Structural polymorphism of two-dimensional lattices assembled from baculoviral capsid proteins

Kexing Tian; Heya Na; Yan Fu; Tingting Chong; Chao Leng; Fanxing Meng; Yaozhou Liang; Manli Wang; Zhihong Hu; Xi Wang; Guibo Rao; Sheng Cao

doi:10.1016/j.virs.2025.11.005

December 2025

Citation: Kexing Tian, Heya Na, Yan Fu, Tingting Chong, Chao Leng, Fanxing Meng, Yaozhou Liang, Manli Wang, Zhihong Hu, Xi Wang, Guibo Rao, Sheng Cao. Structural polymorphism of two-dimensional lattices assembled from baculoviral capsid proteins .VIROLOGICA SINICA, 2025, 40(6) : 935-945. http://dx.doi.org/10.1016/j.virs.2025.11.005

Structural polymorphism of two-dimensional lattices assembled from baculoviral capsid proteins

Kexing Tian ^a,b ,
Heya Na ^a,b ,
Yan Fu ^a ,
Tingting Chong ^a,b ,
Chao Leng ^a,b ,
Fanxing Meng ^a,b ,
Yaozhou Liang ^a,b ,
Manli Wang ^a ,
Zhihong Hu ^a ,
Xi Wang ^{a
,,} ,
Guibo Rao ^{a
,,} ,
Sheng Cao ^{a
,,}

a. State Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China;
b. University of Chinese Academy of Sciences, Beijing 100049, China

Corresponding author: Xi Wang, wangxi@wh.iov.cn
Guibo Rao, raoguibo@wh.iov.cn
Sheng Cao, caosheng@wh.iov.cn
Received Date: 25 July 2025
Accepted Date: 14 November 2025
Available online: 19 November 2025

Abstract

Protein nanotubes (PNTs) can be regarded as two-dimensional (2D) lattices with p1 or p2 symmetry rolled into tubes. However, attempts to re-assemble their building blocks into stable 2D nanomaterials often fail. Here, starting from two baculoviral capsid proteins, we screened protein variants for the in vitro assembly of various nanotubes and nanosheets. These high-order assemblies were structurally characterized by cryo-electron microscopy techniques. Interfacial analysis of three groups of PNTs revealed that helical heterogeneity is largely the result of the redundancy of p2 symmetry-related contacting interfaces. The assembled nanosheets showed similar interfacial networks to their nanotubular counterparts. In addition, foreign macromolecules could be efficiently displayed on the size-controllable double-layered nanosheets. This study sheds light on the rational design of flexible nanosheets, and it also provides novel 2D protein scaffolds for developing biocompatible materials.
- Baculovirus
- , Nanotube
- , Nanosheet
- , Cryo-electron microscopy
- , Helical symmetry

