Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology.
| Original language | English |
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| Pages (from-to) | 523-535 |
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| Number of pages | 13 |
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| Journal | Nature Methods |
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| Volume | 20 |
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| Issue number | 4 |
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| DOIs | |
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| Publication status | Published - 2023 |
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| Externally published | Yes |
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Work in the laboratory of T.C. was financed by a European Research Council (ERC) Starting grant (no. ERC-StG 638536—SM-IMPORT), German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) within grant nos. GRK2062 (project C03) and SFB863 (project A13) and an Alexander von Humboldt postdoctoral fellowship (to N.Z.). T.C., P.T. and D.C.L. acknowledge the support of the Center for integrated protein science Munich and the Center for NanoScience. D.C.L. acknowledges the support of the Nanosystems Initiative Munich and LMUinnovative program BioImaging Network. We also acknowledge support via the SFB1035 (DFG, Sonderforschungsbereich 1035 project no. 201302640, project no. A11 to D.C.L. and project no. B03 to M. Sattler). D.C.L. and P.T. acknowledge support by the Federal Ministry of Education and Research (BMBF) and the Free State of Bavaria under the Excellence Strategy of the Federal Government and the Länder through the ONE MUNICH Project Munich Multiscale Biofabrication. C.A.M.S. acknowledges the support by the ERC (grant no. 671208 (hybridFRET)) and by the DFG (grant nos. SE 1195/17-1 and CRC 1208 (project no. A08)). A.B. acknowledges funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 101029907. Research in the contributing authors’ laboratories was financed by the following sources: P.T. acknowledges the support by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project ID 201269156, SFB 1032 (A13) and project ID 267681426. T.D.C. was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) (grant no. BB/T008032/1) and Engineering and Physical Sciences Research Council (EPSRC) (grant no. EP/V034804/1), B.A. was supported by an EPSRC Prize Fellowship. BMBF grant nos. 03Z2EN11 and 03Z22E511 as well as DFG grant no. SCHL 1896/4-1 (to M. Schlierf). SFB960 project A7 (to D.G.) US National Institutes of Health grant no. GM122569 (to T. Ha). J.H. acknowledges the Research Foundation Flanders (FWO) (project nos. G0B4915, G0B9922N and G0H3716N) and is indebted to Johan Hofkens at KU Leuven for the used smFRET infrastructure. ERC grant agreement no. 681891 (Prosint) and DFG under Germany’s Excellence Strategy (CIBSS EXC-2189 project ID 390939984) and the SFB1381 program (project ID 403222702) (to T. Hugel). Royal Society Dorothy Hodgkin Research Fellowship DKR00620 and a Research Grant for Research Fellows no. RGF\R1\180054 (to N.C.R.), by the Wellcome Trust (grant no. 110164/Z/15/Z to A.N.K.). The Israel Science Foundation (grant nos. 556/22 to E.L., 3565/20 to E.L., within the KillCorona – Curbing Coronavirus Research Program), the National Institutes of Health (grant no. R01 GM130942 to S.W. and to E.L. as a subaward), by the Milner Fund (to E.L.) and by the Hebrew University of Jerusalem (start-up funds to E.L.). Agence Nationale pour la Recherche (grant nos. ANR 18-CE11-0004-02, ANR-19-CE44-0009-02, ANR-21-CE11-0034-01, ANR-21-CE11-0026-03 and ANR-10-INBS-04, ‘Investments for the future’ to E.M.). E.A.L. acknowledges funding by the ERC ADG MultiOrganelleDesign and the SFB1551 (project ID 464588647).