Precision and accuracy of single-molecule FRET measurements - a worldwide benchmark study
Abstract
Single-molecule F\"orster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards ensuring both the reproducibility and accuracy of measuring FRET efficiencies are currently lacking. Here we report the results of a worldwide, comparative, blind study, in which 20 labs determined the FRET efficiencies of several dye-labeled DNA duplexes. Using a unified and straightforward method, we show that FRET efficiencies can be obtained with a standard deviation between E = +-0.02 and +-0.05. We further suggest an experimental and computational procedure for converting FRET efficiencies into accurate distances. We discuss potential uncertainties in the experiment and the modelling. Our extensive quantitative assessment of intensity-based smFRET measurements and correction procedures serve as an essential step towards validation of distance networks with the ultimate aim to archive reliable structural models of biomolecular systems obtained by smFRET-based hybrid methods.
Keywords
Cite
@article{arxiv.1710.03807,
title = {Precision and accuracy of single-molecule FRET measurements - a worldwide benchmark study},
author = {Björn Hellenkamp and Sonja Schmid and Olga Doroshenko and Oleg Opanasyuk and Ralf Kühnemuth and Soheila Rezaei Adariani and Anders Barth and Victoria Birkedal and Mark E. Bowen and Hongtao Chen and Thorben Cordes and Tobias Eilert and Carel Fijen and Markus Götz and Giorgos Gouridis and Enrico Gratton and Taekjip Ha and Christian A. Hanke and Andreas Hartmann and Jelle Hendrix and Lasse L. Hildebrandt and Johannes Hohlbein and Christian G. Hübner and Eleni Kallis and Achillefs N. Kapanidis and Jae-Yeol Kim and Georg Krainer and Don C. Lamb and Nam Ki Lee and Edward A. Lemke and Brié Levesque and Marcia Levitus and James J. McCann and Nikolaus Naredi-Rainer and Daniel Nettels and Thuy Ngo and Ruoyi Qiu and Carlheinz Röcker and Hugo Sanabria and Michael Schlierf and Benjamin Schuler and Henning Seidel and Lisa Streit and Philip Tinnefeld and Swati Tyagi and Niels Vandenberk and Keith R. Weninger and Bettina Wünsch and Inna S. Yanez-Orozco and Jens Michaelis and Claus A. M. Seidel and Timothy D. Craggs and Thorsten Hugel},
journal= {arXiv preprint arXiv:1710.03807},
year = {2022}
}
Comments
27 pages, 5 figures, supplementary material, corrections: p20 last sentence; eqn. 13 & 14