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Agam, G.* ; Gebhardt, C.* ; Popara, M.* ; Mächtel, R.* ; Folz, J.* ; Ambrose, B.* ; Chamachi, N.* ; Chung, S.Y.* ; Craggs, T.D.* ; de Boer, M.* ; Grohmann, D.* ; Ha, T.* ; Hartmann, A.* ; Hendrix, J.* ; Hirschfeld, V.* ; Hübner, C.G.* ; Hugel, T.* ; Kammerer, D.* ; Kang, H.-S.* ; Kapanidis, A.N.* ; Krainer, G.* ; Kramm, K.* ; Lemke, E.A.* ; Lerner, E.* ; Margeat, E.* ; Martens, K.* ; Michaelis, J.* ; Mitra, J.* ; Moya Muñoz, G.G.* ; Quast, R.B.* ; Robb, N.C.* ; Sattler, M. ; Schlierf, M.* ; Schneider, J.* ; Schröder, T.* ; Sefer, A.* ; Tan, P.S.* ; Thurn, J.* ; Tinnefeld, P.* ; van Noort, J.* ; Weiss, S.* ; Wendler, N.* ; Zijlstra, N.* ; Barth, A.* ; Seidel, C.A.M.* ; Lamb, D.C.* ; Cordes, T.*

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

Nat. Methods 20, 523-535 (2023)
DOI PMC
Creative Commons Lizenzvertrag
Open Access Green möglich sobald Postprint bei der ZB eingereicht worden ist.
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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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Korrespondenzautor
Schlagwörter Resonance Energy-transfer; Alternating-laser Excitation; Nano-positioning System; Conformational Dynamics; Orientational Freedom; Lifetime Distribution; Photon Distribution; Ligand-binding; Rna; Spectroscopy
ISSN (print) / ISBN 1548-7091
e-ISSN 1548-7105
Zeitschrift Nature Methods
Quellenangaben Band: 20, Heft: 4, Seiten: 523-535 Artikelnummer: , Supplement: ,
Verlag Nature Publishing Group
Verlagsort New York, NY
Nichtpatentliteratur Publikationen
Begutachtungsstatus Peer reviewed
Institut(e) Institute of Structural Biology (STB)
Förderungen Royal Society
Biotechnology and Biological Sciences Research Council (BBSRC)
Laender through the ONE MUNICH Project Munich Multiscale Biofabrication
Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)
LMUinnovative program BioImaging Network
European Union
Center for NanoScience
DFG
European Research Council (ERC) starting grant
ERC
Free State of Bavaria under the Excellence Strategy of the Federal Government
Federal Ministry of Education and Research (BMBF)
Nanosystems Initiative Munich
Center for integrated protein science Munich
Alexander von Humboldt postdoctoral fellowship
German Research Foundation (Deutsche Forschungsgemeinschaft, DFG)
Marie Curie Actions (MSCA)
ERC ADG MultiOrganelleDesign
Agence Nationale pour la Recherche
Hebrew University of Jerusalem
Milner Fund
National Institutes of Health
Israel Science Foundation
Wellcome Trust
Royal Society Dorothy Hodgkin Research Fellowship
Engineering and Physical Sciences Research Council (EPSRC)
DFG under Germany's Excellence Strategy
Research Foundation Flanders (FWO)
US National Institutes of Health
BMBF
EPSRC Prize Fellowship