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Imaging single glycans

Nature volume 582, pages 375–378 (2020)Cite this article

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A Publisher Correction to this article was published on 15 July 2020

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Abstract

Imaging of biomolecules guides our understanding of their diverse structures and functions1,2. Real-space imaging at sub-nanometre resolution using cryo-electron microscopy has provided key insights into proteins and their assemblies3,4. Direct molecular imaging of glycans—the predominant biopolymers on Earth, with a plethora of structural and biological functions5—has not been possible so far6. The inherent glycan complexity and backbone flexibility require single-molecule approaches for real-space imaging. At present, glycan characterization often relies on a combination of mass spectrometry and nuclear magnetic resonance imaging to provide insights into size, sequence, branching and connectivity, and therefore requires structure reconstruction from indirect information7,8,9. Here we show direct imaging of single glycan molecules that are isolated by mass-selective, soft-landing electrospray ion beam deposition and imaged by low-temperature scanning tunnelling microscopy10. The sub-nanometre resolution of the technique enables the visualization of glycan connectivity and discrimination between regioisomers. Direct glycan imaging is an important step towards a better understanding of the structure of carbohydrates.

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Fig. 1: STM topography imaging of linear glycans prepared by ES-IBD.
Fig. 2: STM topographic image of branched hexamanosides 4 and 5.
Fig. 3: STM topography image of undecasaccharide 6.
Fig. 4: Analysis of glycans from mixtures by chemically selective deposition followed by STM imaging.

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Data availability

The data that support the findings of this study are available from the authors on reasonable request; see Author contributions for specific datasets.

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Acknowledgements

We thank the Max Planck Society and Alexander von Humboldt Foundation for financial support. M.D. thanks the Minerva Fast Track Program and the MPG-FhG Cooperation Project Glyco3Dysplay. The authors acknowledge the Emmy-Noether-Program of the Deutsche Forschungsgemeinschaft.

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Author notes

  1. These authors contributed equally: X. Wu, M. Delbianco

Authors and Affiliations

  1. Max Planck Institute for Solid State Research, Stuttgart, Germany

    X. Wu, K. Anggara, T. Michnowicz, S. Sen, M. Pristl, S. Rauschenbach, U. Schlickum, S. Abb & K. Kern

  2. Max Planck Institute for Colloids and Interfaces, Potsdam, Germany

    M. Delbianco, A. Pardo-Vargas, P. Bharate & P. H. Seeberger

  3. Department of Chemistry, University of Oxford, Oxford, UK

    S. Rauschenbach

  4. Institute of Applied Physics, Technische Universität Braunschweig, Braunschweig, Germany

    U. Schlickum

  5. Institute for Chemistry and Biochemistry, Free University Berlin, Berlin, Germany

    P. H. Seeberger

  6. Institut de Physique, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

    K. Kern

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  1. X. Wu
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Contributions

S.R., S.A., P.H.S. and K.K. conceived this project. X.W., K.A., S.R., S.A., U.S., K.K., P.H.S. and M.D. designed the experiments. X.W., K.A., T.M., S.S., M.P., U.S. and S.A. carried out the sample preparation and measurements. X.W., S.A. and T.M. analysed the data. M.D., A.P.V. and P.B. carried out the molecule synthesis. All authors contributed to and discussed the manuscript.

Corresponding authors

Correspondence to S. Rauschenbach, P. H. Seeberger or K. Kern.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature thanks Steven De Feyter and Sabine Flitsch for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Mass spectra of all deposited glycans.

a–f, The mass spectra show absence of major contaminants in the ion beam after mass filtering. For glycans 1–5, the charge state of the molecule is −1, whereas undecasaccharide 6 has a charge state of −2.

Extended Data Fig. 2 STM overview image of a sample of hexamannoside 4.

Single molecules (marked with yellow circles) are observed as the major species on the terrace. The dark spots are due to impurities on the surface, which is typical for depositions at low temperature. Scale bar, 10 nm.

Extended Data Fig. 3 STM topography image of linear glycans.

a–f, STM images of pentamannoside 1 (a, b), hexamannoside 2 (c, d) and hexaglucoside 3 (e, f) showing different conformations on the surface. Scale bars, 1 nm.

Extended Data Fig. 4 STM topography image of branched hexamannosides.

a, b, STM images of adsorbed hexamannoside 4 showing different conformations. Two branches are clearly visible. c, d, STM images of hexamannoside 5 of different conformations on the surface, with branching clearly visible. The assignment of the linkage is based on the position of the linker (low-height feature) and on the interunit distance, as shown in the inset (1-2 linkage in pink). Scale bars, 1 nm.

Extended Data Fig. 5 Statistical analysis of the feature distance of 1-6 linkages in linear glycans.

a, Pentamannoside 1. b, Hexamannoside 2. c, Hexaglucoside 3. d, Combined histogram for all linear glycans. Values for the mean feature distance of the 1-6 linkages were calculated by fitting with Gaussian peaks, resulting in centre and width values of 0.48 ± 0.09 nm, 0.51 ± 0.08 nm and 0.53 ± 0.04 nm, respectively. The total averaging of the 1-6 linkages (including α1-6 and β1-6) yields a measured 1-6 feature distance of 0.52 ± 0.07 nm. Histogram bin size, 0.05 nm.

Extended Data Fig. 6 Statistical analysis of the feature distance of α1-6 and α1-2 linkages in hexamannoside 4.

The distance between the monosaccharide units connected with α1-6 and α1-2 linkages in hexamannoside 4 was identified on the basis of the linker position and the branching points. The histogram (bin size, 0.02 nm) of the α1-6 (grey) and α1-2 (purple) linkages was fitted with Gaussian peaks at 0.53 ± 0.05 nm and 0.61 ± 0.03 nm, respectively. The distributions show little overlap and thus the feature distance is identified as a reliable parameter for distinguishing between α1-6 and α1-2 linkages. The wide distribution of α1-6 (black) linkages compared to the narrow shape of α1-2 linkages (purple) indicates larger conformational variability, probably due to the greater flexibility of the α1-6 links.

Extended Data Fig. 7 STM topography image of branched undecasaccharide 6.

a, STM images of undecasaccharide 6, clearly showing branching points and individually resolved monosaccharide units. The assignment of the branches is made by the number of units in the branch and by the feature distance between the monosaccharide units. Scale bar, 1 nm. b, Line profiles as indicated in a.

Supplementary information

Supplementary Information

This file contains: (1) General Materials and Methods, (2) Automated Glycan Assembly, and (3) additional References.

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Wu, X., Delbianco, M., Anggara, K. et al. Imaging single glycans. Nature 582, 375–378 (2020). https://doi.org/10.1038/s41586-020-2362-1

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