title: Seeing tobacco mosaic virus through direct electron detectors.
abstract: With the introduction of direct electron detectors (DED) to the field of electron cryo-microscopy, a wave of atomic-resolution structures has become available. As the new detectors still require comparative characterization, we have used tobacco mosaic virus (TMV) as a test specimen to study the quality of 3D image reconstructions from data recorded on the two direct electron detector cameras, K2 Summit and Falcon II. Using DED movie frames, we explored related image-processing aspects and compared the performance of micrograph-based and segment-based motion correction approaches. In addition, we investigated the effect of dose deposition on the atomic-resolution structure of TMV and show that radiation damage affects negative carboxyl chains first in a side-chain specific manner. Finally, using 450,000 asymmetric units and limiting the effects of radiation damage, we determined a high-resolution cryo-EM map at 3.35Å resolution. Here, we provide a comparative case study of highly ordered TMV recorded on different direct electron detectors to establish recording and processing conditions that enable structure determination up to 3.2Å in resolution using cryo-EM.
authors.affiliation: EMBL - European Molecular Biology Laboratory, Structural and Computational Biology Unit, Meyerhofstr. 1, 69117 Heidelberg, Germany.
authors.forename: Simon A
authors.initials: SA
authors.lastname: Fromm
authors.affiliation: MRC Laboratory of Molecular Biology, Structural Studies Division, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Tanmay A M
authors.initials: TA
authors.lastname: Bharat
authors.affiliation: EMBL - European Molecular Biology Laboratory, Structural and Computational Biology Unit, Meyerhofstr. 1, 69117 Heidelberg, Germany; EMBL - European Molecular Biology Laboratory, Hamburg Unit, Notkestrasse 85, 22603 Hamburg, Germany.
authors.forename: Arjen J
authors.initials: AJ
authors.lastname: Jakobi
authors.affiliation: EMBL - European Molecular Biology Laboratory, Structural and Computational Biology Unit, Meyerhofstr. 1, 69117 Heidelberg, Germany.
authors.forename: Wim J H
authors.initials: WJ
authors.lastname: Hagen
authors.affiliation: EMBL - European Molecular Biology Laboratory, Structural and Computational Biology Unit, Meyerhofstr. 1, 69117 Heidelberg, Germany. Electronic address: carsten.sachse@embl.de.
authors.forename: Carsten
authors.initials: C
authors.lastname: Sachse
mesh_terms: D036022:Capsid Proteins / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy / Q000295:instrumentation; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D014027:Tobacco Mosaic Virus / Q000648:ultrastructure*
chemical_list: D036022:Capsid Proteins
keywords: Direct electron detectors; High-resolution electron cryo-microscopy; Radiation damage; Single-particle helical reconstruction; Tobacco mosaic virus