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

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, Brie; 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, Andres Manuel; Weninger, Keith R.; Wunsch, Bettina; Yanez-Orozco, Inna S.; Michaelis, Jens; Seidel, Claus A. M.; Craggs, Timothy D. und Hugel, Thorsten (2018): Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study. In: Nature Methods, Bd. 15, Nr. 9: S. 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.

• Dokumententyp: Zeitschriftenartikel
• EU Funded Grant Agreement Number: 638536
• EU-Projekte: Horizon 2020 > ERC Grants > ERC Starting Grant > ERC Grant 638536: SM-IMPORT - Substrate import at work: single-molecule studies of ABC transporters
• Fakultätsübergreifende Einrichtungen: Center for NanoScience (CENS)
• Themengebiete: 500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften
• ISSN: 1548-7091
• Bemerkung: Publisher Correction: https://www.nature.com/articles/s41592-018-0193-x
• Sprache: Englisch