Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

Area of Science:

• Structural Biology
• Biophysics
• Cryo-Electron Microscopy

Background:

• Capturing short-lived molecular states is crucial but challenging for cryo-electron microscopy (cryo-EM).
• Existing time-resolved methods require optimization for efficiency and sample volume.

Purpose of the Study:

• To present a detailed protocol for the Spotiton system, a novel robotic platform for time-resolved cryo-EM.
• To demonstrate Spotiton's capability in interrogating early structural rearrangements from minimal sample volumes.

Main Methods:

• Spotiton utilizes piezoelectric dispensers for picoliter-sized sample droplet delivery.
• Two samples are rapidly mixed on a nanowire EM grid as it plunges into cryogen.
• The system allows for precise temporal and spatial control over sample mixing and grid preparation.

Main Results:

• The Spotiton workflow enables rapid preparation of hundreds of grids from microliters of sample.
• It offers an efficient approach to study transient molecular interactions.
• The protocol includes troubleshooting guidance for grid preparation.

Conclusions:

• Spotiton provides an efficient and robust method for time-resolved cryo-EM.
• This technique facilitates the study of dynamic molecular processes at high resolution.
• The detailed protocol aids researchers in implementing this advanced cryo-EM methodology.