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Expression, Purification, and Cryo-EM Structural Analysis of an Outer Membrane Secretin Channel

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Transmembrane β-Barrel Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2778))

Abstract

Secretin proteins form pores in the outer membranes of Gram-negative bacteria, and as such provide a means of transporting a wide variety of molecules out of or in to the cell. They are important components of several different bacterial secretion systems, surface filament assembly machineries, and virus assembly complexes. Despite accommodating a diverse assortment of molecules, including virulence factors, folded proteins, and whole viruses, the secretin family of proteins is highly conserved, particularly in their membrane-embedded β-barrel domain. We describe here a protocol for the expression, purification and cryo-EM structural determination of the pIV secretin from the Ff family of filamentous bacteriophages.

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Acknowledgments

A Wellcome Trust Seed Award in Science (210363/Z/18/Z) and Leverhulme Trust Research Project Grant (RPG-2023-069) supported R.C. and a BBSRC responsive mode grant (BB/R008639/1) supported M.M, all awarded to V.G. We thank Prof. Jasna Rakonjac for sharing materials and for many helpful discussions. For the pIV structural work, we acknowledge Diamond Light Source for access and support of the cryo-EM facilities at the UK’s national Electron Bio-imaging Centre (eBIC) [under proposal BI25452]. We acknowledge access and support of the GW4 Regional Facility for High-Resolution Electron Cryo-Microscopy, funded by the Wellcome Trust (202904/Z/16/Z and 206181/Z/17/Z) and BBSRC (BB/R000484/1). The deposited dataset was collected at eBIC, and the GW4 facility was used for sample screening. We are grateful to Ufuk Borucu of the GW4 Regional Facility for help with screening.

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Authors and Affiliations

  1. Living Systems Institute, University of Exeter, Exeter, UK

    Rebecca Conners, Mathew McLaren & Vicki A. M. Gold

  2. Faculty of Health and Life Sciences, University of Exeter, Exeter, UK

    Rebecca Conners, Mathew McLaren & Vicki A. M. Gold

  3. Rockefeller University, New York, NY, USA

    Marjorie Russel

Authors
  1. Rebecca Conners
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  2. Mathew McLaren
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  3. Marjorie Russel
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  4. Vicki A. M. Gold
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Corresponding author

Correspondence to Vicki A. M. Gold .

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Editors and Affiliations

  1. Laboratoire de Microbiologie et Génétique Moléculaires, Centre de Biologie Intégrative, CNRS, Université de Toulouse, Toulouse, France

    Raffaele Ieva

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Conners, R., McLaren, M., Russel, M., Gold, V.A.M. (2024). Expression, Purification, and Cryo-EM Structural Analysis of an Outer Membrane Secretin Channel. In: Ieva, R. (eds) Transmembrane β-Barrel Proteins. Methods in Molecular Biology, vol 2778. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3734-0_18

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  • DOI: https://doi.org/10.1007/978-1-0716-3734-0_18

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3733-3

  • Online ISBN: 978-1-0716-3734-0

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