Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM.

Area of Science:

• Structural biology
• Biophysics
• Microscopy

Background:

• Single-particle cryo-electron microscopy (cryo-EM) excels with large, high-symmetry viruses.
• Resolving smaller proteins with lower symmetry using cryo-EM remains a significant challenge.
• Electron beam-induced motion is a major factor limiting resolution in cryo-EM imaging.

Purpose of the Study:

• To overcome resolution limitations in cryo-EM for smaller, low-symmetry protein structures.
• To demonstrate the effectiveness of a novel electron-counting detector system.
• To improve image quality and data acquisition efficiency for broader cryo-EM applications.

Main Methods:

• Utilized a new single electron-counting detector with rapid readout.
• Implemented subpixel accuracy correction for electron beam-induced motion.
• Determined the structure of the Thermoplasma acidophilum 20S proteasome.

Main Results:

• Confirmed electron beam-induced motion significantly degrades resolution.
• Achieved subpixel motion correction, restoring high-resolution image information (Thon rings visible to ~3 Å).
• Determined a 3.3 Å resolution structure of the 700-kDa Thermoplasma acidophilum 20S proteasome, revealing clear side-chain density.

Conclusions:

• The developed method significantly enhances cryo-EM image quality and data acquisition.
• This advancement facilitates near-atomic-resolution cryo-EM for a wider range of protein samples.
• Enables detailed structural analysis of smaller, low-symmetry proteins previously difficult to resolve.