Question

1. Protein crystallography is one of the most common methods used to determine the structure of proteins. However, an emerging method called “Cryo-EM” is used to determine the structure of the spike protein.
In your own words, describe what “Cryo-EM” is, and why it’s a useful for studying protein structures.

2. The spike protein is heavily glycosylated. Briefly describe what glycosylation is, and why it might be useful for the virus.

3. Is the spike protein structure a Primary, Secondary, Tertiary, or Quaternary structure?

4. The spike protein binds to our cells before the virus can infect. This binding is mediated by the “receptor binding domain” (RBD) portion of the spike protein. Which secondary structure is the RBD predominantly comprised of?

Answer 1

1. Protein crystallography and Cryo- electron microscopy are the analytical tools of interest in determining the three dimensional structures of complex macromolecules like proteins.

Protein crystallography	Cryo- electron microscopy
The protein sample is crystallized and then the shape and structure of it is reproduced with the help of X-rays.	The sample is cooled down suddenly in the liquid nitrogen-cooled ethane to frozen condition and then it is exposed to low levels of electron beam to produce images.
The X-ray diffraction produces the image of protein	The electrons received after bombarding with the specimen provide information on protein structure.
The samples need to be crystallized	Doesn't need 3D crystals but can be done with native form of samples
Relatively larger amount of sample is required	Requires smaller amounts of sample and can hold a large variety of samples

Cryo-EM:

Cryo-EM stands for Cryo electron microscopy. It has emerged as a promising tool in macromolecular structure determination at high resolution i., atomic level (<4 A). Its advantages and scope have left other techniques far beyond Cryo-EM.

In Cryo electron microscopy, the sample protein is subjected to flash freezing process making use of cryogens like liquid nitrogen, liquid ethane, etc. It forms a thin layer of amorphous ice coating on the sample surface allowing it to be present in native hydrated form. Inorder to maintain the coating, the temperature of the sample holding column is lowered. Then a beam of electrons is bombarded on the sample in such a level that it doesn't collapse the structure of the protein. The electrons hitting the target surface may be reflected; absorbed or transmitted. These electrons provide valuable information to determine the protein shape, structure and its functioning.

Advantages:

1. High resolution images in the atomic scale.

2. More sensitivity.

3. Analyze dynamics using advanced image processing algorithms.

4. Requires smaller amount of samples.

5. Samples need not be crystallized.

Disadvantage:

1. Lesser the signal to noise ratio in single particle analysis, which can be corrected by taking multiple samples and averaging the results.

Protein samples may find difficulty in crystallizing in case of X-ray crystallography, which are thus more conveniently analyzed by Cryo-EM technique at greater resolution.