Abstract
SummaryRecent innovations in specimen preparation, data collection, and image processing have led to improved structure determination using single-particle electron cryomicroscopy (cryo-EM). Here we explore some of these advances to improve structures determined using electron cryotomography (cryo-ET) and sub-tomogram averaging. We implement a new three-dimensional model for the contrast transfer function, and use this in a regularized likelihood optimization algorithm as implemented in the RELION program. Using direct electron detector data, we apply both single-particle analysis and sub-tomogram averaging to analyze radiation-induced movements of the specimen. As in single-particle cryo-EM, we find that significant sample movements occur during tomographic data acquisition, and that these movements are substantially reduced through the use of ultrastable gold substrates. We obtain a sub-nanometer resolution structure of the hepatitis B capsid, and show that reducing radiation-induced specimen movement may be central to attempts at further improving tomogram quality and resolution.
Highlights
High-resolution structure determination by single-particle analysis of electron cryomicroscopy data is undergoing rapid progress
The main differences are the three-dimensionality of the data, and the way the tomographic missing wedge and the contrast transfer function (CTF) are treated simultaneously in a single 3D model
For all Fourier components of all N experimental sub-tomograms, this noise is assumed to be independent, zero-mean, and Gaussian distributed with variance s2ij
Summary
High-resolution structure determination by single-particle analysis of electron cryomicroscopy (cryo-EM) data is undergoing rapid progress. New substrates reduce specimen movement to improve image quality (Russo and Passmore, 2014a, 2014b) Added to these instrumentation developments, improvements in refinement algorithms have made image alignment and classification more accurate, resulting in higher-resolution structures from lower amounts of cryo-EM data than was previously possible (Bai et al, 2013; Scheres, 2012a). This has led to a number of near-atomic resolution cryo-EM structures by single-particle analysis This has led to a number of near-atomic resolution cryo-EM structures by single-particle analysis (e.g. Allegretti et al, 2014; Bartesaghi et al, 2014; Liao et al, 2013; Wong et al, 2014)
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