The lower γ region supports forward rotation in the latter half of the 80° substep of F1-ATPase.

F1-ATPase achieves unidirectional rotation of its γ shaft through coordinated conformational cycling of the α3β3 ring, yet how shaft-ring interactions contribute to the directionality of rotation remains unclear. Here we analyzed an axle-less TF1, in which the lower γ region is truncated, by combining single-molecule rotation assays with cryo-EM structural analysis under catalysis conditions. Under ATP-saturated conditions, wild-type TF1 exhibited three pauses per turn corresponding to the catalytic dwells, whereas axle-less TF1 exhibited six, revealing an additional intermediate dwell that may be stabilized by γ truncation. This dwell occurred at 40° between the binding and catalytic dwells. High-speed recordings revealed frequent backsteps during the 40° transition between the intermediate and catalytic dwells. Dwell-time analysis indicated an approximately zero free-energy bias between these states, consistent with the absence of a detectable directional bias. Cryo-EM resolved the corresponding binding, intermediate, and catalytic dwell structures, showing a major β conformational change from 0° to 40°, but minimal β rearrangement between 40° and 80°, suggesting that the rotation is not coupled with a large β conformational transition. Thus, the 0-80° step in axle-less TF1 consists of two regimes: a directional 0-40° motion accompanied by a major β conformational change, followed by a 40-80° interval with little detectable β rearrangement and markedly reduced directional bias. These findings suggest that, in axle-less TF1, the latter half of the 80° substep proceeds through thermal diffusion without directional bias, and that the lower γ region may normally help bias this interval toward forward rotation.