BIOL368/S20:Sahil Patel Week 13

Methods/Results

Exploring the Spike protein Structure

Convert the spike protein DNA sequence into a protein sequence using either the NCBI Open Reading Frame Finder or the ExPASY Translate tool. Paste a screenshot of your results into your wiki.

- How do you know which of the six frames is the correct reading frame (without looking up the answer)? The first reading frame is the correct one. We know this because using the ExPASY translate tool, the first reading frame has the most red highlighting.
- Answer checked with the NCBI protein record this is correct.
I found out what is already known about the spike protein in the UniProt Knowledgebase (UniProt KB). UniProt KB has two parts to it, Swis-Prot, which contains entries for proteins that have been manually reviewed, and TrEMBL (which stands for "Translated EMBL"), which are automated translations of all DNA sequences in the EMBL/GenBank/DDBJ databases. SARS-CoV-2 is so new that it has not yet been added to the UniProt database; it is scheduled to be added with the April 22 release.
- If you search on the keywords "SARS-CoV" (which refers to the first SARS virus), in the main UniProt search field, how many results do you get? 70 review, 748 unreview, so 818 total
- Use the entry with accession number "P59594" which corresponds to the reference entry for the SARS-CoV spike protein.
- What types of information are provided about this protein in this database entry? Common name, synonyms, other names, rank, lineage, virus hosts, strains, function & GO terms
We are going to use the PredictProtein server to analyze the SARS-CoV-2 spike protein.
- Paste the SARS-CoV or SARS-CoV-2 spike protein amino acid sequence that was discussed in your paper into the input field and submit (Ask Dr. Dahlquist for help with finding the sequence if you need it.)
- Paste a screenshot of the results into your wiki. Note that you can zoom in on different parts of the protein by using the slider at the top. Explore the types of information provided (in the menu options at the left). How does this information relate to what is stored in the UniProt database for the SARS-CoV spike protein?

View the structure of the SARS-CoV or SARS-CoV-2 spike protein from your assigned journal club article at the Protein Data Bank.
- Walls et al. (2020): 6VXX or 6VYB
- Wan et al. (2020): 2AJF
- Wrapp et al. (2020): 6VSB
- Yan et al. (2020) 6M17
- Click on the "Structure" underneath the image of the structure on the upper left side of the page. This will open a window where you will be able to interact with the structure image.
  - At the bottom right of the screen, you will see a drop-down menu that says "Select a different viewer". Select "JSMol" or "NGL" to access a palette of options for viewing the structure (they are slightly different from each other).

- Alternately, NCBI has a web-based structure viewer called iCn3D. To use it:
  - Go to the NCBI Structure home page and paste the PDB ID for your structure (above) into the search field and click "search".
  - On the results page, you will see an image of the protein structure. Click on the button for the "full-featured 3D viewer" found on the bottom right corner of the structure image.
  - This will open up a window with a similar image viewer to the one found on PDB. The interface is a little clunkier and the images are less elegant, but the iCn3D viewer has the advantange of showing the structure in a way that makes it easier to identify the N- and C-terminus (there are arrows for the beta strands and the helices are also pointed). It also allows you to see the sequence at the same time and find and highlight particular sequences in the structure.
- If you would prefer to work offline, you can install the stand-alone Cn3D viewer on your own computer.
- When answering the following questions, provide a screenshot pasted into your wiki:

- Create a view of the protein from your paper recreates one of the figures from your paper. You may not be able to get this to be exactly the same in terms of colors or backbone style, but you should try to rotate the to be the same view as the article.
- Find the N-terminus and C-terminus of each polypeptide tertiary structure.
- Locate all the secondary structure elements. Do these match the predictions made by the PredictProtein server? Yes, this closely matches the predictions made by the PredictProtein server.
- Locate particular amino acids that were discussed in your paper, show a screenshot that highlights them.

Conclusion

In this assignment, we looked at various tools found online that allow researchers to visualize, explore, and analyze protein sequence and structure. During this time, my homework partners and I have been developing our research question and hypothesis that will further explain the relationships between spike protein sequence, structure and function. We will be sure to use the skills we have acquired during this exercise as well as the relevant online tools in our final project.

Acknowledgments

I communicated with my homework partners Christina and Annika via Zoom to discuss this assignment.
Questions were copied from the Week 13 assignment page.
Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

References

OpenWetWare. (2020). BIOL368/S20:Week 13. Retrieved April 21, 2020, from https://openwetware.org/wiki/BIOL368/S20:Week_13.
Expasy. (2020). ExPASY Translate Tool. Retrieved April 21, 2020, from http://web.expasy.org/translate
NCBI. (2020). NCBI Structure Database. Retrieved April 21, 2020, from https://www.ncbi.nlm.nih.gov/Structure/index.shtml.
Predict Protein Server (2020). Predict Protein Server. Retrieved April 21, 2020, from https://ppopen.informatik.tu-muenchen.de/
Uniprot. (2020). Uniprot. Retrieved April 21, 2020, from http://www.uniprot.org
NCBI.(2020). NCBI Open Reading Frame Finder. Retrieved April 21, 2020, from https://www.ncbi.nlm.nih.gov/orffinder/ NCBI Open Reading Frame Finder.
Wrapp, D., Wang, N., Corbett, K. S., Goldsmith, J. A., Hsieh, C. L., Abiona, O., ... & McLellan, J. S. (2020). Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science, 367(6483), 1260-1263. DOI: 10.1126/science.abb2507