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Abstract

Mycoplasma pneumoniae, a genome-reduced pathogenic bacterium, is as an ideal model for studying the concept of a minimal cell. This organism benefits from a long history of analyses that encompass many biological processes and molecular types, providing a comprehensive foundation for computational studies. The advent of high-resolution cryogenic electron tomography (cryo-ET) has placed M. pneumoniae in a unique position to bridge in-situ imaging with bioinformatics and systems biology. This unprecedented direct visual access to protein complexes and other macromolecular assemblies presents novel opportunities and challenges. While some large and abundant molecular components can be readily identified in the tomograms, deciphering their functions requires sophisticated computational approaches that make use of the wealth of existing data. The recent introduction of AlphaFold2 has revolutionized the field of structural biology by providing high-confidence structural models for virtually every protein. In this thesis, I leverage this new paradigm to annotate the function of a newly identified protein complex observed exclusively in M. pneumoniae cryo-ET data. I show that access to protein structures dramatically improves the accuracy of functional annotations, particularly when structures are segmented into their constituent domains. One of the largest macromolecular assemblies in the cell is the ribosome, with its many interacting partners. Utilizing a novel cryo-ET dataset that captures the abundances of intermediate states of ribosomes engaged in protein synthesis, I build a kinetic model of the translation-elongation cycle. Furthermore, I develop and implement a generalizable method to calibrate the kinetic rates of biological processes by integrating cryo-ET data with know rates from a reference system. This thesis advances structural bioinformatics by designing innovative analytical frameworks downstream of cryo-ET. These frameworks enable a better annotation of the function of proteins from their structure, as well as a better understanding of the dynamics of molecular processes from static cryo-ET snapshots.