PROJECT SUMMARY/ABSTRACT Organelle morphology is specialized for the highly adapted functions found in complex tissues. Mitochondrial ultrastructure is exquisitely tuned to metabolic and physiological state. Abnormal morphology is a hallmark of neurological disorders, cardiac conditions and cancer. With the cryo-EM `resolution revolution', we have developed dramatic new understanding of membrane protein structure and regulation, but our knowledge of organelle structure lags behind. This is due to the pleomorphic nature of organelles, and the challenge of assigning specific morphological features to protein states. The overarching goals of my lab are to understand, at a mechanistic level, how protein conformational change is influenced by subcellular context, how membrane ultrastructure is regulated by protein factors, and the functional interplay of these elements in physiology and disease. Over the next five years, my group will develop a technical platform that combines electron cryo- microscopy (cryo-EM) and biophysical methods to study mitochondrial ultrastructure and its regulation. We will apply our recent developed in vitro reconstitution systems to visualize reconstituted membrane proteins in liposomes and bilayers by single-particle cryo-EM. We will mature our newly established electron cryo- tomography (cryo-ET) pipeline for computational analyses of membrane properties to understand their dependence on protein-protein interactions. We shall develop new structural and biophysical methods to characterize organelle lipid heterogeneity. And finally, we will explore assembly or protein complexes in native contexts. Together these approaches will advance mechanistic understanding of protein conformational state and help us identify the fundamental determinants of organelle shape. We are broadly interested in questions of membrane spacing, composition and curvature. We will develop tools precisely tailored for these questions using mitochondria as a test bed, exploring the structure and function of candidate factors that regulate mitochondrial membrane morphology, which play causal roles in neurodegenerative conditions. Opa1 is the inner-membrane fusogen and cristae remodeler mutated in Dominant Optic Atrophy. SLC25A46 is an outer- membrane member of the solute transporter family that plays important roles in coordinating lipid homeostasis in Leigh Syndrome. MICOS is the stabilizer and regulator of cristae junctions (the `choke-point' to the mitochondrial inner-membrane folds) whose loss results in early-onset fatal mitochondrial encephalopathy with liver disease. This project's immediate impacts include sharing new models to understand mitochondrial shape with cell biologists, equipping pharmacologists with new, highly specific conformational targets for therapeutic development, and providing physiologists with fundamental rules for understanding tissue specialization. The long-term goal is to build an extensible approach generalizable to ot...