Toward high-resolution in situ structural biology with cryo-electron tomography and subtomogram averaging

•Cryo-ET and subtomogram averaging allow visualizing macromolecular complexes in close to physiological conditions.•This offers a significant potential for in situ structural biology.•New developments in cryo-ET and subtomogram averaging allow determining structures at unprecedented resolutions.•Challenges in data acquisition and image processing remain to be overcome for cryo-ET and subtomogram averaging to achieve their full potential. Cryo-electron tomography (cryo-ET) provides unprecedented insights into the molecular constituents of biological environments. In combination with an image processing method called subtomogram averaging (STA), detailed 3D structures of biological molecules can be obtained in large, irregular macromolecular assemblies or in situ, without the need for purification. The contextual meta-information these methods also provide, such as a protein’s location within its native environment, can then be combined with functional data. This allows the derivation of a detailed view on the physiological or pathological roles of proteins from the molecular to cellular level. Despite their tremendous potential in in situ structural biology, cryo-ET and STA have been restricted by methodological limitations, such as the low obtainable resolution. Exciting progress now allows one to reach unprecedented resolutions in situ, ranging in optimal cases beyond the nanometer barrier. Here, I review current frontiers and future challenges in routinely determining high-resolution structures in in situ environments using cryo-ET and STA.