Structural Biochemistry/Membrane Proteins/Methods for Studying Membrane Proteins/Cryo-electron Tomography and 3D Image Averaging

Combining cryo-electron tomography and 3D image classification together allow for better opportunities to determine the structure of membrane proteins in undisturbed cells. It relies on averaging a vast array of images of identical copies of a complex to determine its structure.

Tomography
Tomography is a technique used to assemble a 3D view, called a tomogram, from several images of the structure. First, the cells are frozen very quickly and embedded in plastic. The plastic is then thinly sliced. Using a specialized high-voltage electron microscope, several images of these samples, at different angles (caused by tilting the stage of the microscope) are collected. The assembled images are called the tomogram.

Some protein membranes can be crystallized when they are in the lipid bilayer plane. Electron crystallographic techniques are able to determine structures of these proteins. It combines information from images of thin crystallized proteins whose images are taken at different angles. For proteins high in symmetry that can be purified, single particle cryo-electron microscopic techniques are used to help build 3D structures of these complexes.

Electron tomography is used to determine 3D density of microscopic objects. To minimize damage from electron irradiation, the images captured in this technique are done at cryogenic temperatures. Averaging the 3D images can be used to create density maps of macromolecular complexes. X-Ray Crystallography is also used to determine atomic structures of individual components. Currently, only low resolution images of protein membranes are able to be recorded. Higher resolution images, however, are also possible as this technique gets further developed as there are no significant barriers preventing this.

This technique can also shed light on the conformation of membrane proteins in their native environment. Combining cryo-electron tomography and 3D image classification and averaging also allows for in situ structure determination of non-symmetric viruses. Though X-Ray Crystallography is advancing and getting better at determining the structure of integral protein membranes, it is still very challenging to determine their structure when they are still in the cell.

Role in Cell Function
A computer assembles these images into the 3D view called the tomogram. A tomogram can help us understand cell function because it allows us to view small organelles like the Golgi. You can even produce a movie of a virtual journey through the cell. Howell’s research of the Golgi for example, shows that there are several pathways for proteins and other molecules to exit the Golgi. The findings are revealing, as earlier studies using different methods had suggested that there was only one road out of this organelle.