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Journal or Publication Title: | Nature Methods | ||||
Publisher: | NATURE PORTFOLIO | ||||
Place of Publication: | BERLIN | ||||
Volume: | 20 | ||||
Number of Issue or Book Chapter: | 4 | ||||
Page Range: | pp. 523-535 | ||||
Date: | 2023 | ||||
Institutions: | Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie > Lehrstuhl für Mikrobiologie (Archaeenzentrum) Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie > Lehrstuhl für Mikrobiologie (Archaeenzentrum) > Prof. Dr. Dina Grohmann | ||||
Identification Number: |
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Keywords: | RESONANCE ENERGY-TRANSFER; ALTERNATING-LASER EXCITATION; NANO-POSITIONING SYSTEM; CONFORMATIONAL DYNAMICS; ORIENTATIONAL FREEDOM; LIFETIME DISTRIBUTION; PHOTON DISTRIBUTION; LIGAND-BINDING; RNA; SPECTROSCOPY; | ||||
Dewey Decimal Classification: | 500 Science > 550 Earth sciences | ||||
Status: | Published | ||||
Refereed: | Yes, this version has been refereed | ||||
Created at the University of Regensburg: | Yes | ||||
Item ID: | 76294 |
Abstract
Single-molecule Forster-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 ...

Abstract
Single-molecule Forster-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 angstrom and accuracy of <= 5 angstrom. 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. An international blind study confirms that smFRET measurements on dynamic proteins are highly reproducible across instruments, analysis procedures and timescales, further highlighting the promise of smFRET for dynamic structural biology.
Metadata last modified: 18 Mar 2025 10:11