Dual-Affinity Graphene Sheets for High-Resolution Cryo-Electron Microscopy

Area of Science:

• Structural Biology
• Biophysics
• Materials Science

Background:

• Cryo-electron microscopy (cryo-EM) enables high-resolution macromolecular structure determination.
• Specimen preparation challenges in cryo-EM include protein adsorption at the air-water interface and preferred particle orientation.
• These issues hinder efficient three-dimensional (3D) reconstruction.

Purpose of the Study:

• To develop an improved supporting material for cryo-EM sample preparation.
• To overcome challenges associated with protein adsorption and orientation in vitreous ice.
• To enhance the efficiency and robustness of cryo-EM structural determination.

Main Methods:

• Exploration of dual-affinity graphene (DAG) as a cryo-EM support material.
• DAG is modified with two distinct affinity ligands to bind specific sites on tagged protein particles.
• Comparison of DAG with single-functionalized graphene using protein samples, including SARS-CoV-2 spike glycoprotein.

Main Results:

• DAG demonstrated high binding specificity and affinity for target macromolecules.
• DAG resulted in more balanced particle Euler angular distributions compared to single-functionalized graphene.
• Prevention of protein particle adsorption at the air-water interface was observed.

Conclusions:

• DAG grids offer a robust and generalizable technique for cryo-EM sample preparation.
• This method facilitates facile and efficient 3D reconstruction for structural determination.
• The DAG approach is anticipated to advance future cryo-EM studies.