Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study

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  • Bjoern Hellenkamp, University of Freiburg, Columbia University
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  • Sonja Schmid, University of Freiburg, Delft University of Technology
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  • Olga Doroshenko, Heinrich-Heine-Universität Düsseldorf
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  • Oleg Opanasyuk, Heinrich-Heine-Universität Düsseldorf
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  • Ralf Kuehnemuth, Heinrich-Heine-Universität Düsseldorf
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  • Soheila Rezaei Adariani, Clemson University
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  • Benjamin Ambrose, University of Sheffield
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  • Mikayel Aznauryan
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  • Anders Barth, Ludwig-Maximilians-Universität, München
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  • Victoria Birkedal
  • Mark E. Bowen, Stony Brook University
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  • Hongtao Chen, University of California
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  • Thorben Cordes, University of Groningen, Ludwig-Maximilians-Universität, München
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  • Tobias Eilert, Ulm University
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  • Carel Fijen, Wageningen University & Research
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  • Christian Gebhardt, Ludwig Maximilians Universität München
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  • Markus Goetz, University of Freiburg
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  • Giorgos Gouridis, University of Groningen, Ludwig-Maximilians-Universität München
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  • Enrico Gratton, University of California
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  • Taekjip Ha, John Hopkins University
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  • Pengyu Hao, North Carolina State University
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  • Christian A. Hanke, Heinrich-Heine-Universität Düsseldorf
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  • Andreas Hartmann, TU Dresden
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  • Jelle Hendrix, University of Leuven, Hasselt University
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  • Lasse L. Hildebrandt
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  • Verena Hirschfeld, University of Lübeck
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  • Johannes Hohlbein, Wageningen University & Research
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  • Boyang Hua, John Hopkins University
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  • Christian G. Huebner, University of Lübeck
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  • Eleni Kallis, Ulm University
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  • Achillefs N. Kapanidis, University of Oxford
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  • Jae-Yeol Kim, Seoul National University
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  • Georg Krainer, TU Dresden, Technische Universität Kaiserslautern
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  • Don C. Lamb, Ludwig-Maximilians-Universität, München
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  • Nam Ki Lee, Seoul National University
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  • Edward A. Lemke, Johannes Gutenberg-University Mainz, Institute of Molecular Biology (IMB), Mainz, European Molecular Biology Laboratory (EMBL)
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  • Brie Levesque, Stony Brook University
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  • Marcia Levitus
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  • James J. McCann, Stony Brook University
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  • Nikolaus Naredi-Rainer, Ludwig-Maximilians-Universität, München
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  • Daniel Nettels, University of Zurich
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  • Trang Thuy Ngo, John Hopkins University
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  • Ruoyi Qiu, North Carolina State University
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  • Nicole C. Robb, University of Oxford
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  • Carlheinz Roecker, Ulm University
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  • Hugo Sanabria, Clemson University
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  • Michael Schlierf, TU Dresden
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  • Tim Schroeder, Ludwig-Maximilians-Universität, München
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  • Benjamin Schuler, University of Zurich
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  • Henning Seidel, University of Lübeck
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  • Lisa Streit, Ulm University
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  • Johann Thurn, University of Freiburg
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  • Philip Tinnefeld, Ludwig-Maximilians-Universität, München, Braunschweig University of Technology
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  • Swati Tyagi, European Molecular Biology Laboratory (EMBL)
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  • Niels Vandenberk, University of Leuven
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  • Andres Manuel Vera, Ludwig-Maximilians-Universität München
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  • Keith R. Weninger, North Carolina State University
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  • Bettina Wunsch, Braunschweig University of Technology
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  • Inna S. Yanez-Orozco, Clemson University
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  • Jens Michaelis, Ulm University
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  • Claus A. M. Seidel, Heinrich-Heine-Universität Düsseldorf
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  • Timothy D. Craggs, University of Sheffield, University of Oxford
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  • Thorsten Hugel, University of Freiburg

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.

Original languageEnglish
JournalNature Methods
Volume15
Issue9
Pages (from-to)669-676
Number of pages12
ISSN1548-7091
DOIs
Publication statusPublished - 30 Sep 2018

    Research areas

  • RESONANCE ENERGY-TRANSFER, ALTERNATING-LASER EXCITATION, STRUCTURAL INFORMATION, SPECTROSCOPIC RULER, PHOTON DISTRIBUTION, REFRACTIVE-INDEX, FLUORESCENCE, DNA, DYNAMICS, DEPENDENCE

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