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@article{Dimura.2016,
 author = {Dimura, Mykola and Peulen, Thomas O. and Hanke, Christian A. and Prakash, Aiswaria and Gohlke, Holger and Seidel, Claus A. M.},
 year = {2016},
 title = {Quantitative FRET studies and integrative modeling unravel the structure and dynamics of biomolecular systems},
 pages = {163--185},
 pagination = {page},
 volume = {40},
 issn = {0959-440X},
 journal = {Curr. Opin. Struct. Biol.},
 abstract = {Förster Resonance Energy Transfer (FRET) combined with single-molecule spectroscopy probes macromolecular structure and dynamics and identifies coexisting conformational states. We review recent methodological developments in integrative structural modeling by satisfying spatial restraints on networks of FRET pairs (hybrid-FRET). We discuss procedures to incorporate prior structural knowledge and to obtain optimal distance networks. Finally, a workflow for hybrid-FRET is presented that automates integrative structural modeling and experiment planning to put hybrid-FRET on rails. To test this workflow, we simulate realistic single-molecule experiments and resolve three protein conformers, exchanging at 30μs and 10ms, with accuracies of 1-3Å RMSD versus the target structure. Incorporation of data from other spectroscopies and imaging is also discussed.},
 doi = {10.1016/j.sbi.2016.11.012}
}


@article{Dimura.2020,
 author = {Dimura, Mykola and Peulen, Thomas-Otavio and Sanabria, Hugo and Rodnin, Dmitro and Hemmen, Katherina and Hanke, Christian A. and Seidel, Claus A. M. and Gohlke, Holger},
 year = {2020},
 title = {Automated and optimally FRET-assisted structural modeling},
 keywords = {Automation;Computer Simulation;Fluorescence Resonance Energy Transfer/instrumentation/methods;Models, Structural;Protein Conformation;Proteins/chemistry;Software;Web Browser},
 pages = {5394},
 pagination = {page},
 volume = {11},
 number = {1},
 issn = {2041-1723},
 journal = {Nat. Commun.},
 abstract = {FRET experiments can provide state-specific structural information of complex dynamic biomolecular assemblies. However, to overcome the sparsity of FRET experiments, they need to be combined with computer simulations. We introduce a program suite with (i) an automated design tool for FRET experiments, which determines how many and which FRET pairs should be used to minimize the uncertainty and maximize the accuracy of an integrative structure, (ii) an efficient approach for FRET-assisted coarse-grained structural modeling, and all-atom molecular dynamics simulations-based refinement, and (iii) a quantitative quality estimate for judging the accuracy of FRET-derived structures as opposed to precision. We benchmark our tools against simulated and experimental data of proteins with multiple conformational states and demonstrate an accuracy of {\~{}}3 Å RMSDCα against X-ray structures for sets of 15 to 23 FRET pairs. Free and open-source software for the introduced workflow is available at https://github.com/Fluorescence-Tools . A web server for FRET-assisted structural modeling of proteins is available at http://nmsim.de .},
 doi = {10.1038/s41467-020-19023-1}
}


@article{Eilert.2017,
 author = {Eilert, Tobias and Beckers, Maximilian and Drechsler, Florian and Michaelis, Jens},
 year = {2017},
 title = {Fast-NPS —A Markov Chain Monte Carlo-based analysis tool to obtain structural information from single-molecule FRET measurements},
 pages = {377--389},
 pagination = {page},
 volume = {219},
 issn = {0010-4655},
 journal = {Comput. Phys. Commun.},
 doi = {10.1016/j.cpc.2017.05.027}
}


@article{Hellenkamp.2018,
 author = {Hellenkamp, Björn and Schmid, Sonja and Doroshenko, Olga and Opanasyuk, Oleg and Kühnemuth, Ralf and {Rezaei Adariani}, Soheila and Ambrose, Benjamin and Aznauryan, Mikayel and Barth, Anders and Birkedal, Victoria and Bowen, Mark E. and Chen, Hongtao and Cordes, Thorben and Eilert, Tobias and Fijen, Carel and Gebhardt, Christian and Götz, Markus and Gouridis, Giorgos and Gratton, Enrico and Ha, Taekjip and Hao, Pengyu and Hanke, Christian A. and Hartmann, Andreas and Hendrix, Jelle and Hildebrandt, Lasse L. and Hirschfeld, Verena and Hohlbein, Johannes and Hua, Boyang and Hübner, Christian G. and Kallis, Eleni and Kapanidis, Achillefs N. and Kim, Jae-Yeol and Krainer, Georg and Lamb, Don C. and Lee, Nam Ki and Lemke, Edward A. and Levesque, Brié and Levitus, Marcia and McCann, James J. and Naredi-Rainer, Nikolaus and Nettels, Daniel and Ngo, Thuy and Qiu, Ruoyi and Robb, Nicole C. and Röcker, Carlheinz and Sanabria, Hugo and Schlierf, Michael and Schröder, Tim and Schuler, Benjamin and Seidel, Henning and Streit, Lisa and Thurn, Johann and Tinnefeld, Philip and Tyagi, Swati and Vandenberk, Niels and Vera, Andrés Manuel and Weninger, Keith R. and Wünsch, Bettina and Yanez-Orozco, Inna S. and Michaelis, Jens and Seidel, Claus A. M. and Craggs, Timothy D. and Hugel, Thorsten},
 year = {2018},
 title = {Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study},
 pages = {669--676},
 pagination = {page},
 volume = {15},
 number = {9},
 issn = {1548-7091},
 journal = {Nat. Methods},
 abstract = {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.},
 doi = {10.1038/s41592-018-0085-0}
}


