Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins [article]

Ganesh Agam, Christian Gebhardt, Milana Popara, Rebecca Maechtel, Julian Folz, Ben Ambrose, Neharika Chamachi, Sang Yoon Chung, Timothy D. Craggs, Marijn de Boer, Dina Grohmann, Taekjip Ha (+34 others)
2022 bioRxiv   pre-print
Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the
more » ... FRET efficiency of less than 0.06, corresponding to an interdye distance precision of less than 0.2 nm and accuracy of less than 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to less than 10% of the Foerster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.
doi:10.1101/2022.08.03.502619 fatcat:6zy4znfomfcvjpn4chvgfo5toe