Addressing preferred specimen orientation in single-particle cryo-EM through tilting

Area of Science:

• Structural Biology
• Biophysics
• Biochemistry

Background:

• Preferred specimen orientation is a major challenge in single-particle cryo-electron microscopy (cryo-EM).
• This orientation bias limits the achievable resolution and structural detail in the final 3D reconstruction.
• Overcoming this is crucial for determining the structures of many important biological macromolecules.

Purpose of the Study:

• To present a novel strategy for addressing preferred specimen orientation in cryo-EM.
• To introduce a computational tool for quantifying directional resolution.
• To demonstrate the application of these methods on biologically relevant samples.

Main Methods:

• Implementing specimen tilts during data collection in cryo-EM.
• Utilizing 3D Fourier Shell Correlation (FSC) volumes to assess directional resolution.
• Applying the strategy to influenza hemagglutinin and ribosomal biogenesis intermediates.

Main Results:

• Successfully determined near-atomic resolution structures of influenza hemagglutinin trimer, a protein with highly preferred orientation.
• Achieved high-resolution structures for ribosomal biogenesis intermediates with moderately preferred orientations.
• Demonstrated the effectiveness of tilt-based data collection and 3D FSC analysis.

Conclusions:

• The presented tilt strategy effectively mitigates preferred specimen orientation in cryo-EM.
• The developed tool provides a reliable measure of directional resolution.
• This approach significantly advances the capability of cryo-EM for solving complex molecular structures.