Laser Flash Melting Cryo-EM Samples to Overcome Preferred Orientation

Area of Science:

• Structural Biology
• Biophysics
• Biochemistry

Background:

• Protein adsorption to the air-water interface during sample preparation is a major challenge in cryo-electron microscopy (cryo-EM).
• This preferred orientation limits the ability to obtain high-resolution structures and can lead to project failure.
• Previous work showed laser flash melting and revitrification reduced preferred orientation for large, symmetric particles.

Purpose of the Study:

• To demonstrate the broad applicability of laser flash melting and revitrification for scrambling protein orientation across various sizes and symmetries.
• To investigate methods for enhancing the orientation scrambling effect.
• To elucidate the underlying mechanism of orientation scrambling.

Main Methods:

• Application of laser flash melting and subsequent revitrification to cryo-EM samples containing proteins of diverse characteristics.
• Systematic variation of laser heating rates during flash melting.
• Deposition of amorphous ice onto samples prior to revitrification.
• Analysis of resulting cryo-EM data to assess particle orientation distribution.

Main Results:

• The laser flash melting and revitrification method successfully scrambled the orientation of proteins across a range of sizes and symmetries.
• Increasing the heating rate during flash melting enhanced the scrambling effect for certain proteins.
• Pre-deposition of amorphous ice also improved orientation scrambling for some samples.
• These modifications provide insights into the mechanism of orientation scrambling.

Conclusions:

• Laser flash melting and revitrification is a versatile tool for overcoming preferred orientation in cryo-EM sample preparation.
• Adjustable parameters like heating rate and amorphous ice deposition offer further optimization.
• These techniques can be readily integrated into existing cryo-EM workflows to improve data quality and success rates.