Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

Area of Science:

• Structural Biology
• Biophysics
• Microscopy

Background:

• Cryo-electron microscopy (cryo-EM) is a key technique for protein structure determination, increasingly contributing to structural data.
• Advances in transmission electron microscopy (TEM) technology and automation enhance data collection speed and image quality for cryo-EM.
• High-resolution cryo-EM maps are achievable with 200 kV TEM systems, particularly when equipped with an energy filter.

Purpose of the Study:

• To demonstrate the implementation of technological advances and automation on a 200 kV cryo-TEM for routine high-resolution data collection.
• To provide a protocol for collecting cryo-EM data sufficient for de novo atomic model building.
• To detail best practices, critical variables, and common issues for high-resolution cryo-EM dataset generation.

Main Methods:

• Automation of microscope alignments and data collection with real-time image quality assessment.
• Optimization of optical parameters for high-quality, high-throughput data collection.
• Energy filter tuning for zero-loss imaging and selection of suitable areas for data acquisition.

Main Results:

• Demonstration of routine high-resolution cryo-EM data collection using a 200 kV TEM with an energy filter and direct electron detector.
• Successful reconstruction of apo-ferritin to 1.6 Å and Thermoplasma acidophilum 20S proteasome to 2.1 Å resolution.
• Improved achievable resolution and increased yield of high-quality images through automation and energy filtration.

Conclusions:

• Recent technological advancements and automation enable routine high-resolution cryo-EM structure determination on 200 kV systems.
• The use of an energy filter significantly enhances resolution achievable with 200 kV cryo-TEM.
• This protocol facilitates the generation of high-quality cryo-EM datasets for de novo atomic model building.