Developing minimal purification cryo-EM to understand mitochondrial myopathies

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ABSTRACT Single particle cryoEM structure determination is now a widely used methodology that has revealed the detailed mechanisms underlying a wide range of biological systems. High-resolution single particle cryoEM studies have helped us understand how environmental or genetic factors perturb normal biological function, and how these factors can give rise to disease. Insights gained through such structural studies of cellular machinery have greatly benefited drug discovery efforts, as well as expanded our understanding of drug resistance and therapeutic relapse. However, successful single particle cryoEM structure determination continues to be dependent on the production and purification of highly homogeneous, biochemically stable samples for imaging. Here, we plan to harness the unique strengths of single particle cryoEM technologies - minimal sample requirements and an exceptional capacity for structural characterization of highly heterogeneous data - to move beyond this traditional approach. Precedence for such studies have been set by previous high-resolution cryoEM structures that were determined from heterogeneous mixtures of soluble or membrane-associated proteins extracted from single-cell lysates. We plan to extend these approaches to elucidate structures of endogenous mammalian mitochondrial complexes. In particular, the methodologies developed by this work will establish an avenue to perform structural investigation of mitochondrial complexes derived from mitochondrial myopathy patients. Mitochondrial dysfunction in skeletal muscle cells can have severe pathological outcomes, and is associated with a variety of muscle-wasting diseases and numerous neuromuscular disorders. One in 5000 individuals in the U.S. suffers from mitochondrial myopathies due to genetic mutation, and while substantial effort has been placed on understanding the genetics of these diseases, we lack an underlying molecular description of the specific perturbations responsible for pathology. Directly visualizing the endogenous mitochondrial complexes that carry mutations implicated in disease states enables us to inspect how missense mutations impact macromolecular assembly and interactions. We will develop mitochondrial isolation and structure determination methodologies to enable detailed structural assessment of the endogenous complexes involved in human mitochondrial proteostasis and the mitochondrial OXPHOS system, without the need for extensive protein purification. We have shown that mitochondrial lysates can be directly applied to EM grids and imaged to yield high-resolution structures of abundant complexes. We will further develop this pipeline to produce high-resolution structures of mitochondrial complexes and interaction partners from the distinct mitochondrial subcompartments, providing important molecular insights into how mutations associated with mitochondrial myopathies perturb protein structure and function. The results will advance our understanding of skeletal muscle myopathies through direct visualization of the machines involved in disease progression, unveiling never-before-seen mitochondrial assemblies, providing a molecular explanation for disease states, and laying the groundwork for future therapies.