The rotor stoichiometry of F-ATPases has been revealed by the combined approaches of X-ray diffraction (XRD), electron crystallography, and atomic force microscopy (AFM). XRD showed the rotor from the yeast mitochondrial F-ATPase to contain 10 subunits. AFM was used to visualize the tetradecameric chloroplast rotors, and electron crystallography and AFM together revealed the rotors from Ilyobacter tartaricus to be composed of 11 subunits. While biochemical methods had determined an approximate stoichiometric value, precise measurements and new insights into a species-dependent rotor stoichiometry became available by applying the three structural tools together. The structures of AQP1, a water channel, and G1pF, a glycerol channel, were determined by electron crystallography and XRD. The combination of both of these structural tools with molecular dynamics simulations gave a differentiated description of the mechanisms determining the selectivity of water and glycerol channels. This illustrates that the combination of different methods in structural biology reveals more than each method alone.