Cryogenic Electron Microscopy (Cryo-EM)

Epitope mapping is an important method to determine the binding site or epitope of natural autoantibodies (NAA) to their target antigens and contributes to the discovery and development of new therapies, vaccines, and disease diagnosis. Cryo-EM is one of the NAA epitope mapping methods. Creative Biolabs offers well-established and innovative One-Stop-Shop NAA solutions, and has built a strong technology platform to provide a full range of NAA services and products.

Overview of Cryo-EM

Cryo-EM is a structural biological method, which can be used to determine the 3D structures of biomacromolecules. Cryo-EM combined with 3D reconstruction has become an effective method to study the structure and dynamics of flexible molecular assemblies. The basic principle of Cryo-EM is to image biological macromolecules frozen and immobilized in glassy ice, thus obtaining projections of protein molecules in all directions. A large number of two-dimensional images are then processed and computed by computer, and the three-dimensional structure of biological macromolecules is reconstructed.

Fig.1 Cryo-EM structure determination. (Fernandez-Leiro, 2016)

The Procedure of Structure Analysis by Cryo-EM

Protein expression and purification
It is necessary to obtain samples with high purity, uniformity, and integrity, and the molecular sieve should be symmetrically distributed with a single peak.
Negative staining
The sample molecules are embedded in a solution of a heavy metal salt (usually uranium acetate) that surrounds them.
Data collection of negative staining sample
Freezing sample
This is very important and usually involves two steps, first loading the sample on the grid to form a thin film of water, and then rapidly freezing.
Load the sample in the cryo-EM
Screen sample
Before data collection, samples need to be screened to check whether the water in the sample is in a glass state and whether the ice thickness is appropriate.
Data collection
The well selected particles must be photographed using the minimum exposure technique because high-energy electrons can cause radiation damage to the sample.
Three-dimensional reconstruction

Problems of Structure Analysis by Cryo-EM

The sample is unstable, degraded, or aggregated
Some ligands with small molecular weight, may not be seen in the density map.
There may be organics such as sugar, DMSO, or glycerol in the buffer, resulting in reduced contrast and resolution of the sample.
The purity of the sample may be good or even very good, but the homogeneity is poor, which greatly reduces the resolution.
The target area may have greater flexibility after 2D or 3D averaging, the resolution of the target area becomes very poor.
Samples may be destroyed during freezing.
Optimize more parameters, such as sample concentration, clogging time, temperature, mesh size, etc.

Applications of Cryo-EM

Gene expression and regulation related complexes
Cryo-EM is mainly used to determine the structures of macromolecules and macromolecule supercomplexes. Spliceosomes and chromosomes are the most typical examples.
Protein synthesis and degradation related complexes
The most typical examples are the ribosome and the proteasome.
Membrane proteins
These include various transporters, ion channels, and supermembrane protein complexes.
Viruses and related protein complexes
Neurodegenerative disease-related proteins
The innate immune system related proteins

As a leading international biotechnology company, Creative Biolabs is flexible to meet the unique needs of clients' projects. You can count on our skilled and passionate workforce to find the most suitable path. We have expertise and capabilities in providing high-quality NAA solutions. If you are interested in our services and products, please contact us for more information.

Reference:

Fernandez-Leiro, R.; Scheres, S.H.W. Unravelling biological macromolecules with cryo-electron microscopy. Nature. 2016, 537(7620): 339-346.

For Research Use Only | Not For Clinical Use

Related Services:

Products:

NAA Detection Kits

Resource:

Pathways

Applications:

Online Inquiry