Customising the plunge-freezing workflow for challenging conditions

Area of Science:

• Structural Biology
• Biophysics
• Materials Science

Background:

• Cryo-transmission electron microscopy (cryo-TEM) requires optimal grid freezing for high-resolution structure determination.
• Modern plunge-freezers incorporate temperature and humidity control, typically using cold (4°C) humid conditions.
• Standard conditions may not suit all biological or polymeric samples, presenting a bottleneck in cryo-EM workflows.

Purpose of the Study:

• To describe modifications to a Leica GP2 plunge-freezer for accommodating samples with unique handling requirements.
• To present methods for plunge-freezing sensitive proteins and temperature-dependent gelling polymers.
• To offer solutions for improving cryo-EM sample preparation reproducibility for challenging specimens.

Main Methods:

• Adaptations were made to a Leica GP2 plunge-freezer system.
• Methods were developed for plunge-freezing proteins sensitive to light and oxygen.
• Protocols were established for maintaining samples at 37°C during plunge-freezing and for freezing a polymer at its gelling temperature.

Main Results:

• Successful plunge-freezing protocols were established for samples with specific environmental needs.
• The adaptations allowed for the preservation of sample integrity under non-standard conditions.
• The modified system demonstrated flexibility in handling diverse sample types.

Conclusions:

• Standard plunge-freezing conditions are not universally applicable for cryo-EM sample preparation.
• System modifications can overcome challenges posed by sensitive or temperature-dependent samples.
• Sharing these adaptable methods can aid researchers in optimizing cryo-EM grid preparation for difficult specimens.