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Abstract

To structurally and quantitatively delineate molecular processes inside the cell with atomic detail is the ultimate goal of structural biology. Cryo-electron tomography (cryo-ET) and sub-tomogram analysis provide currently the most promising approach to achieve this goal. In this thesis, I aim to push the limits of in-cell structural biology and achieve its full potential, using the small genome-reduced bacterium Mycoplasma pneumoniae as a cell model. Translation, the fundamental process of protein synthesis, is mediated by ribosomes in all living cells. With comprehensive ribosome sub-tomogram datasets extracted in silico from tomograms of intact M. pneumoniae cells, I determined the in-cell ribosome structure at 3 Å resolution, the highest for complex structures resolved directly inside cells to date. The built atomic model represents the first ribosome model for mycoplasma species, revealing distinct ribosomal protein extensions. I then performed extensive sub-tomogram classification, and resolved over 13 translation intermediate structures at 4-10 Å resolutions. The analysis not only visualizes structural dynamics of translation elongation in native cells but also provides unique insights into its energy landscapes. Moreover, the spatial organization of translating ribosomes revealed by mapping back structures into the cell volume unraveled a local elongation coordination mechanism within polysomes, which is mediated by the ribosomal protein L9. Furthermore, I resolved more than 17 ribosome intermediate structures in three antibiotic-treated datasets and demonstrated how the drug molecules fundamentally reshape the energy and spatial landscapes of translation inside the cell. The two fundamental processes of gene expression, transcription and translation, can be coupled in prokaryotes. By combining cryo-ET, in-cell crosslinking mass spectrometry and integrative structural modeling, I determined the architecture of an actively transcribing and translating expressome complex inside the cell. In the integrative model, the transcription factor NusA was pinpointed at the interface between the NusG-bound RNA polymerase and the ribosome to mediate transcription-translation coupling. Inhibiting translation by antibiotics dissociated the expressome, whereas inhibiting transcription stalled and rearranged it, demonstrating that the active expressome assembly requires ongoing translation and transcription within the native cellular context. These works provide unprecedented insights into molecular machines in action within the cell, and herald a new era in the emerging field of integrative in-cell structural biology. This thesis demonstrates its possibility to visualize dynamic molecular machineries, and eventually the molecular sociology, with atomic detail inside cells.