title: Cryo-EM single particle analysis with the Volta phase plate.
abstract: We present a method for in-focus data acquisition with a phase plate that enables near-atomic resolution single particle reconstructions. Accurate focusing is the determining factor for obtaining high quality data. A double-area focusing strategy was implemented in order to achieve the required precision. With this approach we obtained a 3.2 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome. The phase plate matches or slightly exceeds the performance of the conventional defocus approach. Spherical aberration becomes a limiting factor for achieving resolutions below 3 Å with in-focus phase plate images. The phase plate could enable single particle analysis of challenging samples in terms of small size, heterogeneity and flexibility that are difficult to solve by the conventional defocus approach.
authors.affiliation: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Radostin
authors.identifier: 0000-0001-6406-8993
authors.initials: R
authors.lastname: Danev
authors.affiliation: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Baumeister
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods*; D046988:Proteasome Endopeptidase Complex / Q000648:ultrastructure*; D013822:Thermoplasma / Q000201:enzymology*
chemical_list: D046988:Proteasome Endopeptidase Complex
keywords: biophysics; cryo-EM; none; phase plate; single particle analysis; structural biology