期刊
JOURNAL OF STRUCTURAL BIOLOGY
卷 194, 期 3, 页码 383-394出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jsb.2016.03.018
关键词
Cryo-electron tomography (cryoET); Single particle cryo-electron tomography (cryoSPT); Subtomogram averaging; Direct detection device (DDD); Contrast transfer function (CTF); EMAN2
资金
- National Institutes of Health (NIH, United States) [P41GM103832, R01GM080139, AI057156]
- Robert Welch Foundation (Texas, United States) [Q1242]
- Computational Cancer Biology Training Program of the Gulf Coast Consortia (Cancer Prevention Research Institute of Texas, CPRIT, in the United States) [RP140113]
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. (C) 2016 Elsevier Inc. All rights reserved.
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