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

• Published in: Nature methods Vol. 20; no. 4; pp. 523 - 535
• Main Authors: Agam, Ganesh, Gebhardt, Christian, Popara, Milana, Mächtel, Rebecca, Folz, Julian, Ambrose, Benjamin, Chamachi, Neharika, Chung, Sang Yoon, Craggs, Timothy D, de Boer, Marijn, Grohmann, Dina, Ha, Taekjip, Hartmann, Andreas, Hendrix, Jelle, Hirschfeld, Verena, Hübner, Christian G, Hugel, Thorsten, Kammerer, Dominik, Kang, Hyun-Seo, Kapanidis, Achillefs N, Krainer, Georg, Kramm, Kevin, Lemke, Edward A, Lerner, Eitan, Margeat, Emmanuel, Martens, Kirsten, Michaelis, Jens, Mitra, Jaba, Moya Muñoz, Gabriel G, Quast, Robert B, Robb, Nicole C, Sattler, Michael, Schlierf, Michael, Schneider, Jonathan, Schröder, Tim, Sefer, Anna, Tan, Piau Siong, Thurn, Johann, Tinnefeld, Philip, van Noort, John, Weiss, Shimon, Wendler, Nicolas, Zijlstra, Niels, Barth, Anders, Seidel, Claus A M, Lamb, Don C, Cordes, Thorben
• Format: Journal Article
• Language: English
• Published: United States Nature Publishing Group 01.04.2023; Nature Publishing Group US
• Subjects: Analysis; Biochemistry, Molecular Biology; Biology; Biophysics; Dynamic structural analysis; Dynamics; Energy transfer; Fluorescence Resonance Energy Transfer; Fluorescence Resonance Energy Transfer - methods; In vivo methods and tests; Laboratories; Laboratory tests; Life Sciences; Molecular Conformation; Perturbation; Proteins; Proteins - chemistry; Reproducibility of Results; Structural Biology; Uncertainty
• ISSN: 1548-7091; 1548-7105
• DOI: 10.1038/s41592-023-01807-0

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.