Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study

Björn Hellenkamp; Sonja Schmid; Olga Doroshenko; Oleg Opanasyuk; Ralf Kühnemuth; Soheila Rezaei Adariani; Benjamin Ambrose; Mikayel Aznauryan; Anders Barth; Victoria Birkedal; null More Authors

doi:10.1038/s41592-018-0085-0

Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study

Björn Hellenkamp, Sonja Schmid, Olga Doroshenko, Oleg Opanasyuk, Ralf Kühnemuth, Soheila Rezaei Adariani, Benjamin Ambrose, Mikayel Aznauryan, Anders Barth, Victoria Birkedal, More Authors

BN/Cees Dekker Lab

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Abstract

Single-molecule Förster 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 to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Original language	English
Pages (from-to)	669-676
Journal	Nature Methods
Volume	15
Issue number	9
DOIs	https://doi.org/10.1038/s41592-018-0085-0
Publication status	Published - 2018

Bibliographical note

Later disappeared an erratum with: 10.1038/s41592-018-0193-x : This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed. Een 2e scopuslink (erratum opgenomen)

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s41592-018-0085-0Final published version, 4.1 MBLicence: CC BY

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abstract = "Single-molecule F{\"o}rster 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 to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.",

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AU - Kühnemuth, Ralf

AU - Rezaei Adariani, Soheila

AU - Ambrose, Benjamin

AU - Aznauryan, Mikayel

AU - Barth, Anders

AU - Birkedal, Victoria

AU - More Authors, null

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N2 - Single-molecule Förster 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 to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

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Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study

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