Bioactive Functionalized Monolayer Graphene for High-Resolution Cryo-Electron Microscopy.

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.