Cryo-Electron Microscopy

Cryo-electron microscopy or cryo-EM is used to determine the 3D structures of biological macromolecules at low temperatures. It is based on the principle that frozen, fixed macromolecules can be viewed from all directions by obtaining the projection through 3D imaging. A large number of 2D images are reconstructed by a computer to form a 3D image, based on the central section theorem. Unlike X-ray crystallography, cryo-EM uses purified samples in solution, enabling the study of complexes and proteins that are difficult to crystallize. Single-particle cryo-EM can also identify individual conformations of a molecule.

The only limitations of this method are the minimum size of the structures and the low resolution. Recent developments like direct electron detectors and more effective computational image analysis techniques are reducing these limitations. The minimum molecular weight limit of structures is 52 kDa. The resolution and freezing methods are also being continuously improved. The advantages of cryo-EM over traditional methods include: i) there is no need for crystallization; ii) it can be used to analyze proteins and complexes of high molecular weight; iii) it reduces radiation damage; iv) it can be used to analyze the structure of membrane proteins like GPCR; v) multiple conformations can be analyzed in a single experiment etc. However, a major limitation is the resolution of only 3 Å even though pharmaceutical development demands a resolution of 2 Å.

Procedure: i) Purified and homogeneous samples of proteins need to be obtained. ii) The molecules are embedded in a layer of heavy metal salt solution. iii) The sample is then loaded on the grid to form a thin film of water and quickly frozen. Water can be made glassy by immersing the grid into liquid ethane quickly. iv) Minimum exposure technology is used in photography of selected particles. v) The 3D image is reconstructed.

However, biochemical reactions often occur rapidly. In order to study the reaction intermediate in such cases, the fast mixing method is used. The two molecules are mixed in milliseconds and frozen quickly. This process can be used to study short-term biological processes.

APPLICATIONS

Cryo-EM is used to study the following categories of biomolecules:

Gene expression related complexes like chromosomes and spliceosomes (enzyme complexes that splice RNA).

Virus particles and related complexes. They are larger than proteins so cryo-EM is suitable for studying their structures.

Protein synthesis related complexes like the ribosome and proteasome, specifically the eukaryotic mitochondrial ribosomes.

Membrane proteins including ion channels and supermembrane protein complexes involved in material transport. They are difficult to crystallize so cryo-EM is useful in studying them.

Immune system related protein complexes

Neurodegenerative disease related proteins

Cryo-electron tomography (cryo-ET) can obtain the 3D structure of a sample by tilting it in multiple directions in the microscope and reconstructing using calculations and the 2D projections. Future prospects for cryo-EM include studying structures of low molecular weight, obtaining better resolution to about 2 Å, improving the efficiency and using it for drug discovery and development in the pharmaceutical industry.