Skip to main content

Thank you for visiting 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.

Design of a hyperstable 60-subunit protein icosahedron

A Corrigendum to this article was published on 19 October 2016

This article has been updated


The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport1,2. There has been considerable interest in repurposing such structures3,4,5 for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery6, vaccine design7 and synthetic biology8.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



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

Figure 1: Design methodology and biochemical characterization.
Figure 2: Cryo-EM.
Figure 3: Tuning nanocage structure and function with genetic fusions.

Change history


  1. Zandi, R., Reguera, D., Bruinsma, R. F., Gelbart, W. M. & Rudnick, J. Origin of icosahedral symmetry in viruses. Proc. Natl Acad. Sci. USA 101, 15556–15560 (2004)

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  2. Ritsert, K. et al. Studies on the lumazine synthase/riboflavin synthase complex of Bacillus subtilis: crystal structure analysis of reconstituted, icosahedral beta-subunit capsids with bound substrate analogue inhibitor at 2.4 Å resolution. J. Mol. Biol. 253, 151–167 (1995)

    Article  CAS  PubMed  Google Scholar 

  3. Howorka, S. Rationally engineering natural protein assemblies in nanobiotechnology. Curr. Opin. Biotechnol. 22, 485–491 (2011)

    Article  CAS  PubMed  Google Scholar 

  4. Roldão, A., Mellado, M. C. M., Castilho, L. R., Carrondo, M. J. T. & Alves, P. M. Virus-like particles in vaccine development. Expert Rev. Vaccines 9, 1149–1176 (2010)

    Article  PubMed  Google Scholar 

  5. Effio, C. L. & Hubbuch, J. Next generation vaccines and vectors: designing downstream processes for recombinant protein-based virus-like particles. Biotechnol. J. 10, 715–727 (2015)

    Article  CAS  PubMed  Google Scholar 

  6. Ma, Y., Nolte, R. J. M. & Cornelissen, J. J. L. M. Virus-based nanocarriers for drug delivery. Adv. Drug Deliv. Rev. 64, 811–825 (2012)

    Article  CAS  PubMed  Google Scholar 

  7. Smith, M. L. et al. Modified tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications. Virology 348, 475–488 (2006)

    Article  CAS  PubMed  Google Scholar 

  8. Bauler, P., Huber, G., Leyh, T. & McCammon, J. A. Channeling by proximity: the catalytic advantages of active site colocalization using Brownian dynamics. J. Phys. Chem. Lett. 1, 1332–1335 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Brodin, J. D. et al. Metal-directed, chemically tunable assembly of one-, two- and three-dimensional crystalline protein arrays. Nat. Chem. 4, 375–382 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Der, B. S. et al. Metal-mediated affinity and orientation specificity in a computationally designed protein homodimer. J. Am. Chem. Soc. 134, 375–385 (2012)

    Article  CAS  PubMed  Google Scholar 

  11. Fletcher, J. M. et al. Self-assembling cages from coiled-coil peptide modules. Science 340, 595–599 (2013)

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  12. Usui, K. et al. Nanoscale elongating control of the self-assembled protein filament with the cysteine-introduced building blocks. Protein Sci. 18, 960–969 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Raman, S., Machaidze, G., Lustig, A., Aebi, U. & Burkhard, P. Structure-based design of peptides that self-assemble into regular polyhedral nanoparticles. Nanomedicine 2, 95–102 (2006)

    Article  CAS  PubMed  Google Scholar 

  14. Raman, S. et al. Design of peptide nanoparticles using simple protein oligomerization domains. Open Nanomed. J. 2, 15–26 (2009)

    Article  CAS  Google Scholar 

  15. Sinclair, J. C., Davies, K. M., Vénien-Bryan, C. & Noble, M. E. M. Generation of protein lattices by fusing proteins with matching rotational symmetry. Nat. Nanotechnol. 6, 558–562 (2011)

    Article  CAS  PubMed  ADS  Google Scholar 

  16. Boyle, A. L. et al. Squaring the circle in peptide assembly: from fibers to discrete nanostructures by de novo design. J. Am. Chem. Soc. 134, 15457–15467 (2012)

    Article  CAS  PubMed  Google Scholar 

  17. Lai, Y.-T. et al. Structure of a designed protein cage that self-assembles into a highly porous cube. Nat. Chem. 6, 1065–1071 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. King, N. P. et al. Computational design of self-assembling protein nanomaterials with atomic level accuracy. Science 336, 1171–1174 (2012)

