Resolving structural heterogeneity in situ through cryogenic electron tomography

Cryogenic electron tomography (cryoET) has emerged as a powerful tool for studying the structural heterogeneity of proteins and their complexes, offering insights into macromolecular dynamics directly within cells. Driven by recent computational advances, including powerful machine learning frameworks, researchers can now resolve both discrete structural states and continuous conformational changes from 3D subtomograms and stacks of 2D particle-images acquired across tilt-series. In this review, we survey recent innovations in particle classification and heterogeneous 3D reconstruction methods, focusing specifically on the relative merits of workflows that operate on reconstructed 3D subtomogram volumes compared to those using extracted 2D particle-images. We additionally highlight how these methods have provided specific biological insights into the organization, dynamics, and structural variability of cellular components. Finally, we advocate for the development of benchmarking datasets collected in vitro and in situ to enable a more objective comparison of existent and emerging methods for particle classification and heterogeneous 3D reconstruction.