A New Support Film for Cryo Electron Microscopy Protein Structure Analysis Based on Covalently Functionalized Graphene

Protein adsorption at the air-water interface is a serious problem in cryogenic electron microscopy (cryoEM) as it restricts particle orientations in the vitrified ice-film and promotes protein denaturation. To address this issue, the preparation of a graphene-based modified support film for coverage of conventional holey carbon transmission electron microscopy (TEM) grids is presented. The chemical modification of graphene sheets enables the universal covalent anchoring of unmodified proteins via inherent surface-exposed lysine or cysteine residues in a one-step reaction. Langmuir-Blodgett (LB) trough approach is applied for deposition of functionalized graphene sheets onto commercially available holey carbon TEM grids. The application of the modified TEM grids in single particle analysis (SPA) shows high protein binding to the surface of the graphene-based support film. Suitability for high resolution structure determination is confirmed by SPA of apoferritin. Prevention of protein denaturation at the air-water interface and improvement of particle orientations is shown using human 20S proteasome, demonstrating the potential of the support film for structural biology.

Automated summary

This study presents a method for preparing a graphene-based modified support film to address the problem of protein adsorption at the air-water interface in cryoEM. The chemical modification of graphene sheets allows for the covalent anchoring of unmodified proteins, which improves particle orientations and prevents protein denaturation. The application of the modified support film in single particle analysis demonstrates its potential for high resolution structure determination.