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  19. King, N. P. et al. Accurate design of co-assembling multi-component protein nanomaterials. Nature 510, 103–108 (2014)

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  20. Leaver-Fay, A. et al. ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol. 487, 545–574 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. DiMaio, F., Leaver-Fay, A., Bradley, P., Baker, D. & André, I. Modeling symmetric macromolecular structures in Rosetta3. PLoS One 6, e20450 (2011)

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  22. Lawrence, M. C. & Colman, P. M. Shape complementarity at protein/protein interfaces. J. Mol. Biol. 234, 946–950 (1993)

    Article  CAS  PubMed  Google Scholar 

  23. Griffiths, J. S. et al. Cloning, isolation and characterization of the Thermotoga maritima KDPG aldolase. Bioorg. Med. Chem. 10, 545–550 (2002)

    Article  CAS  PubMed  Google Scholar 

  24. Fullerton, S. W. B. et al. Mechanism of the class I KDPG aldolase. Bioorg. Med. Chem. 14, 3002–3010 (2006)

    Article  CAS  PubMed  Google Scholar 

  25. Perlmutter, J. D. & Hagan, M. F. Mechanisms of virus assembly. Annu. Rev. Phys. Chem. 66, 217–239 (2015)

    Article  CAS  PubMed  ADS  Google Scholar 

  26. Pédelacq, J.-D., Cabantous, S., Tran, T., Terwilliger, T. C. & Waldo, G. S. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88 (2006)

    Article  PubMed  CAS  Google Scholar 

  27. Andrews, B. T., Schoenfish, A. R., Roy, M., Waldo, G. & Jennings, P. A. The rough energy landscape of superfolder GFP is linked to the chromophore. J. Mol. Biol. 373, 476–490 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Cortese, K., Diaspro, A. & Tacchetti, C. Advanced correlative light/electron microscopy: current methods and new developments using Tokuyasu cryosections. J. Histochem. Cytochem. 57, 1103–1112 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Huang, P.-S. et al. High thermodynamic stability of parametrically designed helical bundles. Science 346, 481–485 (2014)

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  30. Zhou, Z. et al. Genetically encoded short peptide tags for orthogonal protein labeling by Sfp and AcpS phosphopantetheinyl transferases. ACS Chem. Biol. 2, 337–346 (2007)

    Article  CAS  PubMed  Google Scholar 

  31. Baalousha, M. & Lead, J. R. Nanoparticle dispersity in toxicology. Nat. Nanotechnol. 8, 308–309 (2013)

    Article  CAS  PubMed  ADS  Google Scholar 

  32. Zulauf, M. & D’Arcy, A. Light scattering of proteins as a criterion for crystallization. J. Cryst. Growth 122, 102–106 (1992)

    Article  CAS  ADS  Google Scholar 

  33. Nannenga, B. L., Iadanza, M. G., Vollmar, B. S. & Gonen, T. in Current Protocols in Protein Science (eds Coligan, J. E., Dunn, B. M., Speicher, D. W. & Wingfield, P. T. ) Ch. 17.15 (John Wiley & Sons, 2013)

  34. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007)

    Article  CAS  PubMed  Google Scholar 

  35. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. van Heel, M., Harauz, G., Orlova, E. V., Schmidt, R. & Schatz, M. A new generation of the IMAGIC image processing system. J. Struct. Biol. 116, 17–24 (1996)

    Article  CAS  PubMed  Google Scholar 

  38. Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    Article  CAS  PubMed  Google Scholar 

  39. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996)

    Article  CAS  PubMed  Google Scholar 

  40. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012)

    Article  CAS  PubMed  Google Scholar 

  41. Huang, P.-S. et al. RosettaRemodel: a generalized framework for flexible backbone protein design. PLoS One 6, e24109 (2011)

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  42. Muller, E. G. D. et al. The organization of the core proteins of the yeast spindle pole body. Mol. Biol. Cell 16, 3341–3352 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Shimogawa, M. M., Wargacki, M. M., Muller, E. G. & Davis, T. N. Laterally attached kinetochores recruit the checkpoint protein Bub1, but satisfy the spindle checkpoint. Cell Cycle 9, 3619–3628 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


