Collaborative Innovation Center of Quantum Matter , Beijing 100871 , China.
J Am Chem Soc. 2019 Mar 6;141(9):4016-4025. doi: 10.1021/jacs.8b13038. Epub 2019 Feb 20.
Single-particle cryo-electron microscopy (cryo-EM) has become one of the most essential tools to understand biological mechanisms at molecular level. A major bottleneck in cryo-EM technique is the preparation of good specimens that embed biological macromolecules in a thin layer of vitreous ice. In the canonical cryo-EM specimen preparation method, biological macromolecules tend to be adsorbed to the air-water interface, causing partial denaturation and/or preferential orientations. In this work, we have designed and produced a new type of cryo-EM grids using bioactive-ligand functionalized single-crystalline monolayer graphene membranes as supporting films. The functionalized graphene membrane (FGM) grids exhibit specific binding affinity to histidine (His)-tagged proteins and complexes. In cryo-EM, the FGM grids generate relatively low background for imaging and selectively anchor 20S proteasomes to the supporting film surface, enabling near-atomic-resolution 3D reconstruction of the complex. We envision that the FGM grids could benefit single particle cryo-EM specimen preparation with high reproducibility and robustness, therefore enhancing the efficiency and throughput of high-resolution cryo-EM structural determination.
单颗粒冷冻电子显微镜（cryo-EM）已成为理解分子水平生物机制的最重要工具之一。cryo-EM 技术的一个主要瓶颈是制备良好的样品，将生物大分子嵌入薄的玻璃态冰层中。在典型的 cryo-EM 样品制备方法中，生物大分子容易被吸附到气-水界面，导致部分变性和/或优先取向。在这项工作中，我们设计并制作了一种新型的 cryo-EM 网格，使用生物活性配体功能化的单晶单层石墨烯膜作为支撑膜。功能化石墨烯膜（FGM）网格对组氨酸（His）标记的蛋白质和复合物具有特定的结合亲和力。在 cryo-EM 中，FGM 网格产生相对较低的背景用于成像，并选择性地将 20S 蛋白酶体锚定到支撑膜表面，从而能够对复合物进行近原子分辨率的 3D 重构。我们设想 FGM 网格可以通过高重现性和稳健性受益于单颗粒 cryo-EM 样品制备，从而提高高分辨率 cryo-EM 结构测定的效率和通量。
