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

• Main Authors: Hellenkamp, Björn, Schmid, Sonja, Doroshenko, Olga, Opanasyuk, Oleg, Kühnemuth, Ralf, Adariani, Soheila Rezaei, Barth, Anders, Birkedal, Victoria, Bowen, Mark E, Chen, Hongtao, Cordes, Thorben, Eilert, Tobias, Fijen, Carel, Götz, Markus, Gouridis, Giorgos, Gratton, Enrico, Ha, Taekjip, Hanke, Christian A, Hartmann, Andreas, Hendrix, Jelle, Hildebrandt, Lasse L, Hohlbein, Johannes, 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, Röcker, Carlheinz, Sanabria, Hugo, Schlierf, Michael, Schuler, Benjamin, Seidel, Henning, Streit, Lisa, Tinnefeld, Philip, Tyagi, Swati, Vandenberk, Niels, Weninger, Keith R, Wünsch, Bettina, Yanez-Orozco, Inna S, Michaelis, Jens, Seidel, Claus A. M, Craggs, Timothy D, Hugel, Thorsten
• Format: Journal Article
• Language: English
• Published: 29-12-2017
• Subjects: Physics - Biological Physics; Quantitative Biology - Quantitative Methods
• DOI: 10.48550/arxiv.1710.03807

Natue Methods, vol. 15, pages 669-676, 2018 Single-molecule F\"orster 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
${\Delta}$E = +-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.