Project Abstract Bacterial pathogens are a threat to global health and have evolved elaborate strategies to infect their hosts. One potent bacterial weapon in the war between host and pathogen is the Type IV secretion system (T4SS), found in a wide variety of bacterial species, including those that cause human disease, such as Legionella pneumophila, Helicobacter pylori, Bordetella pertussis, Brucella, Bartonella, and Coxiella. The L. pneumophila, T4SS translocates 100s of effector proteins into the host cell and is essential for pathogenesis. T4SSs are challenging complexes to work with because they span the inner and outer membranes of Gram-negative bacteria and contain a minimum of 12 proteins. Studies in prototype systems, such as E. coli plasmids and A. tumefaciens show that T4SSs are generally organized into an outer membrane core complex (OMCC), an inner membrane complex (IMC), and in some species an extracellular pilus. While there are no published high- resolution (sub-3.5 Å) structures of an entire T4SS; there are sub-3.5 Å structures of OMCCs from both the Xanthomonas citri and conjugation system that translocate DNA. In contrast to the DNA-translocating T4SSs, the L. pneumophila T4SS is composed of ~26 components and low resolution (~30 Å) cryo-electron tomography studies show that this T4SS has an enlarged OMCC and contains periplasmic densities not seen in the structures of minimized E. coli and X. citri T4SSs. The lack of detailed structural information limits our mechanistic understanding of how the L. pneumophila T4SS functions and contributes to pathogenesis. The focus of this proposal is to use a combination of structural and genetic approaches to construct the first detailed molecular map of the organization of the L. pneumophila T4SS. These high resolution models will be used to address fundamental questions such as whether there are conserved structural components shared among T4SSs that can be exploited for new anti-bacterial treatment strategies, how T4SSs are tuned to export proteins versus nucleic acids, and how export of effectors is regulated.