![]() ![]() This ratiometric approach is often sufficient to distinguish different conformations of a biomolecule (e.g., an open conformation with low FRET efficiency versus a closed conformation with high FRET efficiency) and to determine their interconversion kinetics. So far most intensity-based smFRET studies have characterized relative changes in FRET efficiency. Here we focus on intensity-based measurements in which the FRET efficiency E is determined from donor and acceptor photon counts and subsequently used to calculate the interfluorophore distance according to Förster’s theory. Various fluorescence-intensity- and lifetime-based procedures have been proposed with the aim of determining FRET efficiencies 10, 14, 15, 16, 17, 18, 19, 20. The two most popular smFRET approaches for use in determining distances are confocal microscopy of freely diffusing molecules in solution and total internal reflection fluorescence (TIRF) microscopy of surface-attached molecules. In its single-molecule implementation, FRET largely overcomes ensemble-averaging and time-averaging and can uncover individual species in heterogeneous and dynamic biomolecular complexes, as well as transient intermediates 5. Accordingly, FRET has been termed a ‘spectroscopic ruler’ for measurements on the molecular scale 2, capable of determining distances in vitro, and even in cells 13, with potentially ångström-level accuracy and precision. The transfer efficiency depends on the interdye distance, which is well described by Förster’s theory for distances > 30 Å 11, 12. The fluorophores are usually attached via flexible linkers to defined positions of the system under investigation. In such experiments, the energy transfer between donor and acceptor fluorophores is quantified with respect to their proximity 1. 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.įRET 1, also known as fluorescence resonance energy transfer, is a well-established method for studying biomolecular conformations and dynamics at both the ensemble 2, 3, 4 and the single-molecule level 5, 6, 7, 8, 9, 10. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. 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. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. Nature Methods volume 15, pages 669–676 ( 2018) Cite this article Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study ![]()
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