Laser flash melting cryo-EM samples to overcome preferred orientation.

Area of Science:

• Structural Biology
• Biophysics
• Biochemistry

Background:

• Sample preparation is a critical bottleneck in cryo-electron microscopy (cryo-EM) for protein structure determination.
• Proteins often adsorb to the air-water interface, leading to preferred orientations that hinder high-resolution reconstruction.
• This preferred orientation issue can cause cryo-EM projects to fail.

Purpose of the Study:

• To demonstrate that laser flash melting and revitrification can scramble 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 revitrification to cryo-electron microscopy (cryo-EM) samples.
• Systematic variation of heating rates during flash melting.
• Deposition of amorphous ice onto samples prior to revitrification.

Main Results:

• Laser flash melting and revitrification effectively scramble the orientation of proteins of diverse sizes and symmetries.
• Increasing heating rates during flash melting enhanced the scrambling effect for certain proteins.
• Pre-depositing amorphous ice further improved orientation scrambling for some proteins.
• Experiments provided insights into the mechanism behind orientation scrambling.

Conclusions:

• Laser flash melting and revitrification offer a versatile toolkit for overcoming preferred orientation in cryo-EM sample preparation.
• These methods are easily integrated into existing cryo-EM workflows.
• The developed tools facilitate improved high-resolution structure determination by reducing orientation bias.