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Title: | Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study. | Authors: | Hellenkamp, Björn Schmid, Sonja Doroshenko, Olga Opanasyuk, Oleg Kühnemuth, Ralf Adariani, Soheila Rezaei Ambrose, Benjamin Aznauryan, Mikayel Barth, Anders Birkedal, Victoria Bowen, Mark E. Chen, Hongtao Cordes, Thorben Eilert, Tobias Fijen, Carel Gebhardt, Christian Götz, Markus Gouridis, Giorgos Gratton, Enrico Ha, Taekjip Hao, Pengyu Hanke, Christian A. Hartmann, Andreas HENDRIX, Jelle Hildebrandt, Lasse L. Hirschfeld, Verena Hohlbein, Johannes Hua, Boyang Hübner, Christian G. Kallis, Eleni Kapanidis, Achillefs N. Kim, Jae-Yeol Krainer, Georg Lamb, Don C. Lee, Nam Ki Lemke, Edward A. Levesque, Brié Levitus, Marcia McCann, James J. Naredi-Rainer, Nikolaus Nettels, Daniel Ngo, Thuy Qiu, Ruoyi Robb, Nicole C. Röcker, Carlheinz Sanabria, Hugo Schlierf, Michael Schröder, Tim Schuler, Benjamin Seidel, Henning Streit, Lisa Thurn, Johann Tinnefeld, Philip Tyagi, Swati Vandenberk, Niels Vera, Andrés Manuel Weninger, Keith R. Wünsch, Bettina Yanez-Orozco, Inna S. Michaelis, Jens Seidel, Claus A. M. Craggs, Timothy D. Hugel, Thorsten |
Issue Date: | 2018 | Source: | Nature methods, 15(9), p. 669-676 | Abstract: | Single-molecule Forster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between +/- 0.02 and +/- 0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods. | Notes: | Hugel, T (reprint author), Univ Freiburg, Inst Phys Chem, Freiburg, Germany. Seidel, CAM (reprint author), Heinrich Heine Univ Dusseldorf, Mol Phys Chem, Dusseldorf, Germany. Craggs, TD (reprint author), Univ Sheffield, Dept Chem, Sheffield, S Yorkshire, England. Michaelis, J (reprint author), Ulm Univ, Inst Biophys, Ulm, Germany. Craggs, TD (reprint author), Univ Oxford, Dept Phys, Clarendon Lab, Gene Machines Grp, Oxford, England. Hugel, T (reprint author), Univ Freiburg, BIOSS Ctr Biol Signalling Studies, Freiburg, Germany, Univ Freiburg, BIOSS Ctr Biol Signalling Studies, Freiburg, Germany. | Document URI: | http://hdl.handle.net/1942/27257 | ISSN: | 1548-7091 | e-ISSN: | 1548-7105 | DOI: | 10.1038/s41592-018-0085-0 | ISI #: | 000443439700020 | Rights: | Open Access Tis 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. Te 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/. | Category: | A1 | Type: | Journal Contribution | Validations: | ecoom 2019 |
Appears in Collections: | Research publications |
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s41592-018-0085-0.pdf | Published version | 4.2 MB | Adobe PDF | View/Open |
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