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

TLDR

Single‑particle cryo‑electron microscopy is a key tool for molecular biology, but specimen preparation is limited by the tendency of macromolecules to adsorb to the air–water interface, causing denaturation and preferential orientation. The study aims to design and produce new cryo‑EM grids that incorporate bioactive‑ligand functionalized single‑crystalline monolayer graphene membranes. These grids use the functionalized graphene membranes as supporting films to bind macromolecules through specific ligand interactions. The functionalized graphene grids bind His‑tagged proteins, generate low background, selectively anchor 20S proteasomes, and enable near‑atomic‑resolution 3D reconstruction, promising higher reproducibility and throughput in cryo‑EM.

Abstract

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.