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
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
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
Similar content being viewed by others
References
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).
Sugimoto, Y. et al. Chemical identification of individual surface atoms by atomic force microscopy. Nature 446, 64–67 (2007).
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).
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).
Ido, S. et al. Beyond the helix pitch: direct visualization of native DNA in aqueous solution. ACS Nano 7, 1817–1822 (2013).
Cerreta, A., Vobornik, D. & Dietler, G. Fine DNA structure revealed by constant height frequency modulation AFM imaging. Eur. Polym. J. 49, 1916–1922 (2013).
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).
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).
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).
Dong, M., Husale, S. & Sahin, O. Determination of protein structural flexibility by microsecond force spectroscopy. Nature Nanotech. 4, 514–517 (2009).
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).
Jones, N. Crystallography: atomic secrets. Nature 505, 602 (2014).
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).
Garcia, R. & Herruzo, E. T. The emergence of multifrequency force microscopy. Nature Nanotech. 7, 217–226 (2012).
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).
Platz, D., Forchheimer, D., Tholén, E. A. & Haviland, D. B. Interaction imaging with amplitude-dependence force spectroscopy. Nature Commun. 4, 1360 (2013).
Tetard, L., Passian, A. & Thundat, T. New modes for subsurface atomic force microscopy through nanomechanical coupling. Nature Nanotech. 5, 105–109 (2010).
Dong, M. & Sahin, O. A nanomechanical interface to rapid single-molecule interactions. Nature Commun. 2, 247 (2011).
Hinterdorfer, P. & Dufrene, Y. F. Detection and localization of single molecular recognition events using atomic force microscopy. Nature Methods 3, 347–355 (2006).
Lee, G. U., Chrisey, L. A. & Colton, R. J. Direct measurement of the forces between complementary strands of DNA. Science 266, 771–773 (1994).
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).
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).
Cocco, S., Monasson, R. & Marko, J. F. Force and kinetic barriers to initiation of DNA unzipping. Phys. Rev. E 65, 041907 (2002).
Bansal, P. & Ardell, A. Average nearest-neighbour distances between uniformly distributed finite particles. Metallography 5, 97–111 (1972).
Pertsinidis, A., Zhang, Y. & Chu, S. Subnanometre single-molecule localization, registration and distance measurements. Nature 466, 647–651 (2010).
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).
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).
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
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
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
About this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nnano.2014.335
This article is cited by
-
Diamagnetically levitated nanopositioners with large-range and multiple degrees of freedom
Nature Communications (2022)
-
Closed-loop atomic force microscopy-infrared spectroscopic imaging for nanoscale molecular characterization
Nature Communications (2020)
-
Imaging modes of atomic force microscopy for application in molecular and cell biology
Nature Nanotechnology (2017)
-
Atomic force microscopy-based characterization and design of biointerfaces
Nature Reviews Materials (2017)
-
Peering inside protein complexes with AFM
Nature Methods (2015)