In situ structure determination of Respiratory Supercomplexes and ATP synthase oligomers in mammalian mitochondrial inner membrane.
To understand how membrane protein complexes function within biological membranes, it is essential to determine their structure in their natural membrane environment. Here, we employed cryoEM structure analysis to elucidate the structures of ATP synthase FoF1 and respiratory Supercomplexes (SCs) on sub-mitochondrial particles (SMPs) isolated from bovine heart mitochondria. On SMPs, the majority of FoF1 was identified as dimers bound by the regulatory factor dimeric IF1. In addition, a tetrameric structure formed by association of FoF1 IF1 dimers and with a linear arrangement of the F1 head were also identified. These structures induced a steep membrane curvature, indicating the presence of a structure on SMPs similar to that found on the tips of mitochondrial cristae. High-resolution structures of the respiratory complexes were also determined, and sub-class structures of both CI and CIII2 were resolved. Most SCs were of the CI1CIII2CIV3 structure, although the presence of the CI2CIII2CIV6 mega complex was also identified. Our study enabled rapid in situ structural determination of SCs and FoF1 ATP synthase from small amount of membrane fractions, paving the way for elucidation of the molecular basis of metabolic disorders and mitochondrial diseases at the level of higher-order architecture.