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  Subtomogram analysis using the Volta phase plate

Khoshouei, M., Pfeffer, S., Baumeister, W., Förster, F., & Danev, R. (2017). Subtomogram analysis using the Volta phase plate. Journal of Structural Biology, 197(2), 94-101. doi:10.1016/j.jsb.2016.05.009.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002C-ED7B-0 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002C-ED7C-E
Genre: Journal Article

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 Creators:
Khoshouei, Maryam1, Author              
Pfeffer, Stefan1, Author              
Baumeister, Wolfgang1, Author              
Förster, Friedrich1, Author              
Danev, Radostin1, Author              
Affiliations:
1Baumeister, Wolfgang / Molecular Structural Biology, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565142              

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Free keywords: BIOLOGY IN-SITU; ANGSTROM RESOLUTION; CRYOELECTRON TOMOGRAPHY; CRYSTAL-STRUCTURE; CRYO-EM; ELECTRON CRYOTOMOGRAPHY; EUKARYOTIC RIBOSOME; BACTERIAL; ORGANIZATION; COMPLEXBiochemistry & Molecular Biology; Biophysics; Cell Biology; Ciyo-electron tomography; Subtomogram analysis; Volta phase plate; Ribosome;
 Abstract: Cryo-electron tomography (CET) and subtomogram analysis allow studying the structures of macro molecular complexes in their natural context. The radiation sensitivity of vitrified biological specimens and the resulting low signal-to-noise ratio (SNR) in CET limit the amount of structural information that can be mined from tomographic data. The Volta phase plate (VPP) has emerged as an effective means to increase the SNR and hence contrast compared to 'conventional' defocus-based phase contrast transmission electron microscopy (CTEM). Here, we assess the performance of the VPP compared to CTEM in subtomogram analysis, using the mammalian 80S ribosome as a test case. Accurate focusing is the major factor for achieving high resolution with the VPP, as highlighted by a comparison of slightly different focusing strategies. From only 1400 subtomograms, the VPP yields a subtomogram average of the mammalian 80S ribosome at 9.6 angstrom resolution without laborious contrast transfer function (CTF) correction. The subtomogram averages obtained using CTEM approaches are comparable, but suffer from lower signal transfer in certain frequency bands due to the oscillations of the CTF. Our study demonstrates that the VPP is a valuable tool for subtomogram analysis, because it enables improved performance and efficiency in terms of structure localization and number of subtomograms required for a given resolution. (C) 2016 Elsevier Inc. All rights reserved.

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Language(s): eng - English
 Dates: 2016-05-252017-02
 Publication Status: Published in print
 Pages: 8
 Publishing info: -
 Table of Contents: -
 Rev. Method: -
 Identifiers: ISI: 000393268200004
DOI: 10.1016/j.jsb.2016.05.009
 Degree: -

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Project name : German Research Council (DFG 716/4-1)
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Title: Journal of Structural Biology
  Abbreviation : J. Struct. Biol.
Source Genre: Journal
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Publ. Info: Orlando, Fla. : Academic Press
Pages: - Volume / Issue: 197 (2) Sequence Number: - Start / End Page: 94 - 101 Identifier: ISSN: 1047-8477
CoNE: https://pure.mpg.de/cone/journals/resource/954922650160