Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins.

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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.

Fluorescence Resonance Energy Transfer, Reproducibility of Results, Proteins, Molecular Conformation, Laboratories
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Nat Methods
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Springer Science and Business Media LLC
Agence Nationale de la Recherche (French National Research Agency) (ANR 18-CE11-0004-02, ANR-21-CE11-0034-01, ANR-21-CE11-0026-03)
Deutsche Forschungsgemeinschaft (German Research Foundation) (Center for integrated protein science Munich (CiPSM), SCHL 1896/4-1, Nanosystems Initiative Munich (NIM), SFB863 (project A13), SE 1195/17-1, SFB1035 Projektnummer 201302640 Project B03, CIBSS EXC-2189 Project ID 390939984, Project ID 403222702, Project-ID 267681426, Project-ID 201269156 - SFB 1032 (A13), SFB960 Project A7)
Fonds Wetenschappelijk Onderzoek (Research Foundation Flanders) (G0H3716N, G0B9922N)
Israel Science Foundation (ISF) (3565/20)
Biotechnology and Biological Sciences Research Council (BB/T008032/1)
Wellcome Trust (110164/Z/15/Z)
European Research Council (638536, 671208, 681891, grant No. 671208 (hybridFRET), 681891 (Prosint), ERC-StG 638536-SM-IMPORT)
Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research) (03Z2EN11, 03Z22E511)
RCUK | Engineering and Physical Sciences Research Council (EPSRC) (EPSRC Prize Fellowship, EP/V034804/1)
Ludwig-Maximilians-Universität München (Ludwig Maximilians University Munich) (LMUinnovative program BioImaging Network (BIN))
NIGMS NIH HHS (R01 GM130942)
Hebrew University of Jerusalem (Hebrew University) (Start-up funds)
Royal Society (Dorothy Hodgkin Research Fellowship DKR00620)
EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020) (101029907, ERXC ADG Multiorganelldesign)
U.S. Department of Health & Human Services | National Institutes of Health (NIH) (GM122569)