Thicker Ice Improves the Integrity and Angular Distribution of CDC48A Hexamers on Cryo-EM Grids

Area of Science:

• Structural Biology
• Biophysics
• Biochemistry

Background:

• Sample preparation is a critical bottleneck in cryogenic electron microscopy (cryo-EM) single particle analysis.
• Issues like particle aggregation, dissociation, and preferential orientation often hinder high-resolution structure determination.
• CDC48A, a hexameric AAA ATPase from Arabidopsis thaliana, presents challenges due to particle instability during grid freezing.

Purpose of the Study:

• To overcome sample preparation challenges in cryo-EM for difficult protein targets.
• To optimize cryo-EM data acquisition for improved particle integrity, orientation, and resolution.
• To develop a generally applicable strategy for high-quality cryo-EM data collection.

Main Methods:

• Improved purification protocol for CDC48A to enhance hexamer stability.
• Comparison of classical blotting and blot-free Chameleon methods for vitrification.
• Iterative data acquisition across varying ice thicknesses to correlate particle distribution, orientation, and image resolution.
• Optimization of cryo-EM data acquisition conditions.

Main Results:

• Classical blotting led to CDC48A disassembly, while the Chameleon method caused preferential orientation.
• Thicker ice improved particle distribution and orientation but reduced image resolution.
• An intermediate ice thickness was identified that balanced particle integrity, random orientation, and high-resolution data.
• Improved purification yielded more homogeneous and stable CDC48A hexamers.

Conclusions:

• A combined strategy of improved protein purification and iterative cryo-EM data acquisition across ice thicknesses can overcome common sample preparation limitations.
• This approach yields high-resolution structural information with a complete distribution of particle orientations.
• The method is simple, fast, generally applicable, and valuable when resources are limited.