`Mix-it-up': accessible time-resolved cryo-EM on the millisecond timescale

Area of Science:

• Structural Biology
• Biochemistry
• Cryo-electron Microscopy (cryo-EM)

Background:

• Biological macromolecules undergo rapid, substrate-induced conformational changes crucial for function.
• Traditional structural methods (X-ray crystallography, cryo-EM) struggle to capture these transient states due to slow sample preparation.
• Time-resolved cryo-EM offers potential for visualizing microsecond-to-millisecond dynamics but faces technical hurdles in sample handling and cost.

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 fast biochemical events.
• 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 cryo-EM sample preparation.
• Manual application of one sample, blotting, and subsequent spraying of a second sample onto the EM grid for on-grid mixing.
• Vitrification achieved with a delay as low as approximately 120 milliseconds.

Main Results:

• Demonstrated successful on-grid mixing and vitrification with a significantly reduced delay time.
• Achieved high-resolution structure determination of mixed samples, showcasing MIU's capability.
• Visualized dynamic processes including pH-induced viral capsid contraction and ligand-dependent complex formation.

Conclusions:

• The Mix-it-up (MIU) device provides a cost-effective and versatile solution for rapid sample preparation in cryo-EM.
• MIU facilitates the study of rapid biochemical processes and transient conformational changes.
• This approach lays the foundation for broader applications of time-resolved cryo-EM in structural biology.