Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins
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Published:2023-03-27
Issue:4
Volume:20
Page:523-535
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ISSN:1548-7091
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Container-title:Nature Methods
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language:en
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Short-container-title:Nat Methods
Author:
Agam GaneshORCID, Gebhardt ChristianORCID, Popara MilanaORCID, Mächtel Rebecca, Folz JulianORCID, Ambrose BenjaminORCID, Chamachi NeharikaORCID, Chung Sang Yoon, Craggs Timothy D.ORCID, de Boer Marijn, Grohmann DinaORCID, Ha TaekjipORCID, Hartmann AndreasORCID, Hendrix JelleORCID, Hirschfeld Verena, Hübner Christian G., Hugel ThorstenORCID, Kammerer Dominik, Kang Hyun-SeoORCID, Kapanidis Achillefs N., Krainer GeorgORCID, Kramm Kevin, Lemke Edward A.ORCID, Lerner EitanORCID, Margeat EmmanuelORCID, Martens Kirsten, Michaelis JensORCID, Mitra Jaba, Moya Muñoz Gabriel G.ORCID, Quast Robert B.ORCID, Robb Nicole C., Sattler Michael, Schlierf MichaelORCID, Schneider Jonathan, Schröder TimORCID, Sefer Anna, Tan Piau Siong, Thurn JohannORCID, Tinnefeld Philip, van Noort JohnORCID, Weiss ShimonORCID, Wendler NicolasORCID, Zijlstra Niels, Barth AndersORCID, Seidel Claus A. M.ORCID, Lamb Don C.ORCID, Cordes ThorbenORCID
Abstract
AbstractSingle-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.
Publisher
Springer Science and Business Media LLC
Subject
Cell Biology,Molecular Biology,Biochemistry,Biotechnology
Reference93 articles.
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