Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

Abstract

Single-molecule Förster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. Thereby, multiple smFRET measurements are used to localize an unknown dye position inside a protein complex by means of trilateration. In order to obtain quantitative information, the Nano-Positioning System (NPS) uses probabilistic data analysis to combine structural information from X-ray crystallography with single-molecule fluorescence data to calculate not only the most probable position but the complete three-dimensional probability distribution, termed posterior, which indicates the experimental uncertainty. The concept was generalized for the analysis of smFRET networks containing numerous dye molecules. The latest version of NPS, Fast-NPS, features a new algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering that allows for the analysis of large smFRET networks in a comparably short time. Moreover, Fast-NPS allows the calculation of the posterior by choosing one of five different models for each dye, that account for the different spatial and orientational behavior exhibited by the dye molecules due to their local environment.Here we present a detailed protocol for obtaining smFRET data and applying the Fast-NPS. We provide detailed instructions for the acquisition of the three input parameters of Fast-NPS: the smFRET values, as well as the quantum yield and anisotropy of the dye molecules. Recently, the NPS has been used to elucidate the architecture of an archaeal open promotor complex. This data is used to demonstrate the influence of the five different dye models on the posterior distribution.

Structural reorganization of the chromatin remodeling enzyme Chd1 upon engagement with nucleosomes

“…After acquiring 10–15 smFRET movies for mono-nucleosomes in the absence of Chd1, Chd1 was loaded to the nucleosomes at a concentration of 50 nM in smFRET buffer with or without 150 μM AMP-PNP ( Roche ) and approximately 15 more smFRET movies were recorded. Analysis of smFRET data was performed using a workflow described previously ( Dörfler et al, 2017 ) and analyzed using the custom code accessible at https://github.com/TobiasEilert/2017.02.14_SM-FRET-V5.7dc . Kinetic rates were extracted using the HMM toolbox written by Kevin Murphy ( https://github.com/probml/pmtk3 ) ( Sikor et al, 2013 ).…”

Single‐molecule nucleosome remodeling by INO80 and effects of histone tails

“…TIRF microscopy was performed as described in Ref. . Briefly, nucleosomes were diluted in remodeling buffer to a concentration on the order of 10 p m labeled nucleosomes (equivalent to ca.…”

“…The smFRET analysis software was provided by the Lamb Laboratory (LMU Munich) . For calculation of FRET efficiencies, individual γ corrections (accounting for differences in detection efficiencies and quantum yields) and β corrections (accounting for spectral crosstalk) were applied if appropriate, otherwise a mean γ of 0.55 and β of 0.045 were used.…”

Robust μ-Synthesis With Dahl Model Based Feedforward Compensator Design for Piezo-Actuated Micropositioning Stage

“…Due to these numerous advantages, piezo-actuated positioning stages are commonly used in many applications, e.g. in scanning probe microscopy [2], advanced lithography tools for the fabrication of semiconductor integrated circuits [3], servo system of hard disk-drives [4], optical alignment systems [5], manipulation of nanoscale biological process like DNA analysis [6] and also in the manufacturing of small objects [7]. In all these applications, ultra-precise positioning with high speed and long positioning range is desired.…”