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; Mark E. Bowen; Hongtao Chen; Thorben Cordes; Tobias Eilert; Carel Fijen; Christian Gebhardt; Markus Götz; Giorgos Gouridis; Enrico Gratton; Taekjip Ha; Pengyu Hao; Christian A. Hanke; Andreas Hartmann; Jelle Hendrix; Lasse L. Hildebrandt; Verena Hirschfeld; Johannes Hohlbein; Boyang Hua; Christian G. Hübner; Eleni Kallis; Achillefs N. Kapanidis; Jae Yeol Kim; Georg Krainer; Don C. Lamb; Nam Ki Lee; Edward A. Lemke; Brié Levesque; Marcia Levitus; James J. McCann; Nikolaus Naredi-Rainer; Daniel Nettels; Thuy Ngo; Ruoyi Qiu; Nicole C. Robb; Carlheinz Röcker; Hugo Sanabria; Michael Schlierf; Tim Schröder; Benjamin Schuler; Henning Seidel; Lisa Streit; Johann Thurn; Philip Tinnefeld; Swati Tyagi; Niels Vandenberk; Andrés Manuel Vera; Keith R. Weninger; Bettina Wünsch; Inna S. Yanez-Orozco; Jens Michaelis; Claus A.M. Seidel; Timothy D. Craggs; Thorsten Hugel

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
, Mark E. Bowen
, Hongtao Chen
, Thorben Cordes
, Tobias Eilert
, Carel Fijen
, Christian Gebhardt
, Markus Götz
, Giorgos Gouridis
, Enrico Gratton
, Taekjip Ha

Pengyu Hao, Christian A. Hanke, Andreas Hartmann, Jelle Hendrix, Lasse L. Hildebrandt, Verena Hirschfeld, Johannes Hohlbein, Boyang Hua, Christian G. Hübner, Eleni Kallis, Achillefs N. Kapanidis, Jae Yeol Kim, Georg Krainer, Don C. Lamb, Nam Ki Lee, Edward A. Lemke, Brié Levesque, Marcia Levitus, James J. McCann, Nikolaus Naredi-Rainer, Daniel Nettels, Thuy Ngo, Ruoyi Qiu, Nicole C. Robb, Carlheinz Röcker, Hugo Sanabria, Michael Schlierf, Tim Schröder, Benjamin Schuler, Henning Seidel, Lisa Streit, Johann Thurn, Philip Tinnefeld, Swati Tyagi, Niels Vandenberk, Andrés Manuel Vera, Keith R. Weninger, Bettina Wünsch, Inna S. Yanez-Orozco, Jens Michaelis, Claus A.M. Seidel, Timothy D. Craggs, Thorsten Hugel

Physiology and Biophysics

University of Freiburg
Columbia University
Delft University of Technology
Heinrich Heine University Düsseldorf
Clemson University
University of Sheffield
Aarhus University
Ludwig Maximilian University of Munich
University of California at Irvine
University of Groningen
Ulm University
Wageningen University & Research
Johns Hopkins University
North Carolina State University
Technische Universität Dresden
KU Leuven
Hasselt University
University of Lübeck
University of Oxford
Seoul National University
The University of Kaiserslautern-Landau
Johannes Gutenberg University Mainz
Institute of Molecular Biology (IMB)
European Molecular Biology Laboratory
Stony Brook University
Arizona State University
University of Zurich
Technical University of Braunschweig

Research output: Contribution to journal › Article › peer-review

382 Scopus citations

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
Number of pages	8
Journal	Nature Methods
Volume	15
Issue number	9
DOIs	https://doi.org/10.1038/s41592-018-0085-0
State	Published - Sep 1 2018

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Hellenkamp, B., Schmid, S., Doroshenko, O., Opanasyuk, O., Kühnemuth, R., Rezaei Adariani, S., Ambrose, B., Aznauryan, M., Barth, A., Birkedal, V., Bowen, M. E., Chen, H., Cordes, T., Eilert, T., Fijen, C., Gebhardt, C., Götz, M., Gouridis, G., Gratton, E., ... Hugel, T. (2018). Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study. Nature Methods, 15(9), 669-676. https://doi.org/10.1038/s41592-018-0085-0

@article{408d752d4c5040be963ec22de85c9db3,

title = "Precision and accuracy of single-molecule FRET measurements{\textemdash}a multi-laboratory benchmark study",

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.",

author = "Bj{\"o}rn Hellenkamp and Sonja Schmid and Olga Doroshenko and Oleg Opanasyuk and Ralf K{\"u}hnemuth and \{Rezaei Adariani\}, Soheila and Benjamin Ambrose and Mikayel Aznauryan and Anders Barth and Victoria Birkedal and Bowen, \{Mark E.\} and Hongtao Chen and Thorben Cordes and Tobias Eilert and Carel Fijen and Christian Gebhardt and Markus G{\"o}tz and Giorgos Gouridis and Enrico Gratton and Taekjip Ha and Pengyu Hao and Hanke, \{Christian A.\} and Andreas Hartmann and Jelle Hendrix and Hildebrandt, \{Lasse L.\} and Verena Hirschfeld and Johannes Hohlbein and Boyang Hua and H{\"u}bner, \{Christian G.\} and Eleni Kallis and Kapanidis, \{Achillefs N.\} and Kim, \{Jae Yeol\} and Georg Krainer and Lamb, \{Don C.\} and Lee, \{Nam Ki\} and Lemke, \{Edward A.\} and Bri{\'e} Levesque and Marcia Levitus and McCann, \{James J.\} and Nikolaus Naredi-Rainer and Daniel Nettels and Thuy Ngo and Ruoyi Qiu and Robb, \{Nicole C.\} and Carlheinz R{\"o}cker and Hugo Sanabria and Michael Schlierf and Tim Schr{\"o}der and Benjamin Schuler and Henning Seidel and Lisa Streit and Johann Thurn and Philip Tinnefeld and Swati Tyagi and Niels Vandenberk and Vera, \{Andr{\'e}s Manuel\} and Weninger, \{Keith R.\} and Bettina W{\"u}nsch and Yanez-Orozco, \{Inna S.\} and Jens Michaelis and Seidel, \{Claus A.M.\} and Craggs, \{Timothy D.\} and Thorsten Hugel",

