus

Kaplan

Chang

Oikonomou

et al. 2022

Preprint

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“…However, this capability is limited to thin samples (few hundred nanometers thick, like individual bacterial cells of many species) while thicker samples like the central part of eukaryotic cells, thick bacterial cells, or clusters of bacterial cells are not amenable for direct cryo-ET imaging. Such thick samples can be rendered suitable for cryo-ET experiments by thinning them first using different methods including focused ion beam milling and cryosectioning ( Kaplan et al, 2021a ). Cryo-ET has already been invaluable in revealing the structures of several MEs, including S. oneidensis nanowires ( Subramanian et al, 2018 ), H. pylori tubes ( Chang et al, 2018 ), Delftia acidovorans nanopods ( Shetty et al, 2011 ), V. vulnificus OMV chains ( Hampton et al, 2017 ), and more recently cell-cell bridges in the archaeon Haloferax volcanii ( Sivabalasarma et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

In situ imaging of bacterial outer membrane projections and associated protein complexes using electron cryo-tomography

Kaplan

Chreifi

Metskas

et al. 2021

eLife

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The ability to produce outer membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among diderm bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of ~ 90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab. We identified outer MEs and MVs in 13 diderm bacterial species and classified several major ultrastructures: 1) tubes with a uniform diameter (with or without an internal scaffold), 2) tubes with irregular diameter, 3) tubes with a vesicular dilation at their tip, 4) pearling tubes, 5) connected chains of vesicles (with or without neck-like connectors), 6) budding vesicles and nanopods. We also identified several protein complexes associated with these MEs and MVs which were distributed either randomly or exclusively at the tip. These complexes include a secretin-like structure and a novel crown-shaped structure observed primarily in vesicles from lysed cells. In total, this work helps to characterize the diversity of bacterial membrane projections and lays the groundwork for future research in this field.

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“…Technical improvements, such as workflow automatization and simplification render them available to a wider community of biologists. Comprehensive articles reviewing such workflows and their challenges have been published (Bäuerlein & Baumeister, 2021; Hylton & Swulius, 2021; Kaplan et al., 2021). Specific steps of these workflows are particularly relevant for the study of bacterial secretion systems, namely sample preparation, the capacity to find “rare” structures within the sample, dealing with the overall thickness of the sample, and obtaining deeper insights from tomography data by averaging approaches.…”

Section: Electron Microscopy Workflows To Image Secretion Systems In ...mentioning

confidence: 99%

Caught in the act: In situ visualization of bacterial secretion systems by cryo‐electron tomography

Keck,

Enninga,

Swistak

2023

Molecular Microbiology

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Bacterial secretion systems, such as the type 3, 4, and 6 are multiprotein nanomachines expressed at the surface of pathogens with Gram‐negative like envelopes. They are known to be crucial for virulence and to translocate bacteria‐encoded effector proteins into host cells to manipulate cellular functions. This facilitates either pathogen attachment or invasion of the targeted cell. Effector proteins also promote evasion of host immune recognition. Imaging by cryo‐electron microscopy in combination with structure determination has become a powerful approach to understand how these nanomachines work. Still, questions on their assembly, the precise secretion mechanisms, and their direct involvement in pathogenicity remain unsolved. Here, we present an overview of the recent developments in in situ cryo‐electron microscopy. We discuss its potential for the investigation of the role of bacterial secretion systems during the host‐bacterial crosstalk at the molecular level. These in situ studies open new perspectives for our understanding of secretion system structure and function.

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In Situ Imaging and Structure Determination of Biomolecular Complexes Using Electron Cryo-Tomography

Cited by 12 publications

References 54 publications

Dynamic structural adaptations enable the endobiotic predation ofbdellovibrio bacteriovorus

Dynamic structural adaptations enable the endobiotic predation ofbdellovibrio bacteriovorus

In situ imaging of bacterial outer membrane projections and associated protein complexes using electron cryo-tomography

Caught in the act: In situ visualization of bacterial secretion systems by cryo‐electron tomography

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