High-speed high-resolution data collection on a 200 keV cryo-TEM.

Area of Science:

• Structural Biology
• Biophysics
• Microscopy

Background:

• Single-particle cryo-electron microscopy (cryo-EM) is crucial for determining molecular structures.
• Limited microscope time is a significant bottleneck in cryo-EM projects.
• Efficient data collection is essential for accelerating structure determination.

Purpose of the Study:

• To develop and test methodologies for significantly increasing cryo-EM data collection speed.
• To address the limitation of microscope time in single-particle cryo-EM.
• To enable rapid turnover of cryo-EM structure determination.

Main Methods:

• Tested data collection speeds up to 720 movies per hour on a 200 keV Talos Arctica cryo-transmission electron microscope.
• Employed beam-image shift for multiple images per stage, UltrAufoil TEM grids, hardware-binned data, and adjusted SerialEM image shift delay.
• Collected eight EM maps of mouse apoferritin at 1.8-1.9 Å resolution with data collection times from 56 min to 2 hours.

Main Results:

• Achieved high-resolution cryo-EM maps (1.78-1.9 Å) for mouse apoferritin in rapid data collection sessions.
• Demonstrated no significant variation in data quality with image shift distance or delay.
• Successfully obtained a 1.78 Å map from overnight data collection at 500 movies per hour.

Conclusions:

• The developed methodologies significantly enhance cryo-EM data collection efficiency.
• Rapid turnover of single-particle cryo-EM structure determination is achievable.
• These findings provide practical operating procedures for faster cryo-EM analysis.