Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

University of Sheffield

Human flap endonuclease-1 (hFEN1) catalyzes the divalent metal ion-dependent removal of single-stranded DNA protrusions known as flaps during DNA replication and repair. Substrate selectivity involves passage of the 5'-terminus/flap through the arch and recognition of a single nucleotide 3'-flap by the α2-α3 loop. Using NMR spectroscopy, we show that the solution conformation of free and DNA-bound hFEN1 are consistent with crystal structures; however, parts of the arch region and α2-α3 loop are disordered without substrate. Disorder within the arch explains how 5'-flaps can pass under it. NMR and single-molecule FRET data show a shift in the conformational ensemble in the arch and loop region upon addition of DNA. Furthermore, the addition of divalent metal ions to the active site of the hFEN1-DNA substrate complex demonstrates that active site changes are propagated via DNA-mediated allostery to regions key to substrate differentiation. The hFEN1-DNA complex also shows evidence of millisecond timescale motions in the arch region that may be required for DNA to enter the active site. Thus, hFEN1 regional conformational flexibility spanning a range of dynamic timescales is crucial to reach the catalytically relevant ensemble.

Regional conformational flexibility couples substrate specificity and scissile phosphate diester selectivity in human flap endonuclease 1.

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 to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful technique capable of resolving both relative and absolute distances within and between structurally dynamic biomolecules. High instrument costs, and a lack of open-source hardware and acquisition software have limited smFRET's broad application by non-specialists. Here, we present the smfBox, a cost-effective confocal smFRET platform, providing detailed build instructions, open-source acquisition software, and full validation, thereby democratising smFRET for the wider scientific community.

The smfBox is an open-source platform for single-molecule FRET.

We present CHARMM-compatible force field parameters for a series of fluorescent dyes from the Alexa, Atto, and Cy families, commonly used in Förster resonance energy transfer (FRET) experiments. These dyes are routinely used in experiments to resolve the dynamics of proteins and nucleic acids at the nanoscale. However, little is known about the accuracy of the theoretical approximations used in determining the dynamics from the spectroscopic data. Molecular dynamics simulations can provide valuable insights into these dynamics at an atomistic level, but this requires accurate parameters for the dyes. The complex structure of the dyes and the importance of this in determining their spectroscopic properties mean that parameters generated by analogy to existing parameters do not give meaningful results. Through validation relative to quantum chemical calculation and experiments, the new parameters are shown to significantly outperform those that can be generated automatically, giving better agreement in both the charge distributions and structural properties. These improvements, in particular with regard to orientation of the dipole moments on the dyes, are vital for accurate simulation of FRET processes.

CHARMM-DYES: Parameterization of Fluorescent Dyes for Use with the CHARMM Force Field.

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage 'open science' practices.

Benjamin Ambrose

Sheffield Institute for Nucleic Acids,
Department of Chemistry,
Sheffield Institute for Nucleic Acids, Department of Chemistry

University of Sheffield

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

Benjamin Ambrose

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Sheffield Institute for Nucleic Acids,Department of Chemistry,Sheffield Institute for Nucleic Acids, Department of Chemistry

University of Sheffield

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

Sheffield Institute for Nucleic Acids,
Department of Chemistry,
Sheffield Institute for Nucleic Acids, Department of Chemistry