Dr. Julia Mahamid, EMBL Heidelberg

Structural biology has traditionally focused 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 the level of individual macromolecules 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' enable the assignment of macromolecular structures directly from the high-resolution three-dimensional stills captured by cryo-ET when aided by an array of methods, including computational pattern recognition and novel developments of genetically-encoded molecular tags. 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 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 functions of previously unknown macromolecular complexes. Such integration of cutting-edge methods unlocks an enormous potential for mechanistic understanding and system-spanning discovery by in-cell structural biology.