Through-grid wicking enables high-speed cryoEM specimen preparation

Area of Science:

• Structural Biology
• Biophysics
• Biochemistry

Background:

• Conventional cryo-electron microscopy (cryoEM) specimen preparation involves lengthy blotting steps.
• These blotting times can lead to specimen denaturation and preferred orientations, hindering time-resolved studies and structural analysis.
• Developing rapid and efficient specimen preparation methods is crucial for advancing cryoEM applications.

Purpose of the Study:

• To develop a novel, high-speed cryoEM specimen preparation technique.
• To overcome limitations of conventional blotting, such as denaturation and preferred orientations.
• To enable improved data collection for time-resolved cryoEM studies.

Main Methods:

• A new method involving spraying solution onto a holey cryoEM grid and wicking it through with a glass-fiber filter was developed.
• This through-grid wicking process was integrated into a high-speed device named 'Back-it-up' (BIU).
• The BIU device combines ultrasonic specimen application and through-grid wicking for rapid film formation.

Main Results:

• The through-grid wicking method can prepare specimens suitable for vitrification in tens of milliseconds.
• This technique produces large areas of high-quality vitreous ice for both soluble and detergent-solubilized protein complexes.
• The method is relatively insensitive to sample volume due to the high absorption capacity of the glass fiber.

Conclusions:

• The BIU device offers a significantly faster alternative to conventional cryoEM specimen preparation.
• This rapid preparation method mitigates issues like preferred orientation and denaturation, enhancing cryoEM data acquisition.
• The technique is broadly applicable to various biological macromolecules, advancing structural biology research.