Project Summary Pathogenic bacteria employ specialized secretion systems to identify and interact with host cells and to exchange genetic information through horizontal gene transfer. These machines are attractive drug targets because they are surface-exposed, widely conserved, and specific for pathogenicity. Unfortunately, however, the structures of many of these critical systems remain poorly understood. Here we describe how we will continue to use electron cryotomography (cryoET) to dissect the structures and functions of pathogenic nanomachines. CryoET is a revolutionary imaging technique with the power to reveal native structures inside intact cells in 3D with macromolecular (2-5 nm) resolution. Subtomogram averaging of identical structures from one or more cryotomograms can push this resolution to better than 1 nm in the most favorable cases, enabling components to be placed in their context in the complete machine. My group has pioneered the development of this revolutionary imaging technology, and in just under four years of our first award period, we have used cryoET to produce tens of new structures of pathogenic secretion systems and build architectural models of key systems belonging to the type IV pilus (T4P), type VI secretion system (T6SS) and type IV secretion system (T4SS) families, producing a flood of new mechanistic insights. By exploiting new cryoET technologies we have just developed in the past couple years, here we propose to extend our work in the next award period to different functional states of these complexes, key related systems, and a new target: the pathogenic type IX secretion system (T9SS). In addition, we will push the whole body of work to higher resolution. For each target, we will image the entire, intact structure in situ. In most cases, this will be the first high-resolution imaging of these structures. We will then combine subtomogram averaging with difference analysis of mutants in which individual components are knocked out or tagged with additional density in order to produce architectural models of the complexes. In cases where atomic models of components (or homologs) are available, we will dock them into our maps to produce pseudo-atomic models of each machine. By comparing these structures with those of non-pathogenic relatives (solved previously or in the proposed work), we aim to identify adaptations underlying virulence functions. We will also apply state-of-the-art cryogenic correlated light and electron microscopy (cryo-CLEM) to guide cryogenic focused ion beam (FIB) milling to enable us to image pathogenic secretion systems in action: in bacterial cells infecting eukaryotic hosts. This will provide the first such images of critical human pathogens, which we expect to provide invaluable insights into the operation of their virulence machinery in vivo. Together, we expect this project to produce a detailed mechanistic picture of the T4SS, T4P, and T9SS nanomachines that mediate pathogenesis, an...