Cryo-tomography and subtomogram averaging are increasingly popular techniques for structural determination of macromolecular complexes in situ. They have the potential to achieve high-resolution views of native complexes, together with the details of their location relative to interacting molecules. The subtomogram averaging (StA) pipelines are well-established, with current developments aiming to optimise each step by reducing manual intervention and user decisions, following similar trends in single-particle approaches that have dramatically increased their popularity. Here, we review the main steps of typical StA workflows. We focus on considerations arising from the fact that the objects of study are embedded within unique crowded environments, and we emphasise those steps where careful decisions need to be made by the user.
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