Abstract
Cryo-electron microscopy (cryo-EM) single-particle analysis faces significant challenges in resolving the structures of small proteins due to low signal-to-noise ratios and insufficient structural features. Here, we present Trimbody, a simple yet robust method that leverages rigid AI-designed scaffolds to overcome these limitations. Trimbody comprises two components: a trimeric scaffold (H3-PrAC-5350A) and a reformatted nanobody fusion (Nb-TAIL). The de novo 3-helix bundle (H3) and TAIL domains, engineered via RFdiffusion and ProteinMPNN, stabilize the interactions between Nbs and the scaffold and enhance the overall rigidity of the system, thereby enabling high-resolution cryo-EM analysis. Using Trimbody, we resolve the atomic structures of four sub-50 kDa test proteins: human Gal10 (2.62 Å), Aequorea coerulescens GFP (2.29 Å), the IgV domain of human Nectin4 (2.43 Å), and membrane protein Escherichia coli LacY (2.50 Å), demonstrating the method’s versatility. Trimbody’s simplicity, cost-effectiveness, and compatibility with standard cryo-EM workflows position it as a universal tool for structural studies of small proteins, further advancing nanobody-based drug development and life science research.
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Data availability
The cryo-EM density maps generated in this study have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-64015 (hGal10/iTrimbody complex), EMD-63989 (AcGFP/Trimbody complex), EMD-64047 (IgV domain of hNectin4/Trimbody complex), and EMD-67094 (EcLacY10Mu/Trimbody complex). The corresponding atomic coordinates have been deposited in the Protein Data Bank (PDB) under accession codes 9UBR (hGal10/iTrimbody complex), 9UAQ (AcGFP/Trimbody complex), 9UCL (IgV domain of hNectin4/Trimbody complex), and 9XOU (EcLacY10Mu/Trimbody complex). All other published PDB codes cited in this paper are 3OGO, 4JJH, 4FRW, 5GXB, 2V8N, 6I2G, 4W6W, 5C1M, 6P6F, 1WA3, 7RXC, 1HDK and 3LVA. Other data supporting the findings of this study are available within the article and its Supplementary Information. Source data are also provided with this paper. Source data are provided with this paper.
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Acknowledgments
We thank the staff at the Biomedical Research Center for Structural Analysis, Shandong University, for technical assistance during cryo-EM data collection. We also thank all staff members of the Translational Medicine Core Facility of the Advanced Medical Research Institute (AMRI), Shandong University. This work was supported by the National Key Research and Development Program of China (No. 7100, Z.L.), and the National Natural Science Foundation of China (32571436 and 32171207, W.W.; 22377068, X.H.). Additional support was provided by the following: Shandong Provincial Natural Foundation (ZR2024MC199, W.W.), Taishan Scholars Program for Young Experts of Shandong Province (tsqn202408004, W.W.), the Joint Research Grant of Shandong Provincial Third Hospital and Shandong University (GYY202405, W.W.), the Instrument Improvement Fund of Shandong University Public Technology Platform (ts20230203, W.W.), the Innovation Research and Development Special Funds of the Municipality-province-ministry Co-constructed (GJZX-HYSW-2024-02, Z.L.), and the Science and Technology Planning Project of Fujian Province (2022L3022, Z.L.).
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J.S. prepared vectors and protein samples for scaffolds, nanobodies, and target proteins with assistance from Y.L., X.Z., Y.H., Z.Z., and Y.G.; L.Q. and J.S. collected cryo-EM datasets and performed structure determination with assistance from D.H. and M.G.; Z.L. conducted nanobody screening; J.S., X.H., H.F., and W.W. designed nanobody-binding scaffolds and performed computational optimization; J.S. and W.W. built structural models and conducted subsequent analysis; Figures were prepared, and the manuscript was written by W.W. and J.S., with W.W. supervising the entire project.
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The authors declare the following competing interests: Shandong University has filed patent application No. 202512010140.8 relating to the AI-designed scaffold protein constructs (Trimbody) described in this study. W.W., Z.L., and J.S. are listed as inventors on this application. The remaining authors declare no competing interests.
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Song, J., Qi, L., Li, Y. et al. Trimbody with rigid AI-designed scaffolds enables atomic-resolution cryo-EM structure determination of small proteins. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69941-9
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DOI: https://doi.org/10.1038/s41467-026-69941-9
