Cryo-EM performance testing of hardware and data acquisition strategies

Area of Science:

• Structural Biology
• Biophysics
• Microscopy

Background:

• Cryo-electron microscopy (cryo-EM) adoption is rapidly increasing globally.
• New cryo-EM installations require rigorous testing to ensure optimal performance and productivity.
• Limited user experience can impact confidence in equipment capabilities.

Purpose of the Study:

• To present a standardized testing methodology for new cryo-electron microscopy (cryo-EM) hardware and data acquisition strategies.
• To evaluate the performance of a new Krios G4 electron microscope with Gatan K3 and Falcon 4 direct electron detectors.
• To assess different data acquisition strategies and the impact of environmental factors on data quality.

Main Methods:

• Utilized apoferritin as a standard test sample for initial cryo-EM workflow validation.
• Tested a Krios G4 electron microscope equipped with Gatan K3 and Falcon 4 direct electron detector cameras.
• Evaluated three beam-image shift-based data acquisition strategies, including single- and multi-shot per-hole schemes.

Main Results:

• Both Gatan K3 and Falcon 4 cameras demonstrated comparable performance.
• Single- and multi-shot per-hole acquisition strategies yielded similar results.
• Confirmed the effectiveness of Relion's software aberration correction in managing severe coma aberration.

Conclusions:

• Apoferritin is a suitable test sample for validating cryo-EM workflows and new hardware.
• The tested Krios G4 system and acquisition strategies perform reliably.
• This work provides a framework for cryo-EM teams to test and troubleshoot their equipment and data collection methods.