The lower γ region supports forward rotation in the latter half of the 80° substep of F 1 ‐ ATPase

Abstract F 1 ‐ATPase achieves unidirectional rotation of its γ shaft through coordinated conformational cycling of the α 3 β 3 ring, yet how shaft–ring interactions contribute to the directionality of rotation remains unclear. Here we analyzed an axle‐less TF 1 , 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 TF 1 exhibited three pauses per turn corresponding to the catalytic dwells, whereas axle‐less TF 1 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 TF 1 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 TF 1 , 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.