Sub-3 Å resolution protein structure determination by single-particle cryo-EM at 100 keV

Area of Science:

• Structural Biology
• Microscopy Techniques
• Biophysics

Background:

• Cryoelectron microscopy (cryo-EM) is crucial for high-resolution structural biology.
• Advancements in electron microscopy hardware are needed to improve accessibility and data quality.

Purpose of the Study:

• To evaluate the performance of a 100 keV cryo-transmission electron microscope (cryo-TEM) system for high-resolution structure determination.
• To demonstrate the utility of the Falcon C direct electron detector (DED) in achieving sub-3 Å resolution.

Main Methods:

• Utilized a 100 keV Tundra cryo-TEM equipped with an extreme-brightness field emission gun (XFEG) and SP-TWIN lens.
• Employed the Falcon C direct electron detector (DED) with high detective quantum efficiency (DQE).
• Collected and processed cryo-EM data for various biological macromolecules.

Main Results:

• Achieved sub-3 Å resolution structures for multiple biological samples, including apoferritin (2.1 Å), T20S proteasome (2.7 Å), and GABA_A receptor (2.8 Å).
• Demonstrated successful structure determination for proteins ranging from 50 kDa to 3.9 MDa.
• Validated the system's capability for high-resolution protein structure determination at lower voltages.

Conclusions:

• The 100 keV cryo-TEM system with the Falcon C DED enables high-resolution cryo-EM structure determination.
• Lower voltage TEMs can be utilized for both screening and high-resolution structural studies, increasing accessibility.
• This technology broadens the application of cryo-EM in structural biology.