@article{Hoefling.2013,
 author = {Hoefling, Martin and Grubmüller, Helmut},
 year = {2013},
 title = {In silico FRET from simulated dye dynamics},
 pages = {841--852},
 pagination = {page},
 volume = {184},
 number = {3},
 issn = {0010-4655},
 journal = {Comput. Phys. Commun.},
 doi = {10.1016/j.cpc.2012.10.018}
}


@article{Hoefling.2011,
 author = {Hoefling, Martin and Lima, Nicola and Haenni, Dominik and Seidel, Claus A. M. and Schuler, Benjamin and Grubmüller, Helmut},
 year = {2011},
 title = {Structural heterogeneity and quantitative FRET efficiency distributions of polyprolines through a hybrid atomistic simulation and Monte Carlo approach},
 keywords = {Coloring Agents/chemistry;Energy Transfer;Fluorescence Resonance Energy Transfer/methods;Molecular Structure;Monte Carlo Method;Peptides/chemistry},
 pages = {e19791},
 pagination = {page},
 volume = {6},
 number = {5},
 issn = {1932-6203},
 journal = {PLoS One},
 abstract = {Förster Resonance Energy Transfer (FRET) experiments probe molecular distances via distance dependent energy transfer from an excited donor dye to an acceptor dye. Single molecule experiments not only probe average distances, but also distance distributions or even fluctuations, and thus provide a powerful tool to study biomolecular structure and dynamics. However, the measured energy transfer efficiency depends not only on the distance between the dyes, but also on their mutual orientation, which is typically inaccessible to experiments. Thus, assumptions on the orientation distributions and averages are usually made, limiting the accuracy of the distance distributions extracted from FRET experiments. Here, we demonstrate that by combining single molecule FRET experiments with the mutual dye orientation statistics obtained from Molecular Dynamics (MD) simulations, improved estimates of distances and distributions are obtained. From the simulated time-dependent mutual orientations, FRET efficiencies are calculated and the full statistics of individual photon absorption, energy transfer, and photon emission events is obtained from subsequent Monte Carlo (MC) simulations of the FRET kinetics. All recorded emission events are collected to bursts from which efficiency distributions are calculated in close resemblance to the actual FRET experiment, taking shot noise fully into account. Using polyproline chains with attached Alexa 488 and Alexa 594 dyes as a test system, we demonstrate the feasibility of this approach by direct comparison to experimental data. We identified cis-isomers and different static local environments as sources of the experimentally observed heterogeneity. Reconstructions of distance distributions from experimental data at different levels of theory demonstrate how the respective underlying assumptions and approximations affect the obtained accuracy. Our results show that dye fluctuations obtained from MD simulations, combined with MC single photon kinetics, provide a versatile tool to improve the accuracy of distance distributions that can be extracted from measured single molecule FRET efficiencies.},
 doi = {10.1371/journal.pone.0019791}
}


@article{Kalinin.2012,
 author = {Kalinin, Stanislav and Peulen, Thomas and Sindbert, Simon and Rothwell, Paul J. and Berger, Sylvia and Restle, Tobias and Goody, Roger S. and Gohlke, Holger and Seidel, Claus A. M.},
 year = {2012},
 title = {A toolkit and benchmark study for FRET-restrained high-precision structural modeling},
 keywords = {DNA Primers/chemistry;Fluorescence Resonance Energy Transfer/methods;HIV Reverse Transcriptase/chemistry;Models, Molecular;Molecular Docking Simulation;Molecular Dynamics Simulation},
 pages = {1218--1225},
 pagination = {page},
 volume = {9},
 number = {12},
 issn = {1548-7091},
 journal = {Nat. Methods},
 abstract = {We present a comprehensive toolkit for Förster resonance energy transfer (FRET)-restrained modeling of biomolecules and their complexes for quantitative applications in structural biology. A dramatic improvement in the precision of FRET-derived structures is achieved by explicitly considering spatial distributions of dye positions, which greatly reduces uncertainties due to flexible dye linkers. The precision and confidence levels of the models are calculated by rigorous error estimation. The accuracy of this approach is demonstrated by docking a DNA primer-template to HIV-1 reverse transcriptase. The derived model agrees with the known X-ray structure with an r.m.s. deviation of 0.5 Å. Furthermore, we introduce FRET-guided 'screening' of a large structural ensemble created by molecular dynamics simulations. We used this hybrid approach to determine the formerly unknown configuration of the flexible single-strand template overhang.},
 doi = {10.1038/nmeth.2222}
}


