Collection, pre-processing and on-the-fly analysis of data for high-resolution, single-particle cryo-electron...

Area of Science:

• Structural Biology
• Biophysics
• Biochemistry

Background:

• Cryo-electron microscopy (cryo-EM) is revolutionizing structural biology, enabling high-resolution macromolecular complex structure determination.
• Titan Krios microscopes are predominantly used for cryo-EM data collection, with structures deposited in the Electron Microscopy Data Bank (EMDB).
• Current single-particle cryo-EM data collection workflows exhibit variability in setup and data handling, impacting efficiency.

Purpose of the Study:

• To present a standardized operating procedure for cryo-EM single-particle data collection.
• To provide a strategy for efficient data pre-processing and real-time monitoring during data acquisition.
• To optimize the use of Thermo Fisher Scientific EPU software with common direct electron detectors.

Main Methods:

• Development of a standard operating procedure for automated data collection using Thermo Fisher Scientific EPU software.
• Integration of two common direct electron detectors: Thermo Fisher Scientific Falcon 3 (F3EC) and Gatan K2.
• Implementation of a data structuring strategy for streamlined pre-processing and on-the-fly monitoring.

Main Results:

• The presented protocol significantly reduces variability in cryo-EM data collection setup.
• The procedure enables efficient data management, facilitating rapid pre-processing and quality control.
• A typical automated data collection session setup can be completed within 3-6 hours.

Conclusions:

• Standardized cryo-EM data collection protocols enhance reproducibility and efficiency.
• The developed procedure facilitates high-resolution structure determination through optimized data acquisition and management.
• This approach supports the growing demand for structural data in biological research.