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TitleSubtomogram analysis using the Volta phase plate.
Journal, issue, pagesJ Struct Biol, Vol. 197, Issue 2, Page 94-9101, Year 2017
Publish dateMay 25, 2016
AuthorsMaryam Khoshouei / Stefan Pfeffer / Wolfgang Baumeister / Friedrich Förster / Radostin Danev /
PubMed AbstractCryo-electron tomography (CET) and subtomogram analysis allow studying the structures of macromolecular complexes in their natural context. The radiation sensitivity of vitrified biological specimens ...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.
External linksJ Struct Biol / PubMed:27235783
MethodsEM (subtomogram averaging)
Resolution9.6 - 11.2 Å
Structure data

EMDB-3418:
Subtomogram average of 80S ribosomes obtained using the VPP
Method: EM (subtomogram averaging) / Resolution: 9.6 Å

EMDB-3419:
Subtomogram average of 80S ribosomes obtained using CTEM at one defocus
Method: EM (subtomogram averaging) / Resolution: 10.6 Å

EMDB-3420:
Subtomogram average of 80S ribosomes obtained using CTEM with a range of defocus values
Method: EM (subtomogram averaging) / Resolution: 11.2 Å

Source
  • Oryctolagus cuniculus (rabbit)

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