Electronic Supplementary Material

10.1016j.virs.2025.11.005-ESM1.docx
References
1. Afonine, P.V., Poon, B.K., Read, R.J., Sobolev, O.V., Terwilliger, T.C., Urzhumtsev, A.,Adams, P.D., 2018. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr D Struct Biol, 74, 531-544.
2. Audette, G.F., Yaseen, A., Bragagnolo, N.,Bawa, R., 2019. Protein Nanotubes: From Bionanotech towards Medical Applications. Biomedicines, 7, 46.
3. Baneyx, F.,Matthaei, J.F., 2014. Self-assembled two-dimensional protein arrays in bionanotechnology: from S-layers to designed lattices. Curr Opin Biotechnol, 28, 39-45.
4. Ben-Sasson, A.J., Watson, J.L., Sheffler, W., Johnson, M.C., Bittleston, A., Somasundaram, L., Decarreau, J., Jiao, F., Chen, J., Mela, I., et al., 2021. Design of biologically active binary protein 2D materials. Nature, 589, 468-473.
5. Benning, F.M.C., Jenni, S., Garcia, C.Y., Nguyen, T.H., Zhang, X.,Chao, L.H., 2024. Helical reconstruction of VP39 reveals principles for baculovirus nucleocapsid assembly. Nat Commun, 15, 250.
6. Burt, A., Gaifas, L., Dendooven, T.,Gutsche, I., 2021. A flexible framework for multi-particle refinement in cryo-electron tomography. PLoS Biol, 19, e3001319.
7. Chen, V.B., Arendall, W.B., 3rd, Headd, J.J., Keedy, D.A., Immormino, R.M., Kapral, G.J., Murray, L.W., Richardson, J.S.,Richardson, D.C., 2010. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr, 66, 12-21.
8. Eber, F.J., Eiben, S., Jeske, H.,Wege, C., 2015. RNA-controlled assembly of tobacco mosaic virus-derived complex structures: from nanoboomerangs to tetrapods. Nanoscale, 7, 344-355.
9. Eisenstein, F., Yanagisawa, H., Kashihara, H., Kikkawa, M., Tsukita, S.,Danev, R., 2023. Parallel cryo electron tomography on in situ lamellae. Nat Methods, 20, 131-138.
10. Emsley, P., Lohkamp, B., Scott, W.G.,Cowtan, K., 2010. Features and development of Coot. Acta Crystallogr D Biol Crystallogr, 66, 486-501.
11. Fagan, R.P.,Fairweather, N.F., 2014. Biogenesis and functions of bacterial S-layers. Nat Rev Microbiol, 12, 211-222.
12. Goddard, T.D., Huang, C.C., Meng, E.C., Pettersen, E.F., Couch, G.S., Morris, J.H.,Ferrin, T.E., 2018. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci, 27, 14-25.
13. Gonen, S., Dimaio, F., Gonen, T.,Baker, D., 2015. Design of ordered two-dimensional arrays mediated by noncovalent protein-protein interfaces. Science, 348, 1365-1368.
14. Hamley, I.W., 2019. Protein Assemblies: Nature-Inspired and Designed Nanostructures. Biomacromolecules, 20, 1829-1848.
15. Hatlem, D., Trunk, T., Linke, D.,Leo, J.C., 2019. Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins. Int J Mol Sci, 20, 2129.
16. He, S.D.,Scheres, S.H.W., 2017. Helical reconstruction in RELION. Journal of Structural Biology, 198, 163-176.
17. Henderson, R.,Unwin, P.N., 1975. Three-dimensional model of purple membrane obtained by electron microscopy. Nature, 257, 28-32.
18. Jenni, S., Horwitz, J.A., Bloyet, L.M., Whelan, S.P.J.,Harrison, S.C., 2022. Visualizing molecular interactions that determine assembly of a bullet-shaped vesicular stomatitis virus particle. Nat Commun, 13, 4802.
19. Jia, X., Gao, Y., Huang, Y., Sun, L., Li, S., Li, H., Zhang, X., Li, Y., He, J., Wu, W., Venkannagari, H., Yang, K., Baker, M.L.,Zhang, Q., 2023. Architecture of the baculovirus nucleocapsid revealed by cryo-EM. Nat Commun, 14, 7481.
20. Klug, A., 1999. The tobacco mosaic virus particle: structure and assembly. Philos Trans R Soc Lond B Biol Sci, 354, 531-535.
