Paper

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

Craggs, Timothy D, Tinnefeld, Philip, Lamb, Don C, Ambrose, Ben, Kammerer, Dominik, Gebhardt, Christian, Barth, Anders, Wendler, Nicolas, Moya Munoz, Gustavo G, Hyun Seo Kang, Chamachi, Neharika, Quast, Robert, Hirschfeld, Verena, Martens, Kirsten, Michaelis, Jens, Huebner, Christian G, Mitra, Jaba, Schlierf, Michael, Sang Yoon Chung, Schroeder, Tim, Cordes, Thorben, Grohmann, Dina, Kapanidis, Achillefs, De Boer, Marijn, Weiss, Shimon, Maechtel, Rebecca, Kramm, Kevin, Popara, Milana, Hugel, Thorsten, Margeat, Emmanuel, Seidel, Claus A M, Sefer, Anna, Sattler, Michael, Agam, Ganesh, Hartmann, Andreas, Krainer, Georg, Ha, Taekjip, Schneider, Jonathan, Lerner, Eitan, Tan, Piau Siong, Thurn, Johann, Hendrix, Jelle, Folz, Julian, Robb, Nicole, Lemke, Edward, John Van Noort

2022

Overview

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 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. Competing Interest Statement Tim Craggs and Achilles Kapanidis, two of the authors are founders of different companies selling single-molecule fluorescence microscopes (Exciting Instruments, Oxford Nanoimager).

• Publisher: Cold Spring Harbor Laboratory Press,Cold Spring Harbor Laboratory
• Subject: Biophysics / Fluorescence resonance energy transfer / Microscopes