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Cryogenic optical localization provides 3D protein structure data with Angstrom resolution

Nature Methods volume 14, pages 141144 (2017) | Download Citation

Abstract

We introduce Cryogenic Optical Localization in 3D (COLD), a method to localize multiple fluorescent sites within a single small protein with Angstrom resolution. We demonstrate COLD by determining the conformational state of the cytosolic Per-ARNT-Sim domain from the histidine kinase CitA of Geobacillus thermodenitrificans and resolving the four biotin sites of streptavidin. COLD provides quantitative 3D information about small- to medium-sized biomolecules on the Angstrom scale and complements other techniques in structural biology.

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Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

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Acknowledgements

This work was supported by the Max Planck Society and the Alexander von Humboldt Foundation (Humboldt Professor, V.S.). We thank L. Wei for her contribution to the earlier stage of this work. We thank E. Eichhammer, S. Götzinger and T. Utikal for their contributions in the development of the cryostat extension. We also thank I. Schoen and M. Piliarik for fruitful discussions. The work on the PAS domain was supported by the DFG (to C.G., grant no. GR1211/17-1). S.B. thanks G. Sheldrick for advice on refinement using data from twinned crystals. We thank G. Unden (University of Mainz, Germany) for the GtCitA clone.

Author information

Affiliations

  1. Max Planck Institute for the Science of Light, Erlangen, Germany.

    • Siegfried Weisenburger
    • , Daniel Boening
    •  & Vahid Sandoghdar
  2. Department of Physics, Friedrich Alexander University (FAU) Erlangen-Nürnberg, Erlangen, Germany.

    • Siegfried Weisenburger
    •  & Vahid Sandoghdar
  3. Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

    • Benjamin Schomburg
    • , Karin Giller
    • , Stefan Becker
    •  & Christian Griesinger

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Contributions

V.S. conceived and supervised the project. S.W. built the experimental setup. S.W. and D.B. performed the optical measurements and analyzedthe data. B.S., K.G. and S.B. provided GtCitA PASc samples and solved the crystal structure. B.S. and C.G. recorded and analyzed NMR spectra. S.W. and V.S. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Vahid Sandoghdar.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15, Supplementary Tables 1 and 2, and Supplementary Note.

  2. 2.

    Supplementary Protocol

    COLD measurements.

Videos

  1. 1.

    Increasing the quality of the 3D reconstruction with increasing the quality of the input data.

    Demonstration of the influence of the localization precision of the projection data on the 3D reconstruction. We gradually increase the requirement on the data quality by changing the filtering conditions from using only data sets with 20 Å < σLoc < 30 Å to using the best data sets with σLoc < 13 Å while keeping the number of input images similar. The movie shows an overlay of the reconstructed volumes (red) with the crystal structure. Isosurfaces are plotted at an isovalue of 0.68.

  2. 2.

    Raw data recording.

    Example of a wide-field fluorescence image stack recorded at T = 4.3 K with an integration time of 2 s. The movie shows the first hour raw data from a 2-hour recording. A black frame every 15 min indicates when automated refocusing is performed. Brightness has been adjusted for clarity.

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DOI

https://doi.org/10.1038/nmeth.4141

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