Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Imaging and three-dimensional reconstruction of chemical groups inside a protein complex using atomic force microscopy

Abstract

Scanning probe microscopes can be used to image and chemically characterize surfaces down to the atomic scale1,2,3. However, the localized tip–sample interactions in scanning probe microscopes limit high-resolution images to the topmost atomic layer of surfaces1,2,3,4,5,6,7,8,9, and characterizing the inner structures of materials and biomolecules is a challenge for such instruments. Here, we show that an atomic force microscope can be used to image and three-dimensionally reconstruct chemical groups inside a protein complex. We use short single-stranded DNAs as imaging labels that are linked to target regions inside a protein complex, and T-shaped atomic force microscope cantilevers10,11 functionalized with complementary probe DNAs allow the labels to be located with sequence specificity and subnanometre resolution. After measuring pairwise distances between labels, we reconstruct the three-dimensional structure formed by the target chemical groups within the protein complex using simple geometric calculations. Experiments with the biotin–streptavidin complex show that the predicted three-dimensional loci of the carboxylic acid groups of biotins are within 2 Å of their respective loci in the corresponding crystal structure, suggesting that scanning probe microscopes could complement existing structural biological techniques in solving structures that are difficult to study due to their size and complexity12.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Chemically specific imaging and three-dimensional reconstruction using DNA labels.
Figure 2: Tuning the lifetime of DNA interactions enhances target specificity.
Figure 3: Multicolour imaging of target DNA molecules immobilized directly onto the substrate.
Figure 4: Chemically specific imaging and three-dimensional reconstruction in a protein complex.

Similar content being viewed by others

References

  1. Giessibl, F. J., Hembacher, S., Bielefeldt, H. & Mannhart, J. Subatomic features on the silicon (111)-(7×7) surface observed by atomic force microscopy. Science 289, 422–425 (2000).

    Article  CAS  Google Scholar 

  2. Sugimoto, Y. et al. Chemical identification of individual surface atoms by atomic force microscopy. Nature 446, 64–67 (2007).

    Article  CAS  Google Scholar 

  3. Gross, L., Mohn, F., Moll, N., Liljeroth, P. & Meyer, G. The chemical structure of a molecule resolved by atomic force microscopy. Science 325, 1110–1114 (2009).

    Article  CAS  Google Scholar 

  4. San Paulo, A. & Garcia, R. High-resolution imaging of antibodies by tapping-mode atomic force microscopy: attractive and repulsive tip–sample interaction regimes. Biophys. J. 78, 1599–1605 (2000).

    Article  CAS  Google Scholar 

  5. Ido, S. et al. Beyond the helix pitch: direct visualization of native DNA in aqueous solution. ACS Nano 7, 1817–1822 (2013).

    Article  CAS  Google Scholar 

  6. Cerreta, A., Vobornik, D. & Dietler, G. Fine DNA structure revealed by constant height frequency modulation AFM imaging. Eur. Polym. J. 49, 1916–1922 (2013).

    Article  CAS  Google Scholar 

  7. Ido, S. et al. Immunoactive two-dimensional self-assembly of monoclonal antibodies in aqueous solution revealed by atomic force microscopy. Nature Mater. 13, 264–270 (2014).

    Article  CAS  Google Scholar 

  8. Müller, D. J., Fotiadis, D., Scheuring, S., Müller, S. A. & Engel, A. Electrostatically balanced subnanometer imaging of biological specimens by atomic force microscope. Biophys. J. 76, 1101–1111 (1999).

    Article  Google Scholar 

  9. Voïtchovsky, K., Kuna, J. J., Contera, S. A., Tosatti, E. & Stellacci, F. Direct mapping of the solid–liquid adhesion energy with subnanometre resolution. Nature Nanotech. 5, 401–405 (2010).

    Article  Google Scholar 

  10. Dong, M., Husale, S. & Sahin, O. Determination of protein structural flexibility by microsecond force spectroscopy. Nature Nanotech. 4, 514–517 (2009).

    Article  CAS  Google Scholar 

  11. Sahin, O., Magonov, S., Su, C., Quate, C. F. & Solgaard, O. An atomic force microscope tip designed to measure time-varying nanomechanical forces. Nature Nanotech. 2, 507–514 (2007).

