title: Alignment algorithms and per-particle CTF correction for single particle cryo-electron tomography.
abstract: Single particle cryo-electron tomography (cryoSPT) extracts features from cryo-electron tomograms, followed by 3D classification, alignment and averaging to generate improved 3D density maps of such features. Robust methods to correct for the contrast transfer function (CTF) of the electron microscope are necessary for cryoSPT to reach its resolution potential. Many factors can make CTF correction for cryoSPT challenging, such as lack of eucentricity of the specimen stage, inherent low dose per image, specimen charging, beam-induced specimen motions, and defocus gradients resulting both from specimen tilting and from unpredictable ice thickness variations. Current CTF correction methods for cryoET make at least one of the following assumptions: that the defocus at the center of the image is the same across the images of a tiltseries, that the particles all lie at the same Z-height in the embedding ice, and/or that the specimen, the cryo-electron microscopy (cryoEM) grid and/or the carbon support are flat. These experimental conditions are not always met. We have developed a CTF correction algorithm for cryoSPT without making any of the aforementioned assumptions. We also introduce speed and accuracy improvements and a higher degree of automation to the subtomogram averaging algorithms available in EMAN2. Using motion-corrected images of isolated virus particles as a benchmark specimen, recorded with a DE20 direct detection camera, we show that our CTF correction and subtomogram alignment routines can yield subtomogram averages close to 4/5 Nyquist frequency of the detector under our experimental conditions.
authors.affiliation: National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Jesús G
authors.initials: JG
authors.lastname: Galaz-Montoya
authors.affiliation: National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA; Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Corey W
authors.initials: CW
authors.lastname: Hecksel
authors.affiliation: National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA; Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Philip R
authors.initials: PR
authors.lastname: Baldwin
authors.affiliation: Institute for Human Infections and Immunity and Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Eryu
authors.initials: E
authors.lastname: Wang
authors.affiliation: Institute for Human Infections and Immunity and Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Scott C
authors.initials: SC
authors.lastname: Weaver
authors.affiliation: National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Schmid
authors.affiliation: National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Ludtke
authors.affiliation: National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA; Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA. Electronic address: wah@bcm.edu.
authors.forename: Wah
authors.initials: W
authors.lastname: Chiu
mesh_terms: D000465:Algorithms; D020285:Cryoelectron Microscopy / Q000379:methods; D055032:Electron Microscope Tomography / Q000379:methods* / Q000592:standards; D014771:Virion / Q000648:ultrastructure
keywords: Contrast transfer function (CTF); Cryo-electron tomography (cryoET); Direct detection device (DDD); EMAN2; Single particle cryo-electron tomography (cryoSPT); Subtomogram averaging