TY - JOUR
T1 - Alignment algorithms and per-particle CTF correction for single particle cryo-electron tomography
AU - Galaz-Montoya, Jesús G.
AU - Hecksel, Corey W.
AU - Baldwin, Philip R.
AU - Wang, Eryu
AU - Weaver, Scott C.
AU - Schmid, Michael F.
AU - Ludtke, Steven J.
AU - Chiu, Wah
N1 - Publisher Copyright:
© 2016 Elsevier Inc.
PY - 2016/6/1
Y1 - 2016/6/1
N2 - 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.
AB - 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.
KW - Contrast transfer function (CTF)
KW - Cryo-electron tomography (cryoET)
KW - Direct detection device (DDD)
KW - EMAN2
KW - Single particle cryo-electron tomography (cryoSPT)
KW - Subtomogram averaging
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U2 - 10.1016/j.jsb.2016.03.018
DO - 10.1016/j.jsb.2016.03.018
M3 - Article
C2 - 27016284
AN - SCOPUS:84962059565
SN - 1047-8477
VL - 194
SP - 383
EP - 394
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 3
ER -