Challenges in making ideal cryo-EM samples

Area of Science:

• Structural Biology
• Biophysics
• Electron Microscopy

Background:

• The air-water interface (AWI) presents a significant challenge in cryo-electron microscopy (cryo-EM) sample preparation.
• Uncontrolled interaction with the AWI can lead to particle denaturation and aggregation, compromising structural integrity.
• Both single-particle cryo-EM and cryo-electron tomography (cryo-ET) are negatively impacted by AWI effects.

Purpose of the Study:

• To review current methodologies for mitigating air-water interface (AWI) issues in cryo-EM.
• To highlight promising techniques for sample preparation, focusing on particle immobilization and thickness control.
• To explore future applications of prepared samples in time-resolved biochemical assays.

Main Methods:

• Review of existing cryo-EM sample preparation techniques.
• Focus on particle immobilization strategies, particularly using affinity grids.
• Analysis of methods for achieving precise control over sample thickness to prevent AWI contact.

Main Results:

• Particle immobilization on affinity grids is identified as a highly promising approach to avoid AWI.
• Effective control of sample thickness is crucial for preventing immobilized particles from contacting residual buffer AWI.
• The importance of AWI avoidance is underscored for both single-particle cryo-EM and cryo-ET.

Conclusions:

• Avoiding the air-water interface is critical for successful cryo-EM and cryo-ET.
• Immobilization techniques and precise thickness control are key strategies for high-quality cryo-EM data.
• Future directions include using immobilized samples for in-situ time-resolved biochemical experiments on EM grids.