3D reconstruction and flexibility of the hybrid engine Acetobacterium woodii F-ATP synthase

The Na⁺-translocating F₁F_O ATP synthase from Acetobacterium woodii (AwF-ATP synthase) with a subunit stoichiometry of α₃:β₃:γ:δ:ε:a:b₂:(c_2/3)₉:c₁ represents an evolutionary path between ATP-synthases and vacuolar ATPases, by containing a heteromeric rotor c-ring, composed of subunits c₁, c₂ and c₃, and an extra loop (γ_195-211) within the rotary γ subunit. Here, the recombinant AwF-ATP synthase was subjected to negative stain electron microscopy and single particle analysis. The reference free 2D class averages revealed high flexibility of the enzyme, wherein the F₁ and F_O domains distinctively bended to adopt multiple conformations. Moreover, both the F₁ and F_O domains tilted relative to each other to a maximum extent of 28° and 30°, respectively. The first 3D reconstruction of the AwF-ATP synthase was determined which accommodates well the modelled structure of the AwF-ATP synthase as well as the γ_195-211-loop. Molecular simulations of the enzyme underlined the bending features and flexibility observed in the electron micrographs, and enabled assessment of the dynamics of the extra γ_195-211-loop.