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

Publikation: Vorabdruck/Dokumentation/BerichtVorabdruck (Preprint)

Beitragende

  • Ganesh Agam - (Autor:in)
  • Christian Gebhardt - , Ludwig-Maximilians-Universität München (LMU) (Autor:in)
  • Milana Popara - (Autor:in)
  • Rebecca Mächtel - (Autor:in)
  • Julian Folz - (Autor:in)
  • Benjamin Ambrose - , University of Sheffield (Autor:in)
  • Neharika Gajanan Chamachi - , Professur für Molekulare Biophysik, Exzellenzcluster PoL: Physik des Lebens (Autor:in)
  • Sang Yoon Chung - (Autor:in)
  • Timothy D. Craggs - , Oxford Brookes University (OBU) (Autor:in)
  • Marijn de Boer - (Autor:in)
  • Dina Grohmann - (Autor:in)
  • Taekjip Ha - , Johns Hopkins University, Howard Hughes Medical Institute (Autor:in)
  • Andreas Hartmann - , Professur für Molekulare Biophysik, Exzellenzcluster PoL: Physik des Lebens (Autor:in)
  • Jelle Hendrix - , Hasselt University (Autor:in)
  • Verena Hirschfeld - , Universität zu Lübeck (Autor:in)
  • Christian G. Hübner - (Autor:in)
  • Thorsten Hugel - , Albert-Ludwigs-Universität Freiburg (Autor:in)
  • Dominik Kammerer - (Autor:in)
  • Hyun-Seo Kang - (Autor:in)
  • Achillefs N. Kapanidis - , Oxford Brookes University (OBU) (Autor:in)
  • Georg Krainer - , Professur für Molekulare Biophysik, Exzellenzcluster PoL: Physik des Lebens (Autor:in)
  • Kevin Kramm - (Autor:in)
  • Edward A. Lemke - , European Molecular Biology Laboratory (EMBL) Heidelberg (Autor:in)
  • Eitan Lerner - (Autor:in)
  • Emmanuel Margeat - (Autor:in)
  • Kristen Martens - (Autor:in)
  • Jens Michaelis - , Universität Ulm (Autor:in)
  • Jaba Mitra - (Autor:in)
  • Gustavo G. Moya Munoz - (Autor:in)
  • Robert Quast - (Autor:in)
  • Nicole B. Robb - (Autor:in)
  • Michael Sattler - (Autor:in)
  • Michael Schlierf - , Professur für Molekulare Biophysik, Exzellenzcluster PoL: Physik des Lebens (Autor:in)
  • Jonathan Schneider - (Autor:in)
  • Tim Schröder - (Autor:in)
  • Anna Sefer - (Autor:in)
  • Piau Siong Tan - (Autor:in)
  • Johann Thurn - , Albert-Ludwigs-Universität Freiburg (Autor:in)
  • Philip Tinnefeld - (Autor:in)
  • John van Noort - (Autor:in)
  • Shimon Weiss - (Autor:in)
  • Nicolas Wendler - (Autor:in)
  • Niels Zijlstra - (Autor:in)
  • Anders Barth - , Ludwig-Maximilians-Universität München (LMU) (Autor:in)
  • Claus A. M. Seidel - , Heinrich Heine Universität Düsseldorf (Autor:in)
  • Don C. Lamb - , Ludwig-Maximilians-Universität München (LMU) (Autor:in)
  • Thorben Cordes - , Ludwig-Maximilians-Universität München (LMU) (Autor:in)

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

OriginalspracheEnglisch
Seitenumfang34
PublikationsstatusVeröffentlicht - 2022
No renderer: customAssociatesEventsRenderPortal,dk.atira.pure.api.shared.model.researchoutput.WorkingPaper

Externe IDs

unpaywall 10.1101/2022.08.03.502619
Mendeley 991d6cfe-53d8-3513-9846-54a5be20eb6a
ORCID /0000-0002-6209-2364/work/142658677
ORCID /0000-0002-2213-2763/work/142659212
ORCID /0000-0002-4657-9092/work/142659880