note = "Publisher Copyright: {\textcopyright} 2018, The Author(s).",

year = "2018",

month = sep,

day = "1",

doi = "10.1038/s41592-018-0085-0",

language = "English",

volume = "15",

pages = "669--676",

journal = "Nature Methods",

issn = "1548-7091",

number = "9",

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Hellenkamp, B, Schmid, S, Doroshenko, O, Opanasyuk, O, Kühnemuth, R, Rezaei Adariani, S, Ambrose, B, Aznauryan, M, Barth, A, Birkedal, V, Bowen, ME, Chen, H, Cordes, T, Eilert, T, Fijen, C, Gebhardt, C, Götz, M, Gouridis, G, Gratton, E, Ha, T, Hao, P, Hanke, CA, Hartmann, A, Hendrix, J, Hildebrandt, LL, Hirschfeld, V, Hohlbein, J, Hua, B, Hübner, CG, Kallis, E, Kapanidis, AN, Kim, JY, Krainer, G, Lamb, DC, Lee, NK, Lemke, EA, Levesque, B, Levitus, M, McCann, JJ, Naredi-Rainer, N, Nettels, D, Ngo, T, Qiu, R, Robb, NC, Röcker, C, Sanabria, H, Schlierf, M, Schröder, T, Schuler, B, Seidel, H, Streit, L, Thurn, J, Tinnefeld, P, Tyagi, S, Vandenberk, N, Vera, AM, Weninger, KR, Wünsch, B, Yanez-Orozco, IS, Michaelis, J, Seidel, CAM, Craggs, TD & Hugel, T 2018, 'Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study', Nature Methods, vol. 15, no. 9, pp. 669-676. https://doi.org/10.1038/s41592-018-0085-0

TY - JOUR

T1 - Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study

AU - Hellenkamp, Björn

AU - Schmid, Sonja

AU - Doroshenko, Olga

AU - Opanasyuk, Oleg

AU - Kühnemuth, Ralf

AU - Rezaei Adariani, Soheila

AU - Ambrose, Benjamin

AU - Aznauryan, Mikayel

AU - Barth, Anders

AU - Birkedal, Victoria

AU - Bowen, Mark E.

AU - Chen, Hongtao

AU - Cordes, Thorben

AU - Eilert, Tobias

AU - Fijen, Carel

AU - Gebhardt, Christian

AU - Götz, Markus

AU - Gouridis, Giorgos

AU - Gratton, Enrico

AU - Ha, Taekjip

AU - Hao, Pengyu

AU - Hanke, Christian A.

AU - Hartmann, Andreas

AU - Hendrix, Jelle

AU - Hildebrandt, Lasse L.

AU - Hirschfeld, Verena

AU - Hohlbein, Johannes

AU - Hua, Boyang

AU - Hübner, Christian G.

AU - Kallis, Eleni

AU - Kapanidis, Achillefs N.

AU - Kim, Jae Yeol

AU - Krainer, Georg

AU - Lamb, Don C.

AU - Lee, Nam Ki

AU - Lemke, Edward A.

AU - Levesque, Brié

AU - Levitus, Marcia

AU - McCann, James J.

AU - Naredi-Rainer, Nikolaus

AU - Nettels, Daniel

AU - Ngo, Thuy

AU - Qiu, Ruoyi

AU - Robb, Nicole C.

AU - Röcker, Carlheinz

AU - Sanabria, Hugo

AU - Schlierf, Michael

AU - Schröder, Tim

AU - Schuler, Benjamin

AU - Seidel, Henning

AU - Streit, Lisa

AU - Thurn, Johann

AU - Tinnefeld, Philip

AU - Tyagi, Swati

AU - Vandenberk, Niels

AU - Vera, Andrés Manuel

AU - Weninger, Keith R.

AU - Wünsch, Bettina

AU - Yanez-Orozco, Inna S.

AU - Michaelis, Jens

AU - Seidel, Claus A.M.

AU - Craggs, Timothy D.

AU - Hugel, Thorsten

PY - 2018/9/1

Y1 - 2018/9/1

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.

AB - 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.

UR - https://www.scopus.com/pages/publications/85052699968

U2 - 10.1038/s41592-018-0085-0

DO - 10.1038/s41592-018-0085-0

M3 - Article

C2 - 30171252

AN - SCOPUS:85052699968

SN - 1548-7091

VL - 15

SP - 669

EP - 676

JO - Nature Methods

JF - Nature Methods

IS - 9

ER -

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

Abstract

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