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Here we analyzed an axle\u2010less TF\n                    <jats:sub>1<\/jats:sub>\n                    , in which the lower \u03b3 region is truncated, by combining single\u2010molecule rotation assays with cryo\u2010EM structural analysis under catalysis conditions. Under ATP\u2010saturated conditions, wild\u2010type TF\n                    <jats:sub>1<\/jats:sub>\n                    exhibited three pauses per turn corresponding to the catalytic dwells, whereas axle\u2010less TF\n                    <jats:sub>1<\/jats:sub>\n                    exhibited six, revealing an additional intermediate dwell that may be stabilized by \u03b3 truncation. This dwell occurred at 40\u00b0 between the binding and catalytic dwells. High\u2010speed recordings revealed frequent backsteps during the 40\u00b0 transition between the intermediate and catalytic dwells. Dwell\u2010time analysis indicated an approximately zero free\u2010energy bias between these states, consistent with the absence of a detectable directional bias. Cryo\u2010EM resolved the corresponding binding, intermediate, and catalytic dwell structures, showing a major \u03b2 conformational change from 0\u00b0 to 40\u00b0, but minimal \u03b2 rearrangement between 40\u00b0\u00a0and 80\u00b0, suggesting that the rotation is not coupled with a large \u03b2 conformational transition. Thus, the 0\u201380\u00b0 step in axle\u2010less TF\n                    <jats:sub>1<\/jats:sub>\n                    consists of two regimes: a directional 0\u201340\u00b0 motion accompanied by a major \u03b2 conformational change, followed by a 40\u201380\u00b0 interval with little detectable \u03b2 rearrangement and markedly reduced directional bias. These findings suggest that, in axle\u2010less TF\n                    <jats:sub>1<\/jats:sub>\n                    , the latter half of the 80\u00b0 substep proceeds through thermal diffusion without directional bias, and that the lower \u03b3 region may normally help bias this interval toward forward rotation.\n                  <\/jats:p>","DOI":"10.1002\/pro.70803","type":"journal-article","created":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T11:14:32Z","timestamp":1790162072000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["The lower \u03b3 region supports forward rotation in the latter half of the 80\u00b0 substep of\n                    <scp>\n                      F\n                      <sub>1<\/sub>\n                    <\/scp>\n                    \u2010\n                    <scp>ATPase<\/scp>"],"prefix":"10.1002","volume":"35","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-1882-5070","authenticated-orcid":false,"given":"Tomo","family":"Uchiyama","sequence":"first","affiliation":[{"name":"Department of Applied Chemistry Graduate School of Engineering, The University of Tokyo  Bunkyo\u2010ku Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5331-4335","authenticated-orcid":false,"given":"Hiroshi","family":"Ueno","sequence":"additional","affiliation":[{"name":"Department of Applied Chemistry Graduate School of Engineering, The University of Tokyo  Bunkyo\u2010ku Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7086-3467","authenticated-orcid":false,"given":"Meghna","family":"Sobti","sequence":"additional","affiliation":[{"name":"Heart Rhythms Division The Victor Chang Cardiac Research Institute  Sydney New South Wales Australia"},{"name":"St Vincent's Clinical School, Faculty of Medicine UNSW Sydney  Sydney New South Wales Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9086-2979","authenticated-orcid":false,"given":"Emily J.","family":"Furlong","sequence":"additional","affiliation":[{"name":"Heart Rhythms Division The Victor Chang Cardiac Research Institute  Sydney New South Wales Australia"},{"name":"Division of Biomedical Science and Biochemistry, Research School of Biology Australian National University  Canberra Australian Capital Territory Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3997-4614","authenticated-orcid":false,"given":"Simon H. J.","family":"Brown","sequence":"additional","affiliation":[{"name":"School of Science, Molecular Horizons, and the Australian Research Council Centre for Cryo\u2010electron Microscopy of Membrane Proteins University of Wollongong  Wollongong New South Wales Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alastair G.","family":"Stewart","sequence":"additional","affiliation":[{"name":"Heart Rhythms Division The Victor Chang Cardiac Research Institute  Sydney New South Wales Australia"},{"name":"St Vincent's Clinical School, Faculty of Medicine UNSW Sydney  Sydney New South Wales Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8842-6836","authenticated-orcid":false,"given":"Hiroyuki","family":"Noji","sequence":"additional","affiliation":[{"name":"Department of Applied Chemistry Graduate School of Engineering, The University of Tokyo  Bunkyo\u2010ku Tokyo Japan"},{"name":"Research Institute of Planetary Health (RIPH) The University of Tokyo  Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,23]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/370621a0"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2007.05.020"},{"key":"e_1_2_9_4_1","first-page":"531","article-title":"Real\u2010space refinement in PHENIX for cryo\u2010EM and crystallography","volume":"74","author":"Afonine PV","year":"2018","journal-title":"Biol Crystallogr"},{"key":"e_1_2_9_5_1","first-page":"519","article-title":"ISOLDE: a physically realistic environment for model building into low\u2010resolution electron\u2010density maps","volume":"74","author":"Croll TI","year":"2018","journal-title":"Biol 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