Subtomogram Averaging

Subtomogram averaging (STA) is a method to determine macromolecular structures from electron tomograms, typically in their frozen-hydrated state (cryo-ET). Similar to single particle analysis, structures are determined through iterative alignment and averaging of subtomograms containing molecules of interest that exist in the data. The key difference is that in the case of STA, the data to be averaged is already in 3D. This difference gives STA its unique ability to determine structures from complex specimens without the need to purify their molecular components. This enables the structure determination of any repeating features, including pleomorphic assemblies, membrane complexes, and globular proteins, in near-native biologically-relevant environments, such as within cryo-preserved cells. STA is a rapidly developing method, and in this chapter, we provide an overview on the different steps within an STA workflow. These steps are particle picking from tomograms, subtomogram alignment and averaging, classification of particles within datasets, structure-driven refinement of tilt-series parameters, and analysis of the rich meta-data that is determined along with the structure. We provide a review of the aims and methods used at each step, as well as a summary of outstanding problems and future developments. We finish with a summary on the state of the field and an outlook on its future.