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| Title | A large, general and modular DARPin-apoferritin scaffold enables the visualization of small proteins by cryo-EM. |
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| Journal, issue, pages | IUCrJ, Vol. 12, Issue Pt 3, Page 393-402, Year 2025 |
| Publish date | May 1, 2025 |
 Authors | Xin Lu / Ming Yan / Yang Cai / Xi Song / Huan Chen / Mengtan Du / Zhenyi Wang / Jia'an Li / Liwen Niu / Fuxing Zeng / Quan Hao / Hongmin Zhang /  |
| PubMed Abstract | Single-particle cryo-electron microscopy (cryo-EM) has emerged as an indispensable technique in structural biology that is pivotal for deciphering protein architectures. However, the medium-sized ...Single-particle cryo-electron microscopy (cryo-EM) has emerged as an indispensable technique in structural biology that is pivotal for deciphering protein architectures. However, the medium-sized proteins (30-40 kDa) that are prevalent in both eukaryotic and prokaryotic organisms often elude the resolving capabilities of contemporary cryo-EM methods. To address this challenge, we engineered a scaffold strategy that securely anchors proteins of interest to a robust, symmetric base via a selective adapter. Our most efficacious constructs, namely models 4 and 6c, feature a designed ankyrin-repeat protein (DARPin) rigidly linked to an octahedral human apoferritin via a helical linker. By utilizing these large, highly symmetric scaffolds (∼1 MDa), we achieved near-atomic-resolution cryo-EM structures of green fluorescent protein (GFP) and maltose-binding protein (MBP), revealing nearly all side-chain densities of GFP and the distinct structural features of MBP. The modular design of our scaffold allows the adaptation of new DARPins through minor amino-acid-sequence modifications, enabling the binding and visualization of a diverse array of proteins. The high symmetry and near-spherical shape of the scaffold not only mitigates the prevalent challenge of preferred particle orientation in cryo-EM but also significantly reduces the demands of image collection and data processing. This approach presents a versatile solution, breaking through the size constraints that have traditionally limited single-particle cryo-EM. |
 External links | IUCrJ / PubMed:40277178 / PubMed Central |
| Methods | EM (single particle) |
| Resolution | 3.0 - 3.47 Å |
| Structure data | EMDB-60822, PDB-9irv: EMDB-60931, PDB-9ivp: EMDB-61130, PDB-9j48: |
| Source |
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 Keywords | BIOSYNTHETIC PROTEIN / DARPin / dimeric / GFP / scaffold / apoferritin / maltose binding protein / METAL BINDING PROTEIN/LUMINESCENT PROTEIN / METAL BINDING PROTEIN-LUMINESCENT PROTEIN complex |
aequorea victoria (jellyfish)
homo sapiens (human)
escherichia coli (E. coli)