Structural Biochemistry/Membrane Proteins/Study of Protein structure in lipid bilayer: Electron crystallography

Introduction:

Electron crystallography is an excellent technique to study the structures of proteins in their natural bilayer environment and the biological mechanism through freeze-trapping of transitional states. The technique also shows how the proteins in situ are designed and carries out their membrane specific tasks. In 1975, the seven membrane-spanning α-helices of bacteriorhodopsin were resolved, providing the view of polypeptide chains traversing the lipid bilayer. After that, an atomic model of bacteriorhodopsin was obtained.

Sheets and Tubes:

The most informative density maps of membrane protein was studied with Electron crystallographic on protein-lipid arrays in the form of sheets ( 2 dimensional crystals) or tubes( tubular crystals). Two-dimensional crystals may contain thousands of unit cells, providing extensive averaging to improve the signal-to-noise ratio. However, to obtain the three dimensional analysis, the sheets need to be tilted. Tubular crystals contain less unit cells. But the molecules are arranged on the surface lattice with helical symmetry; therefore, give many different views and do not need to be tilted. By averaging the unit cells in each image, the technique enhances the poor signal-to-noise ratio owing to the weak electron does. The lattice and symmetry elements also provide good position and orientation of each molecule. The averaging of isolated membrane proteins to obtain structures is also feasible (>250kDa) The producing of protein-lipid arrays from detergent-solubilised, purified protein, both in Sheet and Tube forms has been developed. Even though the extensive robotic screens developing for Tubes have not been achieved, Tube form has provided many advantages. Ex: many different views of the same molecules that can reconstruct it roughly in three dimensions; there is no ‘missing cone’ of formation, etc...

Freeze-trapping different conformational states: Scientists can use electron crystallography as a direct means to probe the biological mechanism of protein in lipid. The step in preparation is plunge-freezing of the electron microscope grid into liquid nitrogen-cooled ethane, which cools the specimen rapidly( ~106 oC/s). It permits efficient trapping of a transient structure that has a lifetime of a millisecond or longer. Protein is then activated by using light, or spraying the appropriate ligand onto the grid just before the grid hits the ethane surface. This technique has been applied to ACh receptor channel, using tubular crystals,  and yielded a good data collection. The difficult step is the spray-freeze-trapping treatment. So far, freeze-trapping has not been used widely, and still under developed