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. 2021 Dec 10;12:7225. doi: 10.1038/s41467-021-27580-2

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Structure of bR trimer viewed from the cytoplasmic side in the closed (PDB 6RNJ) and open (PDB 6RPH) states. Letters label the 7 helices. Outlines highlight one protomer. b Superposition of the open (green) and closed (red) structural states: The conformational change in helix-F (and to a minor degree in helix-E) are highlighted by arrows. The E-F loop moves by ~5 Å. c HS-AFM setup: Only the main components are shown and labeled: The IR laser (AFM laser) monitors the cantilever position. The green laser (activating laser) is used to excite bR. d Top: Light protocol. Middle: HS-AFM images of bR-D96N patches exposing cytoplasmic and extracellular sides (labeled). Bottom: Averages of the cytoplasmic and extracellular side topographies (labeled cyto and ext in the first pair of images). Only the cytoplasmic side displays measurable conformational changes during and after (bR-D96N has ~10.5 s open state dwell-time) light stimulation. Light-induced conformational changes in bR-D96N subjected to (e) a single, and (f) multiple green laser light periods. Top: Light protocol. Middle: High-resolution HS-AFM images and corresponding correlation averages. bR trimer (white dashed circle) and trefoil, defined as the three nearest-neighbor protomers that gather upon light-activated conformational changes (yellow dashed circle). Bottom: E-F loop displacement (average (black line) ±standard deviation (gray shaded area), n = 12). Gray lines in (e, bottom) show the behavior of three individual protomers. Insets in (e, bottom) are LAFM maps⁴⁰ of molecules in the closed (frames 1–26) and open (frames 28–49) state.