This work was supported by the Howard Hughes Medical Institute (D.B. and T.G.), the JRC visitor programme (S.G.), the National Science Foundation CHE-1332907 (D.B.), a UW/Hutch CCSG Pilot Award NCI 5 P30 CA015704-41 (D.B. and N.P.K.), Takeda Pharmaceutical Company (N.P.K.), the Bill and Melinda Gates Foundation OPP1120319 (D.B. and N.P.K.), the National Institutes of Health (NIH) P41 GM103533 (T.N.D.), the Defense Advanced Research Projects Agency (D.B. and N.P.K., grant no. W911NF-14-1-0162) and the Air Force Office of Scientific Research (AFOSR) AFOSR FA950-12-10112 (D.B.). Y.H. was supported in part by a NIH Molecular Biology Training Grant (T32GM008268). U.N. was supported in part by a PHS National Research Service Award (T32GM007270) from NIGMS. J.B.B. was supported in part by an NSF Graduate Research Fellowship (DGE-0718124). We thank the Janelia Research Campus Cryo-EM Facility and J. de la Cruz for their assistance with the Titan Krios.

Author information

Authors and Affiliations



J.B.B., N.P.K., and W.S. developed the computational design methodology. Y.H. and J.B.B. performed the design of the icosahedra. Y.H. performed all other unlisted experiments. S.G. and D.S. performed the cryo-EM experiments. K.K.F. performed the fluorescence microscopy experiments. U.N. performed the negative-stain electron microscopy experiments. C.X. provided the pentamer sequence for I3-01(HB). P.-S.H. created the computational methodology to model fusions to I3-01. R.R. produced I3-01(HB) proteins. S.Y. produced T33-21 sfGFP fusion proteins.

Corresponding author

Correspondence to David Baker.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 I3-01 tolerance to temperature.

DLS measurements as I3-01 is subjected to heating to 90 °C (solid line), then cooling to 25 °C (dotted line) in TBS (a), 6.7 M GuHCl (b) and 2 M GITC (c). Under all three conditions, any indications of aggregation or increase in size due to temperature appear to be completely reversible.

Extended Data Figure 2 Reproducibility of I3-01 transition in 2 M to 2.25 M GITC.

Four examples each of independent measurements at 2 M (blue) and 2.25 M (red) GITC using DLS show the reproducibility of the cage disassociation. Histograms are plotted offset by 1% intensity from each other for clarity.

Extended Data Figure 3 SEC of T33-21 and I3-01 fused with sfGFP.

Size exclusion chromatography traces for T33-21 (12mer in red and 24mer in blue) and I3-01 (60mer in green and 120mer in purple) sfGFP fusions, display increased particle sizes with increasing copies of GFP, but retain monodispersed populations. The N-terminal fusion of sfGFP (dashed line) is expected to extend mostly outward from the icosahedron, thus greatly increasing the hydrodynamic radius while the C-terminal fusion is predicted to occupy the internal void space. A230, ultraviolet absorbance at 230 nm; mAU, milli-absorbance units.

Extended Data Figure 4 Tolerance of I3-01–sfGFP fusions to GuHCl.

N-terminal (red) and C-terminal (blue) sfGFP fusions were equilibrated to 0–6.4 M GuHCl. Ultraviolet absorbance at 490 nm (A490) monitors the unfolding of sfGFP (top, solid line and crosses). DLS experiments (top, dotted line and dots) reveal as sfGFP unfolds, the hydrodynamic radius increases slightly, and then stabilizes. The bottom panels show that in 1 M GuHCl (solid line) and in 6 M GuHCl (dotted line), the icosahedral assemblies remain relatively monodisperse.

Extended Data Figure 5 I3-01 C-terminal fusions with other fluorescent proteins.

Fluorescent proteins mTurquoise2 (in blue) or sYFP2 (in green) were fused to the C terminus of I3-01. The field of view using widefield fluorescence microscopy shows distinct signals of each type when the two types are mixed together.

Extended Data Figure 6 I3-01 retains native enzyme activity.

Coupled KDPG aldolase assay showing native-like enzymatic activity in I3-01. The K129A knockout shows no enzyme activity, similar to buffer alone. UV339, absorbance at 339 nm; error bars are standard deviation.

Related audio

Supplementary information

Supplementary Information

This zipped file contains the protein sequences, the design structure PDB file and an example script. (ZIP 3559 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hsia, Y., Bale, J., Gonen, S. et al. Design of a hyperstable 60-subunit protein icosahedron. Nature 535, 136–139 (2016).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research