HS-AFM observation was performed in the tapping mode using a laboratory-made HS-AFM apparatus (23) equipped with a dynamic feedback controller, in which the feedback gain was automatically tuned during imaging (32). The A3B3 proteins were immobilized on a mica surface that had been coated with APTES and then treated with glutaraldehyde because the protein tends to move on the mica, and hence, it is difficult to obtain enough good data for analysis. In the previous HS-AFM publication of α3β3 of F1-ATPase (7), the immobilization condition was the same as in this experiment. In that condition, the cooperative unidirectional rotation was observed in the presence of ATP. Therefore, we consider that the immobilization of α3β3 does not affect the protein structure so much (if the symmetry changes due to the immobilization, the protein cannot function properly). Because A3B3 of V1-ATPase is a similar protein, we make the same assumption. The mica substrate was freshly cleaved immediately before use. APTES was diluted 1:100,000 in Milli-Q water, deposited on the mica surface, and incubated for approximately 3 min. The APTES-coated substrate was thoroughly washed with Milli-Q water. Glutaraldehyde diluted to 0.025% was deposited on the APTES-treated mica and incubated for 3 min. The surface was rinsed with an observation buffer [20 mM tris-HCl (pH 7.5), 100 mM NaCl, 2 mM DTT, and 10% glycerol]. A drop (approximately 5 μl) of the A3B3 protein solution diluted to concentrations of 100 to 300 nM was loaded onto the substrate surface for 5 min. The sample was rinsed with the observation buffer and examined. The HS-AFM imaging conditions were as follows: cantilever free oscillation amplitude, 0.8 to 2 nmp-p; set-point amplitude, 80 to 90% of the free oscillation amplitude; frame rate, 3 to 5 frames/s; scan and pixel sizes, 50 nm × 50 nm with 150 × 150 pixels or 50 nm × 40 nm with 150 × 120 pixels.

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