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Nat Methods. 2018 Sep;15(9):669-676. doi: 10.1038/s41592-018-0085-0. Epub 2018 Aug 31.

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

Author information

1
Institute of Physical Chemistry, University of Freiburg, Freiburg im Breisgau, Germany.
2
Engineering and Applied Sciences, Columbia University, New York, NY, USA.
3
Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, the Netherlands.
4
Molecular Physical Chemistry, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany.
5
Department of Physics and Astronomy, Clemson University, Clemson, SC, USA.
6
Department of Chemistry, University of Sheffield, Sheffield, UK.
7
Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, Aarhus University, Aarhus, Denmark.
8
Physical Chemistry, Department of Chemistry, Nanosystems Initiative Munich (NIM), Center for Integrated Protein Science Munich (CiPSM) and Center for Nanoscience (CeNS), Ludwig-Maximilians-Universität München, Munich, Germany.
9
Department of Physiology & Biophysics, Stony Brook University, Stony Brook, NY, USA.
10
Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA.
11
Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen, Groningen, the Netherlands.
12
Physical and Synthetic Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany.
13
Institute for Biophysics, Ulm University, Ulm, Germany.
14
Laboratory of Biophysics, Wageningen University & Research, Wageningen, the Netherlands.
15
Department of Biomedical Engineering, John Hopkins University, Baltimore, MD, USA.
16
Department of Physics, North Carolina State University, Raleigh, NC, USA.
17
B CUBE-Center for Molecular Bioengineering, TU Dresden, Dresden, Germany.
18
Laboratory for Photochemistry and Spectroscopy, Department of Chemistry, University of Leuven, Leuven, Belgium.
19
Dynamic Bioimaging Lab, Advanced Optical Microscopy Center and Biomedical Research Institute, Hasselt University, Hasselt, Belgium.
20
Institute of Physics, University of Lübeck, Lübeck, Germany.
21
Microspectroscopy Research Facility Wageningen, Wageningen University & Research, Wageningen, the Netherlands.
22
Gene Machines Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, UK.
23
School of Chemistry, Seoul National University, Seoul, South Korea.
24
Molecular Biophysics, Technische Universität Kaiserslautern (TUK), Kaiserslautern, Germany.
25
Departments of Biology and Chemistry, Pharmacy and Geosciences, Johannes Gutenberg-University Mainz, Mainz, Germany.
26
Institute of Molecular Biology (IMB), Mainz, Germany.
27
Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
28
School of Molecular Sciences and The Biodesign Institute, Arizona State University, Tempe, AZ, USA.
29
Department of Biochemistry, University of Zurich, Zurich, Switzerland.
30
Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.
31
Institute of Physical & Theoretical Chemistry, Braunschweig Integrated Centre of Systems Biology (BRICS), and Laboratory for Emerging Nanometrology (LENA), Braunschweig University of Technology, Braunschweig, Germany.
32
Institute for Biophysics, Ulm University, Ulm, Germany. jens.michaelis@uni-ulm.de.
33
Molecular Physical Chemistry, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany. cseidel@hhu.de.
34
Department of Chemistry, University of Sheffield, Sheffield, UK. t.craggs@sheffield.ac.uk.
35
Gene Machines Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, UK. t.craggs@sheffield.ac.uk.
36
Institute of Physical Chemistry, University of Freiburg, Freiburg im Breisgau, Germany. thorsten.hugel@pc.uni-freiburg.de.
37
BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg im Breisgau, Germany. thorsten.hugel@pc.uni-freiburg.de.

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.

PMID:
30171252
PMCID:
PMC6121742
[Available on 2019-02-28]
DOI:
10.1038/s41592-018-0085-0
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