Step-by-step guide to efficient subtomogram averaging of virus-like particles with Dynamo

Area of Science:

• Structural Biology
• Biophysics
• Computational Biology

Background:

• Subtomogram averaging (STA) is crucial for determining 3D structures of macromolecular complexes.
• Existing STA protocols often lack practical detail, hindering reproducibility.
• The computational complexity of STA requires standardized, accessible workflows.

Purpose of the Study:

• To provide a comprehensive and reproducible processing protocol for subtomogram averaging.
• To address the lack of detailed guides and practical documentation in STA.
• To facilitate the growth and reproducibility of structural biology research using STA.

Main Methods:

• A command line-based workflow utilizing the Dynamo software package for STA.
• Detailed procedures for particle picking and particle alignment within the STA process.
• Demonstration of streamlining and automation for increased processing throughput.

Main Results:

• A complete, detailed, and fully reproducible STA processing protocol was developed.
• The protocol covers all essential aspects of particle picking and alignment.
• The workflow was successfully validated using HIV-1 virus-like particles from the EMPIAR database.

Conclusions:

• The provided Dynamo-based protocol enhances the reproducibility of subtomogram averaging.
• The streamlined workflow supports users of all levels and can be adapted for specific projects.
• This resource is expected to advance structural biology research by improving STA accessibility and standardization.