Purification and Cryo-Electron Microscopy Analysis of Bacterial Appendages

Area of Science:

• Microbiology
• Structural Biology
• Biophysics

Background:

• Bacterial motility relies on extracellular helical protein polymers, like the flagellum.
• Previous structural studies were limited to straightened filament states.
• Understanding flagellar structure is key to bacterial motility and potential biotechnological applications.

Purpose of the Study:

• To present a robust workflow for high-resolution cryo-electron microscopy (cryo-EM) of biologically relevant bacterial flagellar filaments.
• To enable structural studies of flagella in their native, potentially curved states.
• To facilitate structure-function analyses of filament architecture.

Main Methods:

• A novel purification method using centrifugation to isolate flagellar filaments from *Caulobacter crescentus*.
• Sample quality validation via SDS-PAGE and negative stain transmission electron microscopy (TEM).
• Cryo-EM data collection and helical reconstruction using cryoSPARC algorithms for 3D structure generation.

Main Results:

• Achieved high-resolution cryo-EM structures of bacterial flagellar filaments, with resolutions below 3 Å and one dataset reaching 2.1 Å.
• Successfully reconstructed both straight and curved flagellar filament conformations.
• Demonstrated the workflow's effectiveness for producing high-quality structural data.

Conclusions:

• The developed workflow provides high-resolution structural insights into bacterial flagella in biologically relevant states.
• This method supports structure-function studies to understand filament regulation.
• The workflow lays the foundation for developing novel helical biopolymers for biotechnology.