Quantitative structural information from single-molecule FRET

Abstract

Single-molecule studies can be used to study biological processes directly and in real-time. In particular, the fluorescence energy transfer between reporter dye molecules attached to specific sites on macromolecular complexes can be used to infer distance information. When several measurements are combined, the information can be used to determine the position and conformation of certain domains with respect to the complex. However, data analysis schemes that include all experimental uncertainties are highly complex, and the outcome depends on assumptions about the state of the dye molecules. Here, we present a new analysis algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering termed Fast-NPS that can analyse large smFRET networks in a relatively short time and yields the position of the dye molecules together with their respective uncertainties. Moreover, we show what effects different assumptions about the dye molecules have on the outcome. We discuss the possibilities and pitfalls in structure determination based on smFRET using experimental data for an archaeal transcription pre-initiation complex, whose architecture has recently been unravelled by smFRET measurements.

Single Molecule Data Analysis: An Introduction

“…Conjugate priors do have obvious mathematical appeal and yield analytically tractable forms for posteriors but they are more restrictive. Numerical methods to sample posteriors -including Gibbs sampling and related Markov chain Monte Carlo methods [118,119] -continue to be used [116] and developed [120] for biophysical problems and have somewhat reduced the historical analytical advantage of conjugate priors. However the advantage conferred by the tractability of conjugate priors has turned out to be major advantage for more complex inference problems -such as those involving Dirichlet processes -that we will discuss later.…”

“…To illustrate here that MaxEnt also works on real data, we re-analyzed in vitro data on the diffusion of the small dye Alexa568 (Fig. (6a)) as well as previously published FCS data [19] on as lower-affinity non-specific DNA binding [118]; 2) interactions with other chromatin binding proteins [103]; 3) association with proteins forming subnuclear domains such as nucleoli [119,120]. For instance, the BZip domain interacts with HP1↵ which in turn binds directly to histones (interaction 3) [103,120] and is expected to slow BZip's diffusion.…”

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

“…Here, the orientation factor κ 2 (needed for calculating the characteristic isotropic Förster radius) is set to 2/3. As a result, the computed credible volumes are almost two orders of magnitude smaller as compared to those in the classic model32. In the case that the fluorophore is found in an environment that enables not only fast reorientation, but additionally fast motion all over its accessible volume, the meanpos-iso model should be used.…”

“…To this end, Fast-NPS was introduced32. Fast-NPS uses an advanced sampling algorithm reducing the calculation times drastically.…”

“…For a similar situation in which the dye is not free to rotate the var-meanpos model applies. More details about these models can be found in our recent publication32.…”