@article{Muschielok.2008,
 author = {Muschielok, Adam and Andrecka, Joanna and Jawhari, Anass and Bruckner, Florian and Cramer, Patrick and Michaelis, Jens},
 year = {2008},
 title = {A nano-positioning system for macromolecular structural analysis},
 keywords = {0 (Transcription Factor TFIIB);63231-63-0 (RNA);Computer Simulation;Crystallography, X-Ray/methods;EC 2.7.7.- (RNA Polymerase II);Fluorescence Resonance Energy Transfer;Models, Molecular;Molecular Conformation;Nanotechnology/instrumentation;Protein Binding;RNA Polymerase II/metabolism;RNA/biosynthesis/chemistry/metabolism;Saccharomyces cerevisiae/enzymology/genetics/metabolism;Transcription Factor TFIIB/chemistry/metabolism},
 pages = {965--971},
 pagination = {page},
 volume = {5},
 number = {11},
 issn = {1548-7091},
 journal = {Nat. Methods},
 abstract = {Very often, the positions of flexible domains within macromolecules as well as within macromolecular complexes cannot be determined by standard structural biology methods. To overcome this problem, we developed a method that uses probabilistic data analysis to combine single-molecule measurements with X-ray crystallography data. The method determines not only the most likely position of a fluorescent dye molecule attached to the domain but also the complete three-dimensional probability distribution depicting the experimental uncertainty. With this approach, single-pair fluorescence resonance energy transfer measurements can now be used as a quantitative tool for investigating the position and dynamics of flexible domains within macromolecular complexes. We applied this method to find the position of the 5' end of the nascent RNA exiting transcription elongation complexes of yeast (Saccharomyces cerevisiae) RNA polymerase II and studied the influence of transcription factor IIB on the position of the RNA.},
 doi = {10.1038/nmeth.1259}
}


@article{Peulen.2017,
 author = {Peulen, Thomas-Otavio and Opanasyuk, Oleg and Seidel, Claus A. M.},
 year = {2017},
 title = {Combining Graphical and Analytical Methods with Molecular Simulations To Analyze Time-Resolved FRET Measurements of Labeled Macromolecules Accurately},
 pages = {8211--8241},
 pagination = {page},
 volume = {121},
 number = {35},
 issn = {1520-6106},
 journal = {J. Phys. Chem. B},
 abstract = {Förster resonance energy transfer (FRET) measurements from a donor, D, to an acceptor, A, fluorophore are frequently used in vitro and in live cells to reveal information on the structure and dynamics of DA labeled macromolecules. Accurate descriptions of FRET measurements by molecular models are complicated because the fluorophores are usually coupled to the macromolecule via flexible long linkers allowing for diffusional exchange between multiple states with different fluorescence properties caused by distinct environmental quenching, dye mobilities, and variable DA distances. It is often assumed for the analysis of fluorescence intensity decays that DA distances and D quenching are uncorrelated (homogeneous quenching by FRET) and that the exchange between distinct fluorophore states is slow (quasistatic). This allows us to introduce the FRET-induced donor decay, εD(t), a function solely depending on the species fraction distribution of the rate constants of energy transfer by FRET, for a convenient joint analysis of fluorescence decays of FRET and reference samples by integrated graphical and analytical procedures. Additionally, we developed a simulation toolkit to model dye diffusion, fluorescence quenching by the protein surface, and FRET. A benchmark study with simulated fluorescence decays of 500 protein structures demonstrates that the quasistatic homogeneous model works very well and recovers for single conformations the average DA distances with an accuracy of < 2{\%}. For more complex cases, where proteins adopt multiple conformations with significantly different dye environments (heterogeneous case), we introduce a general analysis framework and evaluate its power in resolving heterogeneities in DA distances. The developed fast simulation methods, relying on Brownian dynamics of a coarse-grained dye in its sterically accessible volume, allow us to incorporate structural information in the decay analysis for heterogeneous cases by relating dye states with protein conformations to pave the way for fluorescence and FRET-based dynamic structural biology. Finally, we present theories and simulations to assess the accuracy and precision of steady-state and time-resolved FRET measurements in resolving DA distances on the single-molecule and ensemble level and provide a rigorous framework for estimating approximation, systematic, and statistical errors.},
 doi = {10.1021/acs.jpcb.7b03441}
}


