Heterogeneous MAC Initiator and Pore Structures in a Lipid Bilayer by Phase-Plate Cryo-electron Tomography

Area of Science:

• Immunology
• Structural Biology
• Biophysics

Background:

• Pore formation in cell membranes is crucial for mammalian immune defense against bacterial invasion.
• The membrane attack complex (MAC) mediates this defense through sequential protein assembly and membrane insertion.

Purpose of the Study:

• To determine the in situ structure of the MAC pore complex (C5b-9) in lipid bilayers.
• To visualize intermediate complexes (C5b-7, C5b-8) and understand the dynamics of pore formation.

Main Methods:

• Cryo-electron tomography with Volta phase plate.
• Subtomogram averaging to analyze C5b-7, C5b-8, and C5b-9 complexes.
• Imaging of protein complexes within lipid bilayers.

Main Results:

• Determined the C5b-9 pore structure at 2.3-nm resolution, revealing a 10- to 11.5-nm cone-shaped pore.
• Identified a poorly closed pore with a seam between C9 and C6, due to variations in beta strands.
• Observed oligomerized C5b-7 and C5b-8 complexes forming ~3.5-nm pores, showing membrane deformation and perforation.

Conclusions:

• The MAC pore structure is dynamic and cone-shaped, with variations in its formation and closure.
• Intermediate complexes demonstrate membrane binding, deformation, and perforation stages.
• The pore formation process appears adaptable to biological membranes under attack.