21. Koch, C., Eber, F.J., Azucena, C., Forste, A., Walheim, S., Schimmel, T., Bittner, A.M., Jeske, H., Gliemann, H., Eiben, S., Geiger, F.C.,Wege, C., 2016. Novel roles for well-known players: from tobacco mosaic virus pests to enzymatically active assemblies. Beilstein J Nanotechnol, 7, 613-629.
22. Krissinel, E.,Henrick, K., 2007. Inference of macromolecular assemblies from crystalline state. J Mol Biol, 372, 774-797.
23. Li, L., Fierer, J.O., Rapoport, T.A.,Howarth, M., 2014. Structural analysis and optimization of the covalent association between SpyCatcher and a peptide Tag. J Mol Biol, 426, 309-317.
24. Li, N., Rao, G., Li, Z., Yin, J., Chong, T., Tian, K., Fu, Y.,Cao, S., 2022. Cryo-EM structure of glycoprotein C from Crimean-Congo hemorrhagic fever virus. Virol Sin, 37, 127-137.
25. Liu, Z., Qiao, J., Niu, Z.,Wang, Q., 2012. Natural supramolecular building blocks: from virus coat proteins to viral nanoparticles. Chem Soc Rev, 41, 6178-6194.
26. Ludtke, S.J., 2016. Single-Particle Refinement and Variability Analysis in EMAN2.1. Methods Enzymol, 579, 159-189.
27. Mastronarde, D.N., 2005. Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol, 152, 36-51.
28. Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C.,Ferrin, T.E., 2004. UCSF chimera - A visualization system for exploratory research and analysis. Journal of Computational Chemistry, 25, 1605-1612.
29. Punjani, A., Rubinstein, J.L., Fleet, D.J.,Brubaker, M.A., 2017. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods, 14, 290-296.
30. Rao, G., Fu, Y., Li, N., Yin, J., Zhang, J., Wang, M., Hu, Z.,Cao, S., 2018. Controllable Assembly of Flexible Protein Nanotubes for Loading Multifunctional Modules. ACS Appl Mater Interfaces, 10, 25135-25145.
31. Scaramuzza, S.,Castano-Diez, D., 2021. Step-by-step guide to efficient subtomogram averaging of virus-like particles with Dynamo. PLoS Biol, 19, e3001318.
32. Schneider, C.A., Rasband, W.S.,Eliceiri, K.W., 2012. NIH Image to ImageJ: 25 years of image analysis. Nat Methods, 9, 671-675.
33. Sleytr, U.B., Schuster, B., Egelseer, E.M.,Pum, D., 2014. S-layers: principles and applications. FEMS Microbiol Rev, 38, 823-864.
34. Speranza, G., 2021. Carbon Nanomaterials: Synthesis, Functionalization and Sensing Applications. Nanomaterials (Basel), 11.
35. Suzuki, Y., Cardone, G., Restrepo, D., Zavattieri, P.D., Baker, T.S.,Tezcan, F.A., 2016. Self-assembly of coherently dynamic, auxetic, two-dimensional protein crystals. Nature, 533, 369-373.
36. Tegunov, D.,Cramer, P., 2019. Real-time cryo-electron microscopy data preprocessing with Warp. Nat Methods, 16, 1146-1152.
37. Tsai, C.J.,Nussinov, R., 2011. A unified convention for biological assemblies with helical symmetry. Acta Crystallogr D Biol Crystallogr, 67, 716-728.
38. Wen, A.M.,Steinmetz, N.F., 2016. Design of virus-based nanomaterials for medicine, biotechnology, and energy. Chem Soc Rev, 45, 4074-4126.
39. Wukovitz, S.W.,Yeates, T.O., 1995. Why protein crystals favour some space-groups over others. Nat Struct Biol, 2, 1062-1067.
40. Yeates, T.O., 2017. Geometric Principles for Designing Highly Symmetric Self-Assembling Protein Nanomaterials. Annu Rev Biophys, 46, 23-42.
41. Zhang, S., Alberstein, R.G., De Yoreo, J.J.,Tezcan, F.A., 2020. Assembly of a patchy protein into variable 2D lattices via tunable multiscale interactions. Nat Commun, 11, 3770.
42. Zhou, K., Si, Z., Ge, P., Tsao, J., Luo, M.,Zhou, Z.H., 2022. Atomic model of vesicular stomatitis virus and mechanism of assembly. Nat Commun, 13, 5980.
43. Zivanov, J., Nakane, T., Forsberg, B.O., Kimanius, D., Hagen, W.J., Lindahl, E.,Scheres, S.H., 2018. New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife, 7, e42166.
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