A practical look at cryo-electron tomography image processing: Key considerations for new biological discoveries

Cryo-electron tomography (cryo-ET) enables 3D visualization of complex biological environments without the need for purification, thereby preserving the native biological context of the specimen. For determining macromolecular structures, repeating molecules can be localized in tomograms and subjected to subtomogram averaging, the 3D analog to single particle analysis. In addition to molecular structure, tomograms have a wealth of other information that can be accessed through image processing, including the analysis of membrane surfaces, cytoskeletal filaments, and the relationships between molecules of interest. Here, we provide an overview of recent developments in cryo-ET image processing with the goal of clarifying key considerations to help new users obtain novel biological findings.