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

Ganesh Agam; Christian Gebhardt; Milana Popara; Rebecca Mächtel; Julian Folz; Benjamin Ambrose; Neharika Chamachi; Sang Yoon Chung; Timothy D Craggs; Marijn de Boer; Dina Grohmann; Taekjip Ha; Andreas Hartmann; Jelle Hendrix; Verena Hirschfeld; Christian G Hübner; Thorsten Hugel; Dominik Kammerer; Hyun-Seo Kang; Achillefs N Kapanidis; Georg Krainer; Kevin Kramm; Edward A Lemke; Eitan Lerner; Emmanuel Margeat; Kirsten Martens; Jens Michaelis; Jaba Mitra; Gabriel G Moya Muñoz; Robert B Quast; Nicole C Robb; Michael Sattler; Michael Schlierf; Jonathan Schneider; Tim Schröder; Anna Sefer; Piau Siong Tan; Johann Thurn; Philip Tinnefeld; John van Noort; Shimon Weiss; Nicolas Wendler; Niels Zijlstra; Anders Barth; Claus a M Seidel; Don C Lamb; Thorben Cordes

doi:10.1038/s41592-023-01807-0

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

Journal article

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Peer reviewed

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

Ganesh Agam, Christian Gebhardt, Milana Popara, Rebecca Mächtel, Julian Folz, Benjamin Ambrose, Neharika Chamachi, Sang Yoon Chung, Timothy D Craggs, Marijn de Boer, …

Nature Methods, Vol.20(4), pp.523 - 535

2023

DOI: https://doi.org/10.1038/s41592-023-01807-0

PMCID: PMC10089922

PMID: 36973549

Abstract

Fluorescence Resonance Energy Transfer

Reproducibility of Results

Proteins

Molecular Conformation

Laboratories

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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Title: Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins
Creators - without role: Ganesh Agam - Ludwig-Maximilians-Universität München
Christian Gebhardt - Ludwig-Maximilians-Universität München
Milana Popara - Heinrich Heine University Düsseldorf
Rebecca Mächtel - Ludwig-Maximilians-Universität München
Julian Folz - Heinrich Heine University Düsseldorf
Benjamin Ambrose - University of Sheffield
Neharika Chamachi - TU Dresden
Sang Yoon Chung - University of California
Timothy D Craggs - University of Sheffield
Marijn de Boer - University of Groningen
Dina Grohmann - University of Regensburg
Taekjip Ha - Howard Hughes Medical Institute
Andreas Hartmann - TU Dresden
Jelle Hendrix - Hasselt University
Verena Hirschfeld - University of Lübeck
Christian G Hübner - University of Lübeck
Thorsten Hugel - University of Freiburg
Dominik Kammerer - University of Oxford
Hyun-Seo Kang - Technical University of Munich
Achillefs N Kapanidis - University of Oxford
Georg Krainer - TU Dresden
Kevin Kramm - University of Regensburg
Edward A Lemke - Johannes Gutenberg University Mainz
Eitan Lerner - Hebrew University of Jerusalem
Emmanuel Margeat - Université de Montpellier, Centre de Biologie Structurale - CBS
Kirsten Martens - Huygens Institute for History and Culture of the Netherlands
Jens Michaelis - Universität Ulm
Jaba Mitra - Howard Hughes Medical Institute
Gabriel G Moya Muñoz - Ludwig-Maximilians University [Munich]
Robert B Quast - Université de Montpellier, Centre de Biologie Structurale - CBS
Nicole C Robb - University of Oxford
Michael Sattler - Technical University of Munich
Michael Schlierf - TU Dresden
Jonathan Schneider - Ludwig-Maximilians-Universität München
Tim Schröder - Ludwig-Maximilians-Universität München
Anna Sefer - Universität Ulm
Piau Siong Tan - Johannes Gutenberg University Mainz
Johann Thurn - University of Freiburg
Philip Tinnefeld - Ludwig-Maximilians-Universität München
John van Noort - Huygens Institute for History and Culture of the Netherlands
Shimon Weiss - University of California, Los Angeles
Nicolas Wendler - Ludwig-Maximilians-Universität München
Niels Zijlstra - Ludwig-Maximilians-Universität München
Anders Barth - Heinrich Heine University Düsseldorf
Claus a M Seidel - Heinrich Heine University Düsseldorf
Don C Lamb - Ludwig-Maximilians-Universität München
Thorben Cordes - Ludwig-Maximilians-Universität München
Publication Details: Nature Methods, Vol.20(4), pp.523 - 535
Identifiers: 9937735609311
Academic Unit: Centre de Biologie Structurale - CBS
Language: English
Resource Type: Journal article
Local Fields: hal-04120543