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 F _o F ₁ and respiratory Supercomplexes (SCs) on sub-mitochondrial particles (SMPs) isolated from bovine heart mitochondria. On SMPs, the majority of F _o F ₁ was identified as dimers bound by the regulatory factor dimeric IF ₁ . In addition, a tetrameric structure formed by association of F _o F ₁ IF ₁ dimers and with a linear arrangement of the F ₁ 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 CIII ₂ were resolved. Most SCs were of the CI ₁ CIII ₂ CIV ₃ structure, although the presence of the CI ₂ CIII ₂ CIV ₆ mega complex was also identified. Our study enabled rapid in situ structural determination of SCs and F _o F ₁ 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.