Co-reconstitution of subunits E and G of the yeast V-ATPase and the α and β subunits of the F₁-ATPase from the thermophilic Bacillus PS3 (TF₁) resulted in an α₃β₃EG hybrid complex showing 53% of the ATPase activity of TF₁. The α₃β₃EG oligomer was characterized by electron microscopy. By processing 40,000 single particle projections, averaged two-dimensional projections at 1.2-2.4-nm resolution were obtained showing the hybrid complex in various positions. Difference mapping of top and side views of this complex with projections of the atomic model of the α ₃β₃ subcomplex from TF₁ (Shirakihara, Y., Leslie, A. G., Abrahams, J. P., Walker, J. E., Ueda, T., Sekimoto, Y., Kambara, M., Saika, K., Kagawa, Y., and Yoshida, M. (1997) Structure 5, 825-836) demonstrates that a seventh mass is located inside the shaft of the α₃β₃ barrel and extends out from the hexamer. Furthermore, difference mapping of the α₃β₃EG oligomer with projections of the A₃B₃E and A ₃B₃EC subcomplexes of the V₁ from Caloramator fervidus (Chaban, Y., Ubbink-Kok, T., Keegstra, W., Lolkema, J. S., and Boekema, E. J. (2002) EMBO Rep. 3, 982-987) shows that the mass inside the shaft is made up of subunit E, whereby subunit G was assigned to belong at least in part to the density of the protruding stalk. The formation of an active α₃β₃EG hybrid complex indicates that the coupling subunit γ inside the α₃β₃ oligomer of F₁ can be effectively replaced by subunit E of the V-ATPase. Our results have also demonstrated that the E and γ subunits are structurally similar, despite the fact that their genes do not show significant homology.