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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. 2 — a Top: Light protocol: Continuous light of predefined intensity. Bottom: HS-AFM-LS raw data kymograph. Red and green arrowheads indicate the closed and open state positions of a single protomer, respectively. Inset: 150 ms of the kymograph illustrating the clarity of the raw data comprising well resolved ~10 ms-events. The protomer E-F loop shifts position in the kymograph. b Height-time traces along the red (closed) and green (open) state E-F loop positions in a. To enhance the signal-to-noise ratio, we subtract the two anticorrelated signals (red and green traces) to get the yellow trace which is used for idealized state assignment (black trace, see Methods). c Schematic of bR trimers and the HS-AFM-LS x-scan line. The x-scan line contours closed (red) and open (green) protomers, giving location depend signatures in the contour profiles (below). The profiles are stacked to give a kymograph (right). Red and green arrowheads indicate the closed and open state positions as in a. d Dwell-time histograms of (a) and many more kymographs with closed state τ_closed = 107.8 ± 0.8 ms (red, n = 483) and open state τ_open = 18.9 ± 0.3 ms (green, n = 471) at 1.9·10¹⁸ photons/cm²·s light intensity. e Response of bR-WT protomers to changing light conditions. Top: Light protocol: Low light – high light – dark. Middle: HS-AFM-LS raw data kymograph. Yellow arrowhead: protomer whose activity is shown below. Bottom: Height-time trace (yellow) and idealized state assignment (black). f HS-AFM-LS raw data kymographs (top) and height-time traces (yellow) and idealized state assignments (black) (bottom) at different light intensities (the green triangle on the right symbolizes increasing light intensity from top to bottom). g Dwell-times in the closed (red, top) and the open (green, bottom) states as a function of light intensity. Error bars in g are smaller than the data points, see d. Black arrowheads in g indicate the data points from d. Insets: At saturating light power <τ>_closed = 40.5 ms and <τ>_open = 13.2 ms. n is the total number of events analyzed. τ denotes dwell-time as the fit of the single exponential fit, $y (x) = C e^{- x / τ}$ .