General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM

Area of Science:

• Structural Biology
• Biophysics
• Materials Science

Background:

• Single-particle cryo-electron microscopy (cryo-EM) requires purified samples for high-resolution structural determination.
• Existing affinity grid strategies for cryo-EM sample preparation have limitations in general utility and efficiency.
• Impure samples often lead to challenges in data collection and analysis, hindering structural studies.

Purpose of the Study:

• To develop a versatile and effective affinity grid strategy for cryo-EM sample preparation.
• To improve the quality of cryo-EM data obtained from crude biological lysates.
• To overcome challenges associated with sample denaturation and preferential orientation in cryo-EM.

Main Methods:

• Chemically functionalizing graphene oxide-coated cryo-EM grids with affinity capture elements.
• Utilizing a protein tagging system with rapid, specific covalent bond formation to a catcher molecule.
• Incorporating a polyethylene glycol (PEG) spacer between the catcher and the grid surface.

Main Results:

• The functionalized grids facilitate rapid affinity capture of proteins of interest from complex mixtures.
• The PEG spacer effectively prevents particle denaturation at the air-water interface and nonspecific binding to the grid.
• High-resolution cryo-EM reconstructions were achieved from significantly cruder samples than previously possible.

Conclusions:

• This novel affinity grid approach enhances cryo-EM sample preparation, enabling high-resolution structural analysis of challenging, impure samples.
• The strategy offers a generalizable solution for improving data quality and reducing preparation time in single-particle cryo-EM.
• The use of PEG spacers is crucial for preserving particle integrity and orientation, leading to more robust structural determination.