@article{Sindbert.2011,
 author = {Sindbert, Simon and Kalinin, Stanislav and Nguyen, Hien and Kienzler, Andrea and Clima, Lilia and Bannwarth, Willi and Appel, Bettina and Müller, Sabine and Seidel, Claus A. M.},
 year = {2011},
 title = {Accurate distance determination of nucleic acids via Förster resonance energy transfer: implications of dye linker length and rigidity},
 keywords = {Carbocyanines/chemistry;DNA/analysis;Fluorescence Resonance Energy Transfer;Hydrazines/chemistry;Molecular Structure;RNA/analysis},
 pages = {2463--2480},
 pagination = {page},
 volume = {133},
 number = {8},
 issn = {0002-7863},
 journal = {J. Am. Chem. Soc.},
 abstract = {In Förster resonance energy transfer (FRET) experiments, the donor (D) and acceptor (A) fluorophores are usually attached to the macromolecule of interest via long flexible linkers of up to 15 Å in length. This causes significant uncertainties in quantitative distance measurements and prevents experiments with short distances between the attachment points of the dyes due to possible dye-dye interactions. We present two approaches to overcome the above problems as demonstrated by FRET measurements for a series of dsDNA and dsRNA internally labeled with Alexa488 and Cy5 as D and A dye, respectively. First, we characterize the influence of linker length and flexibility on FRET for different dye linker types (long, intermediate, short) by analyzing fluorescence lifetime and anisotropy decays. For long linkers, we describe a straightforward procedure that allows for very high accuracy of FRET-based structure determination through proper consideration of the position distribution of the dye and of linker dynamics. The position distribution can be quickly calculated with geometric accessible volume (AV) simulations, provided that the local structure of RNA or DNA in the proximity of the dye is known and that the dye diffuses freely in the sterically allowed space. The AV approach provides results similar to molecular dynamics simulations (MD) and is fully consistent with experimental FRET data. In a benchmark study for ds A-RNA, an rmsd value of 1.3 Å is achieved. Considering the case of undefined dye environments or very short DA distances, we introduce short linkers with a propargyl or alkenyl unit for internal labeling of nucleic acids to minimize position uncertainties. Studies by ensemble time correlated single photon counting and single-molecule detection show that the nature of the linker strongly affects the radius of the dye's accessible volume (6-16 Å). For short propargyl linkers, heterogeneous dye environments are observed on the millisecond time scale. A detailed analysis of possible orientation effects (κ(2) problem) indicates that, for short linkers and unknown local environments, additional κ(2)-related uncertainties are clearly outweighed by better defined dye positions.},
 doi = {10.1021/ja105725e}
}


@article{Steffen.2016,
 author = {Steffen, Fabio D. and Sigel, Roland K. O. and Börner, Richard},
 year = {2016},
 title = {An atomistic view on carbocyanine photophysics in the realm of RNA},
 pages = {29045--29055},
 pagination = {page},
 volume = {18},
 number = {42},
 issn = {1463-9076, 1463-9084},
 journal = {Phys. Chem. Chem. Phys.},
 magnolia = {946dbfe7-de74-4191-93ec-1bb483ab0cbb},
 abstract = {Carbocyanine dyes have a long-standing tradition in fluorescence imaging and spectroscopy, due to their photostability and large spectral separation between individual dye species. Herein, we explore the versatility of cyanine dyes to probe the dynamics of nucleic acids and we report on the interrelation of fluorophores, RNA, and metal ions, namely K+ and Mg2+. Photophysical parameters including the fluorescence lifetime, quantum yield and dynamic anisotropy are monitored as a function of the nucleic acid composition, conformation, and metal ion abundance. Occasional excursions to a non-fluorescent cis-state hint at the remarkable sensitivity of carbocyanines to their local environment. Comparison of time-correlated single photon experiments with all-atom molecular dynamics simulations demonstrate that the propensity of photoisomerization is dictated by sterical constraints imposed on the fluorophore. Structural features in the vicinity of the dye play a crucial role in RNA recognition and have far-reaching implications on the mobility of the fluorescent probe. An atomic level description of the mutual interactions will ultimately benefit the quantitative interpretation of single-molecule FRET measurements on large RNA systems.},
 doi = {10.1039/c6cp04277e}
}


