Developments in cryo-electron tomography for in situ structural analysis

•Improving contrast in cryo-ET.•Direct electron detectors in cryo-ET.•In situ structural studies of macromolecular assemblies.•Focused ion beam milling as a tool for sample preparation.

Structural analysis of macromolecular assemblies and their remodeling during physiological processes is instrumental to defining the fundament of cellular and molecular biology. Recent advances in computational and analytical tools for cryo-electron tomography have enabled the study of macromolecular structures in their native environment, providing unprecedented insights into cell function. Moreover, the recent implementation of direct electron detectors has progressed cryo-electron tomography to a stage where it can now be applied to the reconstruction of macromolecular structures at high resolutions. Here, we discuss some of the recent technical developments in cryo-electron tomography to reveal structures of macromolecular complexes in their physiological medium, focusing mainly on eukaryotic cells.