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

Research output: Contribution to journalResearch articleContributedpeer-review


  • Ganesh Agam - , Ludwig Maximilian University of Munich (Author)
  • Christian Gebhardt - , Ludwig Maximilian University of Munich (Author)
  • Milana Popara - , Heinrich Heine University Düsseldorf (Author)
  • Rebecca Mächtel - , Ludwig Maximilian University of Munich (Author)
  • Julian Folz - , Heinrich Heine University Düsseldorf (Author)
  • Benjamin Ambrose - , University of Sheffield (Author)
  • Neharika Chamachi - , Chair of Molecular Biophysics, DRESDEN-concept Genome Center (CMCB Core Facility) (Author)
  • Sang Yoon Chung - , University of California at Los Angeles (Author)
  • Timothy D. Craggs - , University of Sheffield (Author)
  • Marijn de Boer - , University of Groningen (Author)
  • Dina Grohmann - , University of Regensburg (Author)
  • Taekjip Ha - , Johns Hopkins University (Author)
  • Andreas Hartmann - , Chair of Molecular Biophysics (Author)
  • Jelle Hendrix - , Hasselt University, KU Leuven (Author)
  • Verena Hirschfeld - , University of Lübeck (Author)
  • Christian G. Hübner - , University of Lübeck (Author)
  • Thorsten Hugel - , University of Freiburg (Author)
  • Dominik Kammerer - , University of Oxford (Author)
  • Hyun Seo Kang - , Technical University of Munich (Author)
  • Achillefs N. Kapanidis - , University of Oxford (Author)
  • Georg Krainer - , Center for Molecular Bioengineering (B CUBE), University of Cambridge (Author)
  • Kevin Kramm - , University of Regensburg (Author)
  • Edward A. Lemke - , Johannes Gutenberg University Mainz, Institute of Molecular Biology, European Molecular Biology Laboratory (EMBL) (Author)
  • Eitan Lerner - , Hebrew University of Jerusalem (Author)
  • Emmanuel Margeat - , University of Montpellier (Author)
  • Kirsten Martens - , Leiden University (Author)
  • Jens Michaelis - , Ulm University (Author)
  • Jaba Mitra - , Johns Hopkins University, University of Illinois at Urbana-Champaign (Author)
  • Gabriel G. Moya Muñoz - , Ludwig Maximilian University of Munich (Author)
  • Robert B. Quast - , University of Montpellier (Author)
  • Nicole C. Robb - , University of Oxford, University of Warwick (Author)
  • Michael Sattler - , Technical University of Munich, Helmholtz Centre for Environmental Research (Author)
  • Michael Schlierf - , Chair of Molecular Biophysics, Clusters of Excellence PoL: Physics of Life (Author)
  • Jonathan Schneider - , Ludwig Maximilian University of Munich (Author)
  • Tim Schröder - , Ludwig Maximilian University of Munich (Author)
  • Anna Sefer - , Ulm University (Author)
  • Piau Siong Tan - , Johannes Gutenberg University Mainz, Institute of Molecular Biology (Author)
  • Johann Thurn - , University of Freiburg, German Aerospace Center (DLR) (Author)
  • Philip Tinnefeld - , Ludwig Maximilian University of Munich (Author)
  • John van Noort - , Leiden University (Author)
  • Shimon Weiss - , University of California at Los Angeles, California NanoSystems Institute (Author)
  • Nicolas Wendler - , Ludwig Maximilian University of Munich (Author)
  • Niels Zijlstra - , Ludwig Maximilian University of Munich (Author)
  • Anders Barth - , Heinrich Heine University Düsseldorf, Delft University of Technology (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)


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.


Original languageEnglish
Pages (from-to)523-535
Number of pages24
JournalNature methods
Issue number4
Publication statusPublished - 27 Mar 2023

External IDs

PubMed 36973549
WOS 000961406200002
ORCID /0000-0002-6209-2364/work/142237706
ORCID /0000-0002-2213-2763/work/142239790
ORCID /0000-0002-4657-9092/work/142247779



  • Fluorescence Resonance Energy Transfer/methods, Laboratories, Molecular Conformation, Proteins/chemistry, Reproducibility of Results, Spectroscopy, Nano-positioning system, Orientational freedom, Rna, Conformational dynamics, Lifetime distribution, Ligand-binding, Photon distribution, Alternating-laser excitation, Resonance energy-transfer