6533b85cfe1ef96bd12bca3e

RESEARCH PRODUCT

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

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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. This work was supported by the European Research Council (ERC; grant agreement nos. 261227 (to A.N.K.), 646451 (to E.L.), 638536 (to T.C.), 671208 (to C.A.M.S.), and 681891 (to T. Hugel)), the Deutsche Forschungsgemeinschaft (DFG) (grant MI 749/4-1 to J.M., grant TI 329/10-1 to P.T., and grant SCHL 1896/3-1 to M.S.), the Swiss National Science Foundation (to B.S.), the German Federal Ministry of Education and Research (BMBF; 03Z2EN11 to M.S.), Research Foundation Flanders (FWO; grant G0B4915N to J. Hendrix), the Agency for Innovation by Science and Technology (IWT Flanders; doctoral scholarship to N.V.), the Danish Council for Independent Research (Sapere Aude grant 0602-01670B to V.B.), the Novo Nordisk Foundation (NNF15OC0017956 to V.B.), the UK BBSRC (grant BB/H01795X/1 to A.N.K.),the National Institute of Mental Health (grant MH081923 to M.E.B.), Clemson University (start-up funds to H. Sanabria, S.R.A., and I.S.Y.-O.), the NIH (grants GM109832 and GM118508 to K.R.W.; grant GM112659 to T. Ha), the NSF (Career Award MCS1749778 to H. Sanabria), the Carl-Zeiss-Stiftung (doctoral fellowship to E.K.), the Stipendienstiftung Rheinland-Pfalz (doctoral scholarship to G.K.), the Braunschweig International Graduate School of Metrology (B-IGSM; to B.W.), the DFG Research Training Group (GrK1952/1 "Metrology for Complex Nanosystems" to B.W.), the University of Sheffield (start-up funds to T.D.C.), and the National Research Foundation of Korea funded by the Ministry of Science and ICT (NRF-2017R1A2B3010309 to N.K.L.).