Structures of respiratory supercomplexes and ATP synthase oligomers in mammalian mitochondrial inner membrane

Understanding the functional mechanisms of membrane protein complexes requires structural analysis within their native membrane environment. Here, we applied cryo-electron microscopy to determine the structures of F_oF₁ ATP synthase and respiratory supercomplexes (SCs) on sub-mitochondrial particles (SMPs) isolated from bovine heart mitochondria. Most F_oF₁ complexes were observed as dimers stabilized by the regulatory factor IF₁, and a tetrameric assembly comprising two F_oF₁-IF₁ dimers arranged linearly was also identified. This finding indicates that the tetrameric units of F_oF₁ are present in the mitochondrial inner membrane and contribute to shaping cristae tips in mammalian mitochondria. F_o domain maps resolve the e-subunit- c₈-ring interface and show no discrete density for a tightly bound lipid within the c₈-ring. In addition to the previously reported SCs compositions CI₁CIII₂CIV₁ and CI₁CIII₂CIV₂, our analysis identified an additional assembly with the composition CI₁CIII₂CIV₃, as well as a CI₂CIII₂CIV₆ mega-complex. This approach enables rapid structural determination of F_oF₁ ATP synthase and SCs from minimal membrane fractions, providing a foundation for elucidating the molecular basis of metabolic disorders and mitochondrial diseases at the level of higher-order architecture.