Subtomogram analysis using the Volta phase plate

doi:10.1016/j.jsb.2016.05.009

. 2017 Feb;197(2):94-101.

doi: 10.1016/j.jsb.2016.05.009. Epub 2016 May 25.

Subtomogram analysis using the Volta phase plate

Maryam Khoshouei¹, Stefan Pfeffer¹, Wolfgang Baumeister¹, Friedrich Förster², Radostin Danev³

Affiliations

¹ Max-Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany.
² Max-Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany; Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Electronic address: foerster@biochem.mpg.de.
³ Max-Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address: danev@biochem.mpg.de.

PMID: 27235783
DOI: 10.1016/j.jsb.2016.05.009

Subtomogram analysis using the Volta phase plate

Maryam Khoshouei et al. J Struct Biol. 2017 Feb.

. 2017 Feb;197(2):94-101.

doi: 10.1016/j.jsb.2016.05.009. Epub 2016 May 25.

Authors

Maryam Khoshouei¹, Stefan Pfeffer¹, Wolfgang Baumeister¹, Friedrich Förster², Radostin Danev³

Affiliations

¹ Max-Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany.
² Max-Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany; Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Electronic address: foerster@biochem.mpg.de.
³ Max-Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address: danev@biochem.mpg.de.

PMID: 27235783
DOI: 10.1016/j.jsb.2016.05.009

Abstract

Cryo-electron tomography (CET) and subtomogram analysis allow studying the structures of macromolecular 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 9.6Å 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.

Keywords: Cryo-electron tomography; Ribosome; Subtomogram analysis; Volta phase plate.

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Subtomogram analysis using the Volta phase plate

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Subtomogram analysis using the Volta phase plate

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