FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices

Abstract

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.

From integrative structural biology to cell biology

“…The goal is to create standards and tools for automated data exchange between the PDB and member repositories. Discussions with experts in additional methods, including Foerster resonance energy transfer spectroscopy and microscopy ( 106 ), hydrogen/deuterium exchange by MS, and genome modeling, are in progress. More are expected in the future.…”

Evaluation of FRET X for Single-Molecule Protein Fingerprinting

“…Single-molecule FRET is well suited for this and comes with the benefit that several thousands of molecules can be imaged at the same time, if full length proteins can be immobilized in a microfluidic chamber. 12 Here we verify the feasibility of a single-molecule FRETbased protein fingerprinting method. We first demonstrate that experimentally obtained fingerprints for four model peptides are distinct and are reproduced by our simulation method.…”

Multi-parameter photon-by-photon hidden Markov modeling

“…In the absence of factors correcting for differences in donor and acceptor fluorescence quantum yields and detection efficiencies (better known as the γ factor), acceptor direct excitation, and leakage of donor photons into the acceptor channel, the raw ratio of acceptor photons to donor and acceptor photons, better known as the proximity ratio (PR), serves as the apparent or uncorrected FRET efficiency [26]. smFRET has proven a powerful tool to disentangle sub-populations of biomacromolecules undergoing dynamic transitions over a range of time scales, but remains limited by the time resolution of the apparatus, which in the case of freely diffusing molecules, is often on the order of a few milliseconds, and down to the sub-milliseconds using advanced analyses of photon statis- tics within single-molecule photon bursts, summarized in recent reviews of the field [1,2]. Photon-by-photon hidden Markov modelling (H 2 MM) [27] can extract the number of sub-populations involved in the dynamics, their proximity ratio values and transition rate constants.…”