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

Research output: Preprint/documentation/reportPreprint

Contributors

  • Ganesh Agam - (Author)
  • Christian Gebhardt - , Ludwig Maximilian University of Munich (Author)
  • Milana Popara - (Author)
  • Rebecca Mächtel - (Author)
  • Julian Folz - (Author)
  • Benjamin Ambrose - , University of Sheffield (Author)
  • Neharika Gajanan Chamachi - , DRESDEN-concept Genome Center (CMCB Core Facility), Chair of Molecular Biophysics (Author)
  • Sang Yoon Chung - (Author)
  • Timothy D. Craggs - , Oxford Brookes University (Author)
  • Marijn de Boer - (Author)
  • Dina Grohmann - (Author)
  • Taekjip Ha - , Johns Hopkins University, Univ Illinois, Howard Hughes Medical Institute, University of Illinois System, University of Illinois Urbana-Champaign, Howard Hughes Med Inst (Author)
  • Andreas Hartmann - , Chair of Molecular Biophysics (Author)
  • Jelle Hendrix - , Hasselt University (Author)
  • Verena Hirschfeld - , Univ Lubeck, University of Lubeck, Inst Phys (Author)
  • Christian G. Hübner - (Author)
  • Thorsten Hugel - , University of Freiburg (Author)
  • Dominik Kammerer - (Author)
  • Hyun-Seo Kang - (Author)
  • Achillefs N. Kapanidis - , Oxford Brookes University (Author)
  • Georg Krainer - , Chair of Molecular Biophysics, Nanoanalytical methods (Junior Research Group) (Author)
  • Kevin Kramm - (Author)
  • Edward A. Lemke - , European Molecular Biology Laboratory (Author)
  • Eitan Lerner - (Author)
  • Emmanuel Margeat - (Author)
  • Kristen Martens - (Author)
  • Jens Michaelis - , Ulm University (Author)
  • Jaba Mitra - (Author)
  • Gustavo G. Moya Munoz - (Author)
  • Robert Quast - (Author)
  • Nicole B. Robb - (Author)
  • Michael Sattler - (Author)
  • Michael Schlierf - , Chair of Molecular Biophysics, Physics of Life (Author)
  • Jonathan Schneider - (Author)
  • Tim Schröder - (Author)
  • Anna Sefer - (Author)
  • Piau Siong Tan - (Author)
  • Johann Thurn - , University of Freiburg (Author)
  • Philip Tinnefeld - (Author)
  • John van Noort - (Author)
  • Shimon Weiss - (Author)
  • Nicolas Wendler - (Author)
  • Niels Zijlstra - (Author)
  • Anders Barth - , Ludwig Maximilian University of Munich (Author)
  • Claus A. M. Seidel - , Heinrich Heine University Düsseldorf (Author)
  • Don C. Lamb - , Ludwig Maximilian University of Munich (Author)
  • Thorben Cordes - , Ludwig Maximilian University of Munich (Author)

Abstract

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs 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 that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than ± 0.06, corresponding to an interdye distance precision of ≤ 0.2 nm and accuracy of ≤ 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to ≲ 10% of the Förster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology. ### Competing Interest Statement Tim Craggs and Achilles Kapanidis, two of the authors are founders of different companies selling single-molecule fluorescence microscopes (Exciting Instruments, Oxford Nanoimager).

Details

Original languageEnglish
Publication statusPublished - 2022
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External IDs

unpaywall 10.1101/2022.08.03.502619
Mendeley 991d6cfe-53d8-3513-9846-54a5be20eb6a

Keywords

DFG Classification of Subject Areas according to Review Boards