The Na+-translocating F1FO ATP synthase from Acetobacterium woodii (AwF-ATP synthase) with a subunit stoichiometry of alpha(3):beta(3):gamma:delta:epsilon:a:b(2):(c(2/3))(9):c(1) represents an evolutionary path between ATP-synthases and vacuolar ATPases, by containing a heteromeric rotor c-ring, composed of subunits c(1), c(2) and c(3), and an extra loop (gamma(195-211)) within the rotary g 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 F-1 and F-O domains distinctively bended to adopt multiple conformations. Moreover, both the F-1 and F-O domains tilted relative to each other to a maximum extent of 28 degrees and 30 degrees, 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 gamma(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 gamma(195-211)-loop. (C) 2020 Elsevier Inc. All rights reserved.