Cryo-electron tomography pipeline for plasma membranes.

Area of Science:

• Structural biology
• Cell biology
• Biophysics

Background:

• Cryo-electron tomography (cryoET) offers high-resolution protein structure determination within cellular contexts.
• Current cryoET sample preparation methods struggle to access and visualize proteins at the plasma membrane.
• Understanding plasma membrane protein structure is crucial for cell signaling and function.

Purpose of the Study:

• To present a novel cryo-electron tomography pipeline for enhanced visualization of plasma membrane proteins.
• To enable high-resolution structural analysis of proteins in isolated basal and apical plasma membranes.
• To facilitate targeted structural studies of specific plasma membrane proteins.

Main Methods:

• Developed a correlative cryo-electron tomography pipeline for imaging ultra-thin plasma membrane sections.
• Utilized a genetically-encodable, chemically-induced, electron microscopy visible tag for protein labeling.
• Employed sub-tomogram averaging for angstrom-scale structure determination.

Main Results:

• The pipeline successfully images large areas of isolated basal and apical plasma membranes.
• Achieved angstrom-scale resolution for protein structures using sub-tomogram averaging.
• Demonstrated efficient and targeted labeling of specific plasma membrane proteins.

Conclusions:

• The presented cryo-electron tomography pipeline offers a fast, cost-effective, and distributable method for plasma membrane protein structural studies.
• This approach significantly improves access to and structural determination of proteins at the cell surface.
• Enables detailed structural investigations of key plasma membrane proteins within their native environment.