Mix-it-up: Accessible time-resolved cryo-EM on the millisecond timescale

Area of Science:

• Structural Biology
• Biochemistry
• Biophysics

Background:

• Biological macromolecules undergo rapid, substrate-induced conformational changes crucial for function.
• Capturing these transient states is vital for understanding biological mechanisms.
• Traditional methods like X-ray crystallography and cryo-electron microscopy (cryo-EM) struggle with the speed required for these events.

Purpose of the Study:

• To develop a cost-effective and versatile method for rapid on-grid mixing and vitrification for time-resolved cryo-EM.
• To overcome limitations of existing techniques in capturing microsecond-to-millisecond timescale dynamics.
• To enable high-resolution structural determination of transient states in biological reactions.

Main Methods:

• Development of "Mix-it-up" (MIU), a modified spray device for on-grid sample preparation.
• Manual application of one sample, blotting, followed by spraying of a second sample for rapid mixing.
• Achieving vitrification delays as low as approximately 120 milliseconds.
• Utilizing MIU for time-resolved cryo-EM structure determination.

Main Results:

• Demonstrated successful on-grid mixing and vitrification with a minimal delay of ~120 ms.
• Achieved high-resolution structure determination of mixed samples.
• Visualized dynamic processes including pH-induced viral capsid contraction and ligand-dependent complex formation.
• Established MIU as a viable tool for studying rapid biochemical events.

Conclusions:

• The "Mix-it-up" (MIU) device provides a cost-effective and versatile solution for time-resolved cryo-EM.
• MIU significantly reduces the vitrification delay, enabling the capture of transient macromolecular states.
• This method opens new avenues for investigating rapid biological processes at high resolution.