Download MendeleyA large, general and modular DARPin-apoferritin scaffold enables the visualization of small proteins by cryo-EM.
Lu, X., Yan, M., Cai, Y., Song, X., Chen, H., Du, M., Wang, Z., Li, J., Niu, L., Zeng, F., Hao, Q., Zhang, H.(2025) IUCrJ 12: 393-402
- PubMed: 40277178
- DOI: https://doi.org/10.1107/S2052252525003021
- Primary Citation of Related Structures:
9IRV, 9IVP, 9J48 - 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 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.
Organizational Affiliation:
Spallation Neutron Source Science Center, Chinese Academy of Sciences, Dongguan 523000, People's Republic of China.
