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.
Highlights
Background correctionBackground I(BG) is removed from each uncorrected intensity iI separately, leading to the background-corrected intensities iiI, iiSapp, and iiEapp: iiIDem∣Dex = iIDem∣Dex−ID(BeGm)∣Dex iiIAem∣Aex = iIAem∣Aex−IA(BeGm)∣Aex iiIAem∣Dex = iIAem∣Dex−IA(BeGm)∣Dex (10)For confocal measurements, one can determine the background by averaging the photon count rate for all time bins that are below a certain threshold, which is defined, for example, by the maximum in the frequency-versus-intensity plot
We showed that the factors required for the correction of FRET efficiency can be determined with high precision, regardless of the setup type and acquisition software used
This study shows that the results do not depend heavily on these conditions, as every lab used its own setup and procedure at this stage
Summary
One can determine the background by averaging the photon count rate for all time bins that are below a certain threshold, which is defined, for example, by the maximum in the frequency-versus-intensity plot (the density of bursts should not be too high). Other possibilities include selecting the darkest spots in the illuminated area and subtracting an average background time trace from the data, or using a local background, for example, with a mask around the particle. The latter two options have the advantage that possible (exponential) background bleaching is corrected. We did not investigate the influence of the kind of background correction during this study, but a recent study showed that not all background estimators are suitable for samples with a high molecule surface coverage[41]
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