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. 2023 Dec 15;43(1):1–13. doi: 10.1038/s44318-023-00004-1

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© The Author(s) 2023

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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(A) Confocal volume with illustration of a single proteoliposome (PL) with embedded SecYEG diffusing in and out of the confocal volume alongside. The positions of the dyes are indicted using green/red stars. Note that SecYEG is shown in a single orientation here, but inserts equally in both orientations into the liposomes under the conditions used (Deville et al, 2011). Hence only ~50% of molecules will bind SecA in the experiments presented (see Methods). (B) A representative single-molecule time trace showing photons from the D_exD_em (Donor Excitation, Donor Emission) in green, D_exA_em (Donor Excitation, Acceptor Emission) in red, and A_exA_em (Acceptor Excitation, Acceptor Emission) in purple. (C) A single-molecule photon time trace of a single burst. Photon timestamps are represented by coloured vertical bars (not related to the y-axis). The most likely state path as identified by the Viterbi algorithm as derived by mpH²MM is overlaid as two horizontal coloured lines relating to the y-axis for both E_raw and S_raw.