PROJECT SUMMARY/ABSTRACT Mitochondria form dynamic networks in cells with a regulated cycle of fission and fusion to meet energy demands. An imbalance in the cycle has been implicated in a wide range of diseases—from heart failure to cancer. Dynamin-related protein 1 (Drp1) is the master regulator of fission, and its activity is controlled through several pathways, including self-assembly regulation, post-translational modifications, partner protein interactions, and mitochondria outer membrane contact sites enriched in unsaturated cardiolipin. This project seeks to understand the structure of the Drp1 dimer (the functional unit), and the conformational rearrangements required for helical assembly of these dimers on lipid templates. I propose to use cryo-EM single particle methods to resolve the structures of the WT dimer and an assembly defective mutant. These structures will identify dimer interfaces of the protein in solution and regions of the structure that contribute to the flexibility necessary to assume a wide range of geometries required for fission. While cryo-EM will identify distinct states of the Drp1 dimer, molecular dynamic simulations will be used to elucidate conformational motions through intermediate, or transition, states needed to build larger complexes on mitochondrial membranes. These in silico studies will identify conformation sampling of Drp1 and other dynamin superfamily proteins. The results will inform future studies by identifying specific amino acids that confer functional elasticity. Finally, this proposal seeks to resolve Drp1 helical structures on lipid nanotubes using cryo-EM. The saturation state of lipid acyl chains has been identified as a regulatory factor for Drp1 recruitment and self-assembly. Multimer formation on saturated and unsaturated lipid templates will identify helical structure and polymer diameter changes resulting from lipid membrane perturbations. The conformational changes the dimer must undergo in order to form helical assemblies will provide invaluable insight into the biophysical properties of Drp1 that contribute to mitochondrial membrane fission.