Fig. 4.

Typical approaches used for in situ macromolecular structure determination using cryo-ET and sub-tomogram averaging. The first step is to identify and locate target molecules. This may be achieved using overexpression or deletion and comparison with wild-type cells. The cellular location of molecules can also be ascertained using genetically-encoded tags or cryo-CLEM. Localization and identification strategies can usually be applied either before or after sample thinning, which may be necessary for high quality cryo-ET data collection. Localized macromolecules in thinned specimens can then be imaged using cryo-ET, and densities corresponding to the macromolecule can be used for sub-tomogram averaging refinement and structure determination.