Electrospray-assisted cryo-EM sample preparation to mitigate interfacial effects

Area of Science:

• Structural Biology
• Biophysics
• Cryo-Electron Microscopy

Background:

• Interfacial effects during specimen preparation pose challenges in cryogenic electron microscopy (cryo-EM).
• Macromolecule adsorption at interfaces can lead to protein denaturation and preferred orientation artifacts.
• Existing methods struggle to maintain protein integrity and achieve optimal ice thickness for cryo-EM analysis.

Purpose of the Study:

• To introduce ESI-cryoPrep, a novel specimen preparation method for cryo-EM.
• To address challenges of protein denaturation and preferred orientation in cryo-EM sample preparation.
• To improve the quality of cryo-specimens for high-resolution structural analysis of macromolecules.

Main Methods:

• Developed ESI-cryoPrep, a method integrating electrospray ionization (ESI) with native mass spectrometry principles.
• Optimized ESI spraying parameters to control droplet size, charge, and ice thickness.
• Utilized the protective mechanism of charged droplets to prevent biomolecule adsorption at interfaces.

Main Results:

• Successfully prepared high-quality cryo-specimens of five different proteins using ESI-cryoPrep.
• Achieved near-atomic resolution three-dimensional reconstructions for the analyzed proteins.
• Demonstrated effective confinement of macromolecules within amorphous ice, enabling blotting-free sample preparation.

Conclusions:

• ESI-cryoPrep effectively preserves protein integrity and prevents structural damage during cryo-EM specimen preparation.
• The method mitigates preferred orientation issues by preventing biomolecule adsorption at air-water or graphene-water interfaces.
• ESI-cryoPrep facilitates the preparation of high-quality, vitreous samples suitable for high-resolution cryo-EM structural determination.