ABSTRACT Lipids are synthesized and modified primarily by integral membrane enzymes embedded, at least in part, in the bilayer itself. These enzymatic reactions are essential not only for the biosynthesis of all cellular membranes, but also for lipid-mediated signaling and for the export of soluble molecules as lipid conjugates to outer cellular compartments for a wide array of basic cellular functions, which include protein and lipid glycosylation, and modifications of the chemical properties of outer membranes. However, despite the advances in our understanding of how membrane proteins function, our knowledge of how membrane enzymes interact with their lipidic substrates at atomic level remains scarce, also hindered by the hydrophobicity of the ligands themselves. The main focus of my lab is to use structural biology – mainly single-particle cryo-electron microscopy (cryo-EM) – to investigate at a molecular level the interactions between membrane enzymes and their lipidic substrates. In a structural biology-centered integrated approach, our structures, combined with computational experiments, will produce testable functional hypotheses. We are interested in understanding how hydrophobic and hydrophilic substrates are brought into apposition for catalysis to occur, on how chemical reactions involving charged groups and an aqueous environment can adapt to process lipophilic molecules, on what are the molecular determinants of substrate specificity for hydrophobic ligands, and on the role that the membrane itself plays in these processes. We expect common principles on the interactions between, membrane, membrane enzymes, and lipidic substrates to emerge from our studies. We will focus our attention on the subclass of membrane glycosyltransferases that utilize polyprenyl- mono- and di-phosphate sugar conjugates as substrates. These enzymes play a role in a wide array of biological functions including providing the sugars needed for protein glycosylation, modification of the lipopolysaccharide component of the outer membrane of Gram-negative bacteria in response to antibiotics, biosynthesis of the bacterial peptidoglycan layer, and assembly of the mycobacterial cell well. We will work on individual enzymes deciphering their molecular mechanism across their entire catalytic cycle, as well as understanding how these might function as part of larger macromolecular assemblies, in a physiological context. To succeed, we will combine our expertise in membrane protein production, biochemistry, and structural biology, to that of our collaborators that are leaders in their respective fields. These range from computational biology, to chemical synthesis of sugar-lipid conjugates, to biochemical analysis of LPS, to functional analyses of membrane proteins in reconstituted systems, to the generation of tools to allow cryo-EM analysis of small proteins and time resolved cryo-EM, to native mass spectrometry, to genetic modification of bacterial strains, to in ce...