Single Particle Cryo-Electron Microscopy: From Sample to Structure

Area of Science:

• Structural Biology
• Biophysics
• Biochemistry

Background:

• Cryo-electron microscopy (cryoEM) is essential for determining the structures of macromolecular complexes.
• Single particle analysis (SPA) is a key cryoEM method for high-resolution structure determination.
• Efficient cryoEM workflows are crucial for advancing structural biology.

Purpose of the Study:

• To provide a comprehensive protocol for cryo-electron microscopy single particle analysis.
• To highlight key variables and troubleshooting for each step of the cryoEM workflow.
• To describe variations for remote microscope operation and limited physical access.

Main Methods:

• Specimen vitrification on cryoEM grids.
• Specimen screening for particle distribution and ice quality assessment.
• Single particle dataset collection and image processing to generate EM density maps.

Main Results:

• An overview of the cryoEM SPA workflow, from sample preparation to data analysis.
• Identification of modifiable parameters and common issues encountered during cryoEM.
• Adaptations for efficient remote operation and imaging.

Conclusions:

• This protocol offers a practical guide to cryoEM SPA for researchers.
• Understanding workflow variables and troubleshooting aids in successful structure determination.
• Remote operation protocols enhance accessibility and efficiency in cryoEM facilities.