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

Björn Hellenkamp, Sonja Schmid, Olga Doroshenko, Oleg Opanasyuk, Ralf Kühnemuth, Soheila Rezaei Adariani, Anders Barth, Victoria Birkedal, Mark E. Bowen, Hongtao Chen, Thorben Cordes, Tobias Eilert, Carel Fijen, Markus Götz, Giorgos Gouridis, Enrico Gratton, Taekjip Ha, Christian A. Hanke, Andreas Hartmann, Jelle HendrixLasse L. Hildebrandt, Johannes Hohlbein, Christian G. Hübner, Eleni Kallis, Achillefs N. Kapanidis, Jae Yeol Kim, Georg Krainer, Don C. Lamb, Nam Ki Lee, Edward A. Lemke, Brié Levesque, Marcia Levitus, James J. McCann, Nikolaus Naredi-Rainer, Daniel Nettels, Thuy Ngo, Ruoyi Qiu, Carlheinz Röcker, Hugo Sanabria, Michael Schlierf, Benjamin Schuler, Henning Seidel, Lisa Streit, Philip Tinnefeld, Swati Tyagi, Niels Vandenberk, Keith R. Weninger, Bettina Wünsch, Inna S. Yanez-Orozco, Jens Michaelis*, Claus A.M. Seidel, Timothy D. Craggs, Thorsten Hugel

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Single-molecule Förster 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 ensuring both the reproducibility and accuracy of measuring FRET efficiencies are currently lacking. Here we report the results of a worldwide, comparative, blind study, in which 20 labs determined the FRET efficiencies of several dye-labeled DNA duplexes. Using a unified and straightforward method, we show that FRET efficiencies can be obtained with a standard deviation between ΔE = ±0.02 and ±0.05. We further suggest an experimental and computational procedure for converting FRET efficiencies into accurate distances. We discuss potential uncertainties in the experiment and the modelling. Our extensive quantitative assessment of intensity-based smFRET measurements and correction procedures serve as an essential step towards validation of distance networks with the ultimate aim to archive reliable structural models of biomolecular systems obtained by smFRET-based hybrid methods.

Original languageEnglish (US)
JournalUnknown Journal
StatePublished - Oct 10 2017
Externally publishedYes

ASJC Scopus subject areas

  • General

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