Abstact

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(o)F(1) ATP synthase and respiratory supercomplexes (SCs) on sub-mitochondrial particles (SMPs) isolated from bovine heart mitochondria. Most F(o)F(1) complexes were observed as dimers stabilized by the regulatory factor IF(1), and a tetrameric assembly comprising two F(o)F(1)-IF(1) dimers arranged linearly was also identified. This finding indicates that the tetrameric units of F(o)F(1) are present in the mitochondrial inner membrane and contribute to shaping cristae tips in mammalian mitochondria. F(o) domain maps resolve the e-subunit- c(8)-ring interface and show no discrete density for a tightly bound lipid within the c(8)-ring. In addition to the previously reported SCs compositions CI(1)CIII(2)CIV(1) and CI(1)CIII(2)CIV(2), our analysis identified an additional assembly with the composition CI(1)CIII(2)CIV(3), as well as a CI(2)CIII(2)CIV(6) mega-complex. This approach enables rapid structural determination of F(o)F(1) 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.