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A toolkit and benchmark study for FRET-restrained high-precision structural modeling

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.

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Figure 1: Effect of dye position distributions on FRET efficiency.
Figure 2: FRET-restrained positioning and screening (FPS).
Figure 3: Distance measurements and error estimation.
Figure 4: Cluster analysis of docking results.
Figure 5: Bootstrapping of docked dsDNA dp/dt.
Figure 6: Structure of ssDNA dp/dt overhang obtained by screening of MD trajectories.

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Acknowledgements

We dedicate this paper to the memory of our brilliant colleague, Robert M. Clegg, a pioneer in the application of FRET in the life sciences and a remarkable human being. We would like to thank E. Schweinberger, O. Kensch and B.M. Wöhrl for assistance with the experiments, E. Haustein for analytical software, H. Sanabria for help with data visualization and A. Scheidig for helpful discussions. We acknowledge financial support from the Volkswagen Foundation (to R.S.G. and C.A.M.S.; grant no. I/74470), Max-Planck Society (to R.S.G.), German Federal Ministry of Education and Research (BMBF) (BioFuture grant no. 0311865) and German Science Foundation within SPP 1258 (grant no. SE 1195/12-2) (to C.A.M.S.) and 'Fit for Excellence' initiative of HHU (to H.G.). T.P. thanks the International Helmholtz Research School of Biophysics and Soft Matter (IHRS BioSoft), and S.S. thanks the NRW Research School of Biological Structures in Molecular Medicine and Biotechnology (BioStruct) for scholarships.

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Contributions

S.K. and C.A.M.S. designed studies and assisted in data analysis and interpretation; P.J.R. and S.B. prepared samples and performed experiments; S.K. developed the software; T.P. and S.S. analyzed the data; H.G. performed MD simulations and assisted with data interpretation; T.R. and R.S.G. supervised the biochemical experiments and assisted with data collection and interpretation; S.K., T.P., S.S., T.R., H.G. and C.A.M.S. wrote the manuscript; C.A.M.S. supervised the project.

Corresponding authors

Correspondence to Stanislav Kalinin or Claus A M Seidel.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2, Supplementary Tables 1–5, Supplementary Notes 1–15, Supplementary Data 1–3 and Supplementary Methods (PDF 4054 kb)

Supplementary Data 4

Protein Data Bank file with conformers of the flexible ssDNA template overhang. (ZIP 19103 kb)

Supplementary Software

Software for FRET-restrained positioning and screening with help file and test data. (ZIP 12715 kb)

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Kalinin, S., Peulen, T., Sindbert, S. et al. A toolkit and benchmark study for FRET-restrained high-precision structural modeling. Nat Methods 9, 1218–1225 (2012). https://doi.org/10.1038/nmeth.2222

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