M. Beckers, F. Drechsler, T. Eilert, J. Nagy and J. Michaelis*
Quantitative structural information from single-molecule FRET
Single-molecule studies can be used to study biological processes directly and in realtime. In particular, the fluorescence energy transfer between reporter dye moleculesattached to specific sites on macromolecular complexes can be used to infer distance information. When several measurements are combined, the information can be usedto determine the position and conformation of certain domains with respect to the complex. However, data analysis schemes that include all experimental uncertaintiesare highly complex, and the outcome depends on assumptions about the state of the dye molecules. Here, we present a new analysis algorithm using Bayesian parameterestimation based on Markov Chain Monte Carlo sampling and parallel tempering termed Fast-NPS that can analyse large smFRET networks in a relatively short timeand yields the position of the dye molecules together with their respective uncertainties. Moreover, we show what effects different assumptions about the dyemolecules have on the outcome. We discuss the possibilities and pitfalls in structure determination based on smFRET using experimental data for an archaeal transcriptionpre-initiation complex, whose architecture has recently been unravelled by smFRET measurements.
Faraday Discuss., 2015, 184, 117-129
DOI: 10.1039/c5fd00110b

A. Muschielok, J. Michaelis
Application of the Nano-Positioning System to the Analysis of Fluorescence Resonance Energy Transfer Networks
Single-molecule fluorescence resonance energy transfer (sm-FRET) has been recently applied to distance and position estimation in macromolecular complexes. Here, we generalize the previously published Nano-Positioning System (NPS), a probabilistic method to analyze data obtained in such experiments, which accounts for effects of restricted rotational freedom of fluorescent dyes, as well as for limited knowledge of the exact dye positions due to attachment via flexible linkers. In particular we show that global data analysis of complete FRET networks is beneficial and that the measurement of FRET anisotropies in addition to FRET efficiencies can be used to determine accurately both position and orientation of the dyes. This measurement scheme improves localization accuracy substantially, and we can show that the improvement is a consequence of the more precise information about the transition dipole moment orientation of the dyes obtained by FRET anisotropy measurements. We discuss also rigid body docking of different macromolecules by means of NPS, which can be used to study the structure of macromolecular complexes. Finally, we combine our approach with common FRET analysis methods to determine the number of states of a macromolecule.
Journal physical chemistry B 115 (2011) 11927-11937
Supplementary Material

A. Muschielok, J. Andrecka, A. Jawhari, F. Brückner, P. Cramer, and J. Michaelis
A nano-positioning system for macromolecular structural analysis
By combining single pair FRET data, x-ray crystallographical data and statistical data analysis we have developed a novel method for determining the position of a flexible domain inside of a macromolecule/macromolecular complex. The method shares some similarities to GPS, the global positioning system, which is why we called it NPS, Nano Positioning System.
Nature Methods, 5 (2008 ) 965-971