A simple and robust procedure for preparing graphene-oxide cryo-EM grids.

Area of Science:

• Biophysics
• Materials Science
• Electron Microscopy

Background:

• Graphene oxide (GO) sheets are valuable supporting substrates in cryogenic electron-microscopy (cryo-EM).
• Previous methods for preparing GO-covered holey carbon EM grids have faced challenges, limiting their widespread application.
• High-resolution imaging of biological macromolecules using cryo-EM requires robust and reliable grid preparation techniques.

Purpose of the Study:

• To develop a simple and robust method for preparing graphene oxide-covered holey carbon EM grids.
• To demonstrate the utility of these GO-covered grids for high-resolution single particle cryo-EM.
• To evaluate the benefits of GO substrates in cryo-EM grid preparation and data analysis.

Main Methods:

• A straightforward protocol was established for coating holey carbon EM grids with graphene oxide sheets.
• The prepared GO-covered grids were utilized for single particle cryo-EM experiments.
• Analysis of cryo-EM data obtained using GO grids was performed to assess image quality and resolution.

Main Results:

• The new method successfully produced GO-covered holey carbon EM grids suitable for cryo-EM.
• GO substrates demonstrated enhanced adherence of macromolecules, enabling grid preparation with lower specimen concentrations.
• The GO lattice provided a high-resolution fiducial marker in micrographs, facilitating the evaluation of beam-induced motion correction.

Conclusions:

• The developed method offers a practical solution for preparing GO-covered EM grids for cryo-EM.
• GO substrates enhance cryo-EM grid preparation by improving sample adherence and offering fiducial markers.
• This technique has the potential to advance high-resolution cryo-EM studies of challenging biological macromolecules.