Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins.
Journal
Nature methods
ISSN: 1548-7105
Titre abrégé: Nat Methods
Pays: United States
ID NLM: 101215604
Informations de publication
Date de publication:
04 2023
04 2023
Historique:
received:
02
08
2022
accepted:
31
01
2023
medline:
13
4
2023
pubmed:
28
3
2023
entrez:
27
3
2023
Statut:
ppublish
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.
Identifiants
pubmed: 36973549
doi: 10.1038/s41592-023-01807-0
pii: 10.1038/s41592-023-01807-0
pmc: PMC10089922
doi:
Substances chimiques
Proteins
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
523-535Subventions
Organisme : NIH HHS
ID : GM122569
Pays : United States
Organisme : European Research Council
ID : 681891 (Prosint)
Pays : International
Organisme : Marie Curie
ID : 101029907
Pays : United Kingdom
Organisme : Wellcome Trust
ID : 110164/Z/15/Z
Pays : United Kingdom
Organisme : European Research Council
ID : ERXC ADG Multiorganelldesign
Pays : International
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/T008032/1
Pays : United Kingdom
Organisme : NIGMS NIH HHS
ID : R01 GM130942
Pays : United States
Organisme : European Research Council
ID : ERC-StG 638536-SM-IMPORT
Pays : International
Organisme : European Research Council
ID : grant No. 671208 (hybridFRET)
Pays : International
Organisme : NIGMS NIH HHS
ID : R01 GM130942
Pays : United States
Informations de copyright
© 2023. The Author(s).
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