    Article  Google Scholar 

  12. Jones, N. Crystallography: atomic secrets. Nature 505, 602 (2014).

    Article  CAS  Google Scholar 

  13. Jesse, S., Kalinin, S. V., Proksch, R., Baddorf, A. & Rodriguez, B. The band excitation method in scanning probe microscopy for rapid mapping of energy dissipation on the nanoscale. Nanotechnology 18, 435503 (2007).

    Article  Google Scholar 

  14. Garcia, R. & Herruzo, E. T. The emergence of multifrequency force microscopy. Nature Nanotech. 7, 217–226 (2012).

    Article  CAS  Google Scholar 

  15. Xu, X., Melcher, J., Basak, S., Reifenberger, R. & Raman, A. Compositional contrast of biological materials in liquids using the momentary excitation of higher eigenmodes in dynamic atomic force microscopy. Phys. Rev. Lett. 102, 060801 (2009).

    Article  Google Scholar 

  16. Platz, D., Forchheimer, D., Tholén, E. A. & Haviland, D. B. Interaction imaging with amplitude-dependence force spectroscopy. Nature Commun. 4, 1360 (2013).

    Article  Google Scholar 

  17. Tetard, L., Passian, A. & Thundat, T. New modes for subsurface atomic force microscopy through nanomechanical coupling. Nature Nanotech. 5, 105–109 (2010).

    Article  CAS  Google Scholar 

  18. Dong, M. & Sahin, O. A nanomechanical interface to rapid single-molecule interactions. Nature Commun. 2, 247 (2011).

    Article  Google Scholar 

  19. Hinterdorfer, P. & Dufrene, Y. F. Detection and localization of single molecular recognition events using atomic force microscopy. Nature Methods 3, 347–355 (2006).

    Article  CAS  Google Scholar 

  20. Lee, G. U., Chrisey, L. A. & Colton, R. J. Direct measurement of the forces between complementary strands of DNA. Science 266, 771–773 (1994).

    Article  CAS  Google Scholar 

  21. Strunz, T., Oroszlan, K., Schäfer, R. & Güntherodt, H. J. Dynamic force spectroscopy of single DNA molecules. Proc. Natl Acad. Sci. USA 96, 11277–11282 (1999).

    Article  CAS  Google Scholar 

  22. Kufer, S. K., Puchner, E. M., Gumpp, H., Liedl, T. & Gaub, H. E. Single-molecule cut-and-paste surface assembly. Science 319, 594–596 (2008).

    Article  CAS  Google Scholar 

  23. Cocco, S., Monasson, R. & Marko, J. F. Force and kinetic barriers to initiation of DNA unzipping. Phys. Rev. E 65, 041907 (2002).

    Article  Google Scholar 

  24. Bansal, P. & Ardell, A. Average nearest-neighbour distances between uniformly distributed finite particles. Metallography 5, 97–111 (1972).

    Article  Google Scholar 

  25. Pertsinidis, A., Zhang, Y. & Chu, S. Subnanometre single-molecule localization, registration and distance measurements. Nature 466, 647–651 (2010).

    Article  CAS  Google Scholar 

  26. Murphy, M., Rasnik, I., Cheng, W., Lohman, T. M. & Ha, T. Probing single-stranded DNA conformational flexibility using fluorescence spectroscopy. Biophys. J. 86, 2530–2537 (2004).

    Article  CAS  Google Scholar 

  27. Stenkamp, R. E., Trong, I. L., Klumb, L., Stayton, P. S. & Freitag, S. Structural studies of the streptavidin binding loop. Protein Sci. 6, 1157–1166 (1997).

    Article  Google Scholar 

Download references

Acknowledgements

This work is supported by the NIH Director's New Innovator Award Program (1DP2-EB018657). D.K. is partially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2011-357-C00077).

Author information

Authors and Affiliations

Authors

Contributions

D.K. carried out experiments. O.S. conceived the imaging approach and designed the research. D.K. and O.S. analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Ozgur Sahin.

Ethics declarations

Competing interests

A patent application has been filed by Columbia University. Patent title: Devices and methods for determining molecular structure.

Supplementary information

Supplementary information

Supplementary information (PDF 1782 kb)

Supplementary Moive

Supplementary Moive (MP4 4808 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, D., Sahin, O. Imaging and three-dimensional reconstruction of chemical groups inside a protein complex using atomic force microscopy. Nature Nanotech 10, 264–269 (2015). https://doi.org/10.1038/nnano.2014.335

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nnano.2014.335

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing