title: Subtomogram analysis using the Volta phase plate.
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.
authors.affiliation: Max-Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany.
authors.forename: Maryam
authors.initials: M
authors.lastname: Khoshouei
authors.affiliation: Max-Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Pfeffer
authors.affiliation: Max-Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Baumeister
authors.affiliation: 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.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Förster
authors.affiliation: Max-Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address: danev@biochem.mpg.de.
authors.forename: Radostin
authors.initials: R
authors.lastname: Danev
mesh_terms: D000818:Animals; D020285:Cryoelectron Microscopy / Q000295:instrumentation* / Q000379:methods*; D055032:Electron Microscope Tomography / Q000295:instrumentation / Q000379:methods; D006801:Humans; D046529:Microscopy, Electron, Transmission / Q000295:instrumentation* / Q000379:methods*; D012270:Ribosomes / Q000648:ultrastructure; D059629:Signal-To-Noise Ratio
keywords: Cryo-electron tomography; Ribosome; Subtomogram analysis; Volta phase plate