Alignment algorithms and per-particle CTF correction for single particle cryo-electron tomography - PubMed (original) (raw)

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

Jesús G Galaz-Montoya et al. J Struct Biol. 2016 Jun.

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.

Copyright © 2016 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Drift correction for cryoET images of a tilted specimen collected with a DE20 camera

Examples of VEEV images exhibiting significant drift, taken at different tilt angles, shown before (top) and after (bottom) drift correction.

Figure 2

Figure 2. Quantification of image drift in cryoET images of a tilted specimen

Examples of total drift per frame and average drift per image for all images in (A) a “bad” unidirectional tiltseries and (B) a “good” bidirectional tiltseries.

Figure 3

Figure 3. Interactive CTF fitting in EMAN2

Examples of periodogram averaging by interactive micrograph tiling for power spectrum (PS) computation and CTF fitting directly from the imaging area for cryoET images of a tilted specimen. (A) Tiling of 0° image from a VEEV tiltseries showing clear CTF ripples out to 4/5 Nyquist. (B) Example of per-strip tiling of a high tilt (50°) image from a VEEV tiltseries, showing clear CTF ripples out to 2/3 Nyquist.

Figure 4

Figure 4. Measured defocus variation within and across tiltseries

Plots showing large measured defocus variations within and across six VEEV cryoET tiltseries collected using a JEM3200FSC electron microscope.

Figure 5

Figure 5. Resolution analyses of CTF-Corrected VEEV subtomogram averages

(A) Per-tomogram FSC curves for six CTF-corrected VEEV subtomogram averages from independent tomograms keeping the top 75% best correlating particles in each average. (B) FSCs for CTF-corrected subtomogram averages comprised of different fractions of particles (~100% / 516 particles; ~90% / 464 particles; ~75% / 387 particles; ~30% / 154 particles; ~10% / 51 particles).

Figure 6

Figure 6. CTF-Corrected Subtomogram Average of VEEV

Isosurfaces of VEEV subtomogram average comprised of the top 75% best-correlating subtomograms showing the outer glycoprotein shell (top) and the nucleocapsid (bottom).

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