Electron cryo-tomography (cryo-ET) and sub-tomogram averaging allow structure determination of macromolecules in situ, and are gaining in popularity for initial model generation for single- particle analysis. We describe herein, a protocol for sub-tomogram averaging from cryo-ET data using the RELION software. We describe how to calculate newly developed three-dimensional models for the contrast transfer function and the missing wedge of each sub-tomogram, and how to use these models for regularized-likelihood refinement. This approach has been implemented in the existing workflow for single-particle analysis, so that users may conveniently tap into existing capabilities of the RELION software. As example applications, we present analyses of purified hepatitis B capsid particles and S. cerevisiae 80S ribosomes. In both cases, we show that following initial classification, sub-tomogram averaging in RELION allows de novo generation of initial models, and provides high-resolution maps where secondary structure elements are resolved.
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