title: Advances in Single-Particle Electron Cryomicroscopy Structure Determination applied to Sub-tomogram Averaging.
abstract: Recent innovations in specimen preparation, data collection, and image processing have led to improved structure determination using single-particle electron cryomicroscopy (cryo-EM). Here we explore some of these advances to improve structures determined using electron cryotomography (cryo-ET) and sub-tomogram averaging. We implement a new three-dimensional model for the contrast transfer function, and use this in a regularized likelihood optimization algorithm as implemented in the RELION program. Using direct electron detector data, we apply both single-particle analysis and sub-tomogram averaging to analyze radiation-induced movements of the specimen. As in single-particle cryo-EM, we find that significant sample movements occur during tomographic data acquisition, and that these movements are substantially reduced through the use of ultrastable gold substrates. We obtain a sub-nanometer resolution structure of the hepatitis B capsid, and show that reducing radiation-induced specimen movement may be central to attempts at further improving tomogram quality and resolution.
authors.affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK. Electronic address: tbharat@mrc-lmb.cam.ac.uk.
authors.forename: Tanmay A M
authors.initials: TAM
authors.lastname: Bharat
authors.affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Russo
authors.affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Jan
authors.initials: J
authors.lastname: Löwe
authors.affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Lori A
authors.initials: LA
authors.lastname: Passmore
authors.affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK. Electronic address: scheres@mrc-lmb.cam.ac.uk.
authors.forename: Sjors H W
authors.initials: SHW
authors.lastname: Scheres
mesh_terms: D000465:Algorithms; D001499:Bayes Theorem; D036022:Capsid Proteins / Q000737:chemistry*; D020285:Cryoelectron Microscopy / Q000379:methods*; D006515:Hepatitis B virus / Q000737:chemistry / Q000378:metabolism*; D021621:Imaging, Three-Dimensional / Q000379:methods; D008958:Models, Molecular; D011487:Protein Conformation
chemical_list: D036022:Capsid Proteins