Single-particle electron cryotomography (cryoET)

Schmid, Michael F.

doi:10.1016/b978-0-12-386507-6.00002-6

Cited by 25 publications

(24 citation statements)

References 74 publications

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“…Contrary to previous assertions (Castaño-Díez 2012), boxes > 128 3 voxels are routinely used in SPT on a wide range of specimens such as carboxysomes (Schmid 2011), viruses (Schmid et al 2012) and axonemes (Koyfman 2011). We predict that as SPT strives to achieve higher resolution, finer image sampling will easily result in subtomograms much larger than 128 3 , reaching 512 3 or more for large macromolecular assemblies, such as centrioles (Li 2011) and the nuclear pore complex (Maimon et al 2012).…”

Section: Methodsmentioning

confidence: 99%

Single particle tomography in EMAN2

Galaz-Montoya

Flanagan

Schmid

et al. 2015

Journal of Structural Biology

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108

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Single particle tomography (SPT or subtomogram averaging) offers a powerful alternative to traditional 2-D single particle reconstruction for studying conformationally or compositionally heterogeneous macromolecules. It can also provide direct observation (without labeling or staining) of complexes inside cells at nanometer resolution. The development of computational methods and tools for SPT remains an area of active research. Here we present the EMAN2.1 SPT toolbox, which offers a full SPT processing pipeline, from particle picking to post-alignment analysis of subtomogram averages, automating most steps. Different algorithm combinations can be applied at each step, providing versatility and allowing for procedural cross-testing and specimen-specific strategies. Alignment methods include all-vs-all, binary tree, iterative single-model refinement, multiple-model refinement, and self-symmetry alignment. An efficient angular search, Graphic Processing Unit (GPU) acceleration and both threaded and distributed parallelism are provided to speed up processing. Finally, automated simulations, per particle reconstruction of subtiltseries, and per-particle Contrast Transfer Function (CTF) correction have been implemented. Processing examples using both real and simulated data are shown for several structures.

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Section: Methodsmentioning

confidence: 99%

Single particle tomography in EMAN2

Galaz-Montoya

Flanagan

Schmid

et al. 2015

Journal of Structural Biology

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100

108

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“…This conceptual similarity with Single Particle techniques also shows in the use of cross-correlation as main tool to align data particles to references of increasing quality. Subtomogram averaging is therefore also often referred to as ''Single Particle Tomography'', when the technique is applied on isolated particles (Schmid, 2011).…”

Section: Cryo Electron Tomography and Subtomogram Averagingmentioning

confidence: 99%

Dynamo: A flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments

Castaño-Díez

Kudryashev

Arheit

et al. 2012

Journal of Structural Biology

408

421

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“…Such studies, as well as tools developed for subtomogram averaging (Castaño-Diez et al 2012; Forster et al 2007; Hrabe et al 2012; Nicastro et al 2006), have been comprehensively reviewed (Fernandez 2012; Kudryashev et al 2012; Schmid 2011). …”

Section: Introductionmentioning

confidence: 99%

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

Galaz-Montoya

Hecksel²,

Baldwin

et al. 2016

Journal of Structural Biology

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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 grid and 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.

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Single-particle electron cryotomography (cryoET)

Cited by 25 publications

References 74 publications

Single particle tomography in EMAN2

Single particle tomography in EMAN2

Dynamo: A flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments

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

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