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Multicolor single-particle reconstruction of protein complexes

Abstract

Single-particle reconstruction (SPR) from electron microscopy (EM) images is widely used in structural biology, but it lacks direct information on protein identity. To address this limitation, we developed a computational and analytical framework that reconstructs and coaligns multiple proteins from 2D super-resolution fluorescence images. To demonstrate our method, we generated multicolor 3D reconstructions of several proteins within the human centriole, which revealed their relative locations, dimensions and orientations.

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Fig. 1: Multicolor single-particle reconstruction.
Fig. 2: Multicolor single-particle reconstruction of an asymmetric protein complex.

Data availability

The datasets generated and analyzed in this study are available from the corresponding authors upon reasonable request. Sample datasets are available on Zenodo (https://doi.org/10.5281/zenodo.1288783).

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Acknowledgements

We thank C. Lehmann for growth, purification and AFM characterization of T4 bacteriophages; J.M. de la Rosa-Trevín for support regarding the optimal use of Scipion; and C.G. Morrison (Centre for Chromosome Biology, School of Natural Sciences and National University of Ireland Galway, Galway, Ireland) for providing the Cep164 antibody (1F3G10). We thank B. Huang and X. Shi for providing their 2D particle-alignment code. We also thank R. Laine and C. Kaminski for providing an HSV SMLM dataset. This work was supported by the European Research Council (ERC; grant AdG 340227 to P.G.), the EPFL Fellows postdoctoral fellowship program funded by the European Union’s Horizon 2020 Framework Programme for Research and Innovation (grant agreement 665667 to N.B., MSCA-COFUND), and the National Centre for Competence in Research (NCCR) Chemical Biology (S.M.). We thank M. Daszykowski for providing a public DBSCAN implementation.

Author information

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Authors

Contributions

C.S., N.B., P.G. and S.M. conceived and designed the project. C.S., P.G. and S.M. supervised the project. C.S. and N.B. performed all experiments and data analysis. C.S. and K.M.D. wrote analysis code. All authors wrote, revised and contributed to the final manuscript.

Corresponding authors

Correspondence to Christian Sieben or Suliana Manley.

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The authors declare no competing interests.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–20, Supplementary Notes 1–5 and Supplementary Tables 1–3

Reporting Summary

Supplementary Video 1

Four-color 3D reconstruction of centriolar proteins with a shared symmetry axis. Animated 360° rotation of the four-color 3D model showing Cep152 (magenta), Cep164 (green), Cep57 (cyan) and Cep63 (white mesh).

Supplementary Video 2

3D reconstruction of centriolar proteins without a shared symmetry axis. Animated 360° rotation of 3D reconstructed model of Cep152 (magenta) and Hs-SAS6 (cyan). The 3D reconstructed sum of Cep152 and Hs-SAS-6 is shown as a yellow mesh.

Supplementary Software 1

GUI application and source code.

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Sieben, C., Banterle, N., Douglass, K.M. et al. Multicolor single-particle reconstruction of protein complexes. Nat Methods 15, 777–780 (2018). https://doi.org/10.1038/s41592-018-0140-x

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