Precision and accuracy of single-molecule FRET measurements - a worldwide benchmark study

Abstract

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards ensuring both the reproducibility and accuracy of measuring FRET efficiencies are currently lacking. Here we report the results of a worldwide, comparative, blind study, in which 20 labs determined the FRET efficiencies of several dye-labeled DNA duplexes. Using a unified and straightforward method, we show that FRET efficiencies can be obtained with a standard deviation between $\Delta$E = +-0.02 and +-0.05. We further suggest an experimental and computational procedure for converting FRET efficiencies into accurate distances. We discuss potential uncertainties in the experiment and the modelling. Our extensive quantitative assessment of intensity-based smFRET measurements and correction procedures serve as an essential step towards validation of distance networks with the ultimate aim to archive reliable structural models of biomolecular systems obtained by smFRET-based hybrid methods.

27 pages, 5 figures, supplementary material, corrections: p20 last sentence; eqn. 13 & 14

References in corpus (1)

• Efficient use of single molecule time traces to resolve kinetic rates, models and uncertainties

Cited by in corpus (15)

• The FRET-based structural dynamics challenge -- community contributions to consistent and open science practices
• Unraveling multi-state molecular dynamics in single-molecule FRET experiments- Part I: Theory of FRET-Lines
• Machine learning for faster and smarter fluorescence lifetime imaging microscopy
• A new twist on PIFE: photoisomerisation-related fluorescence enhancement
• Unraveling multi-state molecular dynamics in single-molecule FRET experiments -- Part II: Quantitative analysis of multi-state kinetic networks
• Intramolecular Structural Heterogeneity altered by Long-range Contacts in an Intrinsically Disordered Protein
• Effects of Donor-Acceptor Quantum Coherence and Non-Markovian Bath on the Distance Dependence of Resonance Energy Transfer
• Long-range Single Molecule Förster Resonance Energy Transfer Between Alexa Dyes in Zero-Mode Waveguides
• Excitation Energy Transfer under Strong Laser Drive
• Light Energy Driven Nanocommunications with FRET in Photosynthetic Systems
• QuanTI-FRET: a framework for quantitative FRET measurements in living cells
• Fast ATP-dependent Subunit Rotation in Reconstituted FoF1-ATP Synthase Trapped in Solution
• Time-Heterogeneity of the Förster Radius from Dipole Orientational Dynamics Impacts Single-Molecule FRET Experiments
• Identifying the minimal sets of distance restraints for FRET-assisted protein structural modeling
• Complementary aspects of non-equilibrium thermodynamics