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

Abstract

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.

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Item Type:	Article
Authors/Creators:	Agam, G. https://orcid.org/0000-0003-1623-4920 Gebhardt, C. https://orcid.org/0000-0002-5834-6579 Popara, M. https://orcid.org/0000-0003-2626-6096 Mächtel, R. Folz, J. https://orcid.org/0000-0001-8232-8787 Ambrose, B. https://orcid.org/0000-0002-5046-2532 Chamachi, N. https://orcid.org/0000-0002-4657-9092 Chung, S.Y. Craggs, T.D. https://orcid.org/0000-0002-7121-0609 de Boer, M. Grohmann, D. https://orcid.org/0000-0002-0570-2517 Ha, T. https://orcid.org/0000-0003-2195-6258 Hartmann, A. https://orcid.org/0000-0002-2213-2763 Hendrix, J. https://orcid.org/0000-0001-5731-1297 Hirschfeld, V. Hübner, C.G. Hugel, T. https://orcid.org/0000-0003-3292-4569 Kammerer, D. Kang, H.-S. https://orcid.org/0000-0003-4029-5096 Kapanidis, A.N. Krainer, G. https://orcid.org/0000-0002-9626-7636 Kramm, K. Lemke, E.A. https://orcid.org/0000-0002-0634-0503 Lerner, E. https://orcid.org/0000-0002-3791-5277 Margeat, E. https://orcid.org/0000-0001-6063-6420 Martens, K. Michaelis, J. https://orcid.org/0000-0002-2739-4172 Mitra, J. Moya Muñoz, G.G. https://orcid.org/0000-0003-2797-2005 Quast, R.B. https://orcid.org/0000-0001-8166-6952 Robb, N.C. Sattler, M. Schlierf, M. https://orcid.org/0000-0002-6209-2364 Schneider, J. Schröder, T. https://orcid.org/0000-0002-2474-5395 Sefer, A. Tan, P.S. Thurn, J. https://orcid.org/0000-0001-6370-7052 Tinnefeld, P. van Noort, J. https://orcid.org/0000-0002-9094-5598 Weiss, S. https://orcid.org/0000-0002-0720-5426 Wendler, N. https://orcid.org/0000-0002-5436-3929 Zijlstra, N. Barth, A. https://orcid.org/0000-0003-3671-3072 Seidel, C.A.M. https://orcid.org/0000-0002-5171-149X Lamb, D.C. https://orcid.org/0000-0002-0232-1903 Cordes, T. https://orcid.org/0000-0002-8598-5499
Copyright, Publisher and Additional Information:	© The Author(s) 2023. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Keywords:	Fluorescence resonance energy transfer; Molecular biophysics; Proteins; Single-molecule biophysics; Structure determination
Dates:	Published: April 2023 Published (online): 27 March 2023 Accepted: 31 January 2023
Institution:	The University of Sheffield
Academic Units:	The University of Sheffield > Faculty of Science (Sheffield) > Department of Chemistry (Sheffield)
Funding Information:	Funder Grant number BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL BB/T008032/1 Engineering and Physical Sciences Research Council EP/V034804/1
Depositing User:	Symplectic Sheffield
Date Deposited:	20 Apr 2023 09:46
Last Modified:	20 Apr 2023 09:46
Published Version:	http://dx.doi.org/10.1038/s41592-023-01807-0
Status:	Published
Publisher:	Springer Science and Business Media LLC
Refereed:	Yes
Identification Number:	10.1038/s41592-023-01807-0
Related URLs:	PubMed URL
Open Archives Initiative ID (OAI ID):	oai:eprints.whiterose.ac.uk:198312

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

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