Antibiotic-resistant pathogens pose a significant threat to human health. The NIH identified several Gram- negative species of particular concern due to increasing antibiotic resistances. The cell envelope of Gram- negative bacteria consists of two membranes and lipopolysaccharides (LPS) form an important component of the extracellular leaflet of the outer membrane. LPS are important cell wall components that control diffusion across the outer membrane, stabilize the cell envelope, and assist in escaping host immune defenses, among other functions. Impaired LPS biosynthesis correlates with increased susceptibility to antimicrobial treatments. LPS contain a conserved core, consisting of lipid-A attached to an oligosaccharide backbone, and a hypervariable region, called the O antigen. O antigens are primarily linear complex carbohydrates that reduce the efficacy of complement-mediated cell lysis and phagocytosis as part of the innate immune response. O antigens are synthesized via two fundamentally different mechanisms. One pathway relies on assembling the polymers from short oligosaccharides in the periplasm, the other involves moving fully-assembled O antigens from the cytosolic to the periplasmic side of the inner membrane with the help of an ABC transporter. Not only is ABC transporter-mediated secretion of O antigens an important process for microbial pathogenicity, the transport of a substrate several times the size of the ABC transporter itself is fascinating from a molecular level. Taking advantage of an already determined O antigen-translocating ABC transporter structure, we propose a structural biology approach to unravel the mechanism of O antigen translocation and to identify unique features of the O antigen that regulate transporter activity. ABC transporters use ATP binding and hydrolysis to cycle between conformations that mediate substrate translocation. Our O antigen ABC transporter structure represents a nucleotide-free conformation, in which the transporter forms a continuous channel across the membrane that could accommodate a translocating O antigen. We speculate that conformational changes associated with nucleotide binding induce O antigen translocation by about 1-2 sugar units per ATP hydrolyzed. To reveal the molecular mechanism of O antigen translocation, we seek to determine the structure of the ABC transporter in a nucleotide-bound closed conformation (Aim 1). Further, many bacterial species signal completion of O antigen biosynthesis by modifying the polymer’s growing end with specific groups, such as carbohydrate, phosphate, or methyl moieties. These ‘capped O antigens’ can only be exported by transporte...