A New Support Film for Cryo Electron Microscopy Protein Structure Analysis Based on Covalently Functionalized Graphene. Issue 8 (11th December 2022)

• Record Type: Journal Article
• Authors: Nickl, Philip; Hilal, Tarek; Olal, Daniel; Donskyi, Ievgen Sergeevitch; Radnik, Jörg; Ludwig, Kai; Haag, Rainer
• Is Part Of: Small. Volume 19:Issue 8(2023)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-12-11
• Subjects: cryogenic electron microscopy -- cryogenic electron tomography -- graphene -- single particle analysis -- support films -- x‐ray photoelectron spectroscopy; Nanotechnology -- Periodicals; Nanoparticles -- Periodicals; Microtechnology -- Periodicals; 620.5
• DOI: 10.1002/smll.202205932
• Languages: English
• ISSNs: 1613-6810

Abstract

Abstract: 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. Abstract : The capability of a simple sample support material based on functionalized graphene sheets, enabling the covalent anchoring of proteins, for high‐resolution structure determination in structural cryogenic electron microscopy (cryoEM) analysis is presented and the successful prevention of protein denaturation and limitation of preferential orientation due to air–water interface adsorption is demonstrated.