Functionalized graphene grids with various charges for single-particle cryo-EM

Area of Science:

• Biophysics
• Materials Science
• Structural Biology

Background:

• Single particle cryo-electron microscopy (cryo-EM) faces challenges in specimen preparation due to the air-water interface (AWI) and particle orientation.
• These issues can lead to lower resolution and incomplete structural determination of biological macromolecules.

Purpose of the Study:

• To develop novel functionalized graphene grids for improved cryo-EM specimen preparation.
• To mitigate the detrimental effects of the air-water interface and control particle orientation during cryo-EM.
• To enhance the robustness and resolution of single particle cryo-EM structural determination.

Main Methods:

• Fabrication of graphene grids using a paraffin-assisted method for reduced contamination.
• Functionalization of graphene grids with various charges via a dediazoniation reaction.
• Application of charged graphene grids for cryo-EM specimen preparation of three different macromolecules.

Main Results:

• Paraffin-assisted fabrication resulted in graphene grids with significantly less contamination compared to polymer transfer methods.
• Charged graphene grids effectively repelled macromolecules from the air-water interface.
• Adjustable particle orientation distribution was achieved, leading to more robust cryo-EM structural determination.
• Successful application across three diverse macromolecule types demonstrated the versatility of the method.

Conclusions:

• Functionalized graphene grids offer a promising solution for overcoming critical specimen preparation hurdles in single particle cryo-EM.
• The developed method enhances macromolecule distribution and orientation control, facilitating higher-resolution structural analysis.
• This advancement has the potential to significantly improve the efficiency and success rate of cryo-EM based structural biology studies.