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  Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins

Schaffer, M., Mahamid, J., Engel, B. D., Laugks, T., Baumeister, W., & Plitzko, J. M. (2017). Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins. Journal of Structural Biology, 197(2), 73-82. doi:10.1016/j.jsb.2016.07.010.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002C-ED6B-3 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002C-ED6C-1
Genre: Journal Article

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 Creators:
Schaffer, Miroslava1, Author              
Mahamid, Julia1, Author              
Engel, Benjamin D.1, Author              
Laugks, Tim1, Author              
Baumeister, Wolfgang1, Author              
Plitzko, Jürgen M.1, Author              
Affiliations:
1Baumeister, Wolfgang / Molecular Structural Biology, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565142              

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Free keywords: TRANSMISSION ELECTRON-MICROSCOPY; CRYOELECTRON TOMOGRAPHY; EUKARYOTIC CELLS; ATP SYNTHASE; PHASE PLATE; COMPLEX; CHLAMYDOMONAS; ARCHITECTURE; SPECIMENS; FIBBiochemistry & Molecular Biology; Biophysics; Cell Biology; Cryo-FIB; Sample preparation; Cryo-ET; Volta phase plate; In situ;
 Abstract: While cryo-electron tomography (cryo-ET) can reveal biological structures in their native state within the cellular environment, it requires the production of high-quality frozen-hydrated sections that are thinner than 300 nm. Sample requirements are even more stringent for the visualization of membrane-bound protein complexes within dense cellular regions. Focused ion beam (FIB) sample preparation for transmission electron microscopy (TEM) is a well-established technique in material science, but there are only few examples of biological samples exhibiting sufficient quality for high-resolution in situ investigation by cryo-ET. In this work, we present a comprehensive description of a cryo-sample preparation workflow incorporating additional conductive-coating procedures. These coating steps eliminate the adverse effects of sample charging on imaging with the Volta phase plate, allowing data acquisition with improved contrast. We discuss optimized FIB milling strategies adapted from material science and each critical step required to produce homogeneously thin, non-charging FIB lamellas that make large areas of unperturbed HeLa and Chlamydomonas cells accessible for cryo-ET at molecular resolution. (C) 2016 Elsevier Inc. All rights reserved.

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Language(s): eng - English
 Dates: 2016-07-192017-02
 Publication Status: Published in print
 Pages: 10
 Publishing info: -
 Table of Contents: -
 Rev. Method: -
 Identifiers: ISI: 000393268200002
DOI: 10.1016/j.jsb.2016.07.010
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Project name : ERC-2012-SyG_318987-ToPAG
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Title: Journal of Structural Biology
  Abbreviation : J. Struct. Biol.
Source Genre: Journal
 Creator(s):
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Publ. Info: Orlando, Fla. : Academic Press
Pages: - Volume / Issue: 197 (2) Sequence Number: - Start / End Page: 73 - 82 Identifier: ISSN: 1047-8477
CoNE: /journals/resource/954922650160