“Unique molecular insights into cellular functions by in-cell cryo-electron tomography”
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. As a con-sequence, structural and cell biology have traditionally been separate disciplines and employed techniques that were well defined within the realm of either one or the other. Here, cryo-electron tomography (cryo-ET) provides a unique opportunity for obtaining structural information across a wide range of spatial scales - from whole cells to individual macromolecules. We develop and employ advanced sample preparation techniques for in-cell cryo-electron tomography, including cryo-focused ion beam thinning guided by 3D correlative fluorescence microscopy. Preparations of site-specific ‘electron-transparent windows’ in suitable cellular model systems enable assignment of molecular structures directly from three-dimensional stills of intact cells and reveal their molecular sociology. Using the genome-reduced human pathogen Mycoplasma pneumoniae as a model system, we further developed the synergistic application of whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography and integrative modelling, and determine an in-cell architecture of a transient transcribing and translating expressome at sub-nanometer resolution. Recent computational breakthroughs further allow resolving small molecule antibiotics bound to their active cite within the intact pathogen. These findings highlight the enormous discovery potential of structural cell biology at the single cell level.