Lu Xin, Yan Ming, Cai Yang, Song Xi, Chen Huan, Du Mengtan, Wang Zhenyi, Li Jia'an, Niu Liwen, Zeng Fuxing, Hao Quan, Zhang Hongmin
Spallation Neutron Source Science Center, Chinese Academy of Sciences, Dongguan 523000, People's Republic of China.
Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100000, People's Republic of China.
IUCrJ. 2025 May 1;12(Pt 3):393-402. doi: 10.1107/S2052252525003021.
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.
单颗粒冷冻电子显微镜（cryo-EM）已成为结构生物学中不可或缺的技术，对于解析蛋白质结构至关重要。然而，真核生物和原核生物中普遍存在的中等大小蛋白质（30 - 40 kDa）常常超出当代冷冻电子显微镜方法的分辨能力。为应对这一挑战，我们设计了一种支架策略，通过选择性接头将目标蛋白质牢固地锚定到一个坚固、对称的基座上。我们最有效的构建体，即模型4和6c，其特点是通过螺旋接头将设计的锚蛋白重复蛋白（DARPin）与八面体人脱铁铁蛋白刚性连接。通过利用这些大型、高度对称的支架（约1 MDa），我们获得了绿色荧光蛋白（GFP）和麦芽糖结合蛋白（MBP）的近原子分辨率冷冻电子显微镜结构，揭示了GFP几乎所有的侧链密度以及MBP独特的结构特征。我们支架的模块化设计允许通过微小的氨基酸序列修饰来适配新的DARPin，从而能够结合并可视化多种蛋白质。支架的高对称性和近球形形状不仅减轻了冷冻电子显微镜中普遍存在的优先颗粒取向挑战，还显著降低了图像采集和数据处理的要求。这种方法提供了一种通用的解决方案，突破了传统上限制单颗粒冷冻电子显微镜的尺寸限制。
