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Article Dans Une Revue Nature Methods Année : 2023

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

Benjamin Ambrose
Timothy D Craggs
Dina Grohmann
Taekjip Ha
Thorsten Hugel
Tim Schröder
Johann Thurn
Shimon Weiss
Nicolas Wendler
Claus a M Seidel
Don C Lamb
Thorben Cordes

Résumé

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories 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 with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology.
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Origine : Publication financée par une institution
Licence : CC BY - Paternité

Dates et versions

hal-04120543 , version 1 (07-06-2023)

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Paternité

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Ganesh Agam, Christian Gebhardt, Milana Popara, Rebecca Mächtel, Julian Folz, et al.. Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins. Nature Methods, 2023, 20 (4), pp.523 - 535. ⟨10.1038/s41592-023-01807-0⟩. ⟨hal-04120543⟩
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