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J Struct Biol. 2016 Jun;194(3):383-94. doi: 10.1016/j.jsb.2016.03.018. Epub 2016 Mar 22.

Alignment algorithms and per-particle CTF correction for single particle cryo-electron tomography.

Author information

1
National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA.
2
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.
3
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.
4
Institute for Human Infections and Immunity and Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA.
5
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.

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.

KEYWORDS:

Contrast transfer function (CTF); Cryo-electron tomography (cryoET); Direct detection device (DDD); EMAN2; Single particle cryo-electron tomography (cryoSPT); Subtomogram averaging

PMID:
27016284
PMCID:
PMC4846534
DOI:
10.1016/j.jsb.2016.03.018
[Indexed for MEDLINE]
Free PMC Article

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