Open Access

Alexander Hahn, Kristian Parey, Maike Bublitz, Deryck J. Mills, Volker Zickermann, Janet Vonck, Werner Kühlbrandt, Thomas Kurt Meier

• Highlights • Cryo-EM structure of a yeast F1Fo-ATP synthase dimer • Inhibitor-free X-ray structure of the F1 head and rotor complex • Mechanism of ATP generation by rotary catalysis • Structural basis of cristae formation in the inner mitochondrial membrane Summary We determined the structure of a complete, dimeric F1Fo-ATP synthase from yeast Yarrowia lipolytica mitochondria by a combination of cryo-EM and X-ray crystallography. The final structure resolves 58 of the 60 dimer subunits. Horizontal helices of subunit a in Fo wrap around the c-ring rotor, and a total of six vertical helices assigned to subunits a, b, f, i, and 8 span the membrane. Subunit 8 (A6L in human) is an evolutionary derivative of the bacterial b subunit. On the lumenal membrane surface, subunit f establishes direct contact between the two monomers. Comparison with a cryo-EM map of the F1Fo monomer identifies subunits e and g at the lateral dimer interface. They do not form dimer contacts but enable dimer formation by inducing.