Most structural biology focuses on the structure and function of individual macromolecular complexes, but falls short of revealing how they come together to give rise to cellular functions. Here, cryo-electron tomography (cryo-ET) provides a unique opportunity for obtaining structural information across a wide range of spatial scales - from small model organisms, intact cells and 3D cultures frozen in their close-to-native state, to individual macromolecular assemblies embedded in their native functional environments. We develop and employ advanced sample preparation techniques for in-cell cryo-ET, including cryo-focused ion beam thinning guided by 3D correlative fluorescence microscopy. Preparations of such site-specific ‘electron-transparent windows’ in appropriate cellular model systems visualizes molecular structures directly from three-dimensional stills of intact cells and can reveal their molecular sociology. Using the genome-reduced human pathogen Mycoplasma pneumoniae as a minimal cell model, we further demonstrated the synergistic application of whole-cell crosslinking mass spectrometry and cellular cryo-ET to determine an in-cell integrative model of actively transcribing RNA polymerases coupled to a translating ribosomes. Recent computational breakthroughs now allow resolving these molecular machines to near-atomic resolution directly inside the cell, reveal small molecule antibiotics bound to their active site in ribosomes within the intact pathogen, provide snapshots of their structural dynamics along reaction cycles, and illuminate the existence and hint at functions of previously unknown macromolecular complexes. These cutting-edge methodologies unlock an enormous potential for system-spanning discovery enabled by label-free in-cell structural biology, and have recently enabled us to uncover cellular, molecular and structural basis of how stress to the host triggers viral replication and release during persistent mumps virus infection mediated by biomolecular condensation.
