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

Abstract

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs 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 that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than 0.06, corresponding to an interdye distance precision of less than 0.2 nm and accuracy of less than 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to less than 10% of the Foerster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

Time‐resolved microscopy made easy

“…This allows observation of slower molecular dynamics and measurement of slower diffusion properties of larger particles. In general, "larger confocal volumes, in combination with high irradiances, yield the highest number of photons per burst", according to [5]. Luminosa offers the ability to increase the observation volume from three to six times larger than diffraction limited, depending on the excitation wavelength.…”

Farewell to single-well: An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics

“…Dynamic structural changes of nucleic acids and proteins are central to their functionalities 2 . smFRET is frequently used to gain structural and dynamic insights into conformational changes of nucleic acids and proteins, and to decipher their folding and assembly mechanisms 3,5,8 . In particular changes of solution conditions are an important tool to tune kinetics of structural transitions and to drive molecules into desired conformations.…”

“…Single-molecule Förster resonance energy transfer (smFRET) has become a widely used technique to monitor biomolecular conformations and dynamics on the nanometer scale 1,2 . Advanced data analysis approaches to characterize dynamics from nanoseconds to minutes and hours, together with standardizations across many laboratories and open science initiatives, have pushed smFRET to a routinely accessible technique in biophysical and biochemical research [3][4][5][6][7][8] . smFRET, due to its versatility and sensitivity, is now extensively used to address a wide range of questions in dynamic structural biology and biophysics, including the functional mechanisms of enzymes and membrane proteins [9][10][11] , protein-nucleic acid and small-moleculeprotein interactions 12,13 , and protein or nucleic-acid folding 3,14,15 , to name but a few 2,5,16 .…”

“…Such measurements also often demand a high sampling density to alleviate overparameterization effects and to increase fitting accuracy and precision. Moreover, for the determination of experimental variability, a need for technical and biological replicates arises in general, not least to ascertain reproducibility and enhance scientific rigor 4,8,23 . Hence, rapidly more than 50 conditions need to be probed in smFRET experiments.…”

Deep-Learning Assisted, Single-molecule Imaging analysis (Deep-LASI) of multi-color DNA Origami structures

“…By removing ensemble averaging, it is possible to directly measure the underlying conformational states and molecular dynamics of biomolecules. Its ability to measure accurate distances and kinetics turned smFRET into a powerful tool for deciphering molecular interaction mechanisms and structures of biomolecules 1–3 . Typically, FRET experiments are performed using two colors and used to probe conformational distributions and distance changes.…”