A data-processing strategy of asymmetric reconstruction for tailed phages by Cryo-electron Microscopy

Area of Science:

• Structural Biology
• Virology
• Biophysics

Background:

• Tailed bacteriophages possess complex structures with icosahedral heads and spiral tails.
• Determining the structure of small proteins within the head and flexible tail proteins remains challenging.
• Existing image-processing methods struggle with the asymmetric structures of phages.

Purpose of the Study:

• To develop an effective data-processing strategy for determining the asymmetric structures of tailed phages.
• To improve the resolution of small proteins in the phage head and flexible tail proteins.
• To validate the strategy using cryo-electron microscopy datasets of diverse tailed phages.

Main Methods:

• Developed a four-module data-processing strategy combining icosahedral reconstruction with local refinement.
• Modules include: icosahedral reconstruction, unique vertex selection, local asymmetric reconstruction/refinement, and local defocus refinement.
• Applied the strategy to cryo-electron microscopy datasets of podophage T7, siphophage T1, and myophage Mu.

Main Results:

• Successfully determined the asymmetric structures of multiple tailed phages.
• Resolved small core and scaffolding proteins within the phage head.
• Characterized flexible proteins on the phage tail with improved resolution.
• Local defocus refinement achieved resolution close to the icosahedral capsid's limit.

Conclusions:

• The proposed strategy is a viable method for determining tailed phage asymmetric structures.
• This approach significantly advances the study of small and flexible phage components.
• Particularly effective for podophages, offering new insights into their structural biology.