This journal entry is due on Thursday, October 8, at 12:01am Pacific time.
The learning objectives for this assignment are:
- Learn how to analyze and visualize protein structures.
- Refine your research question for the first mini-project.
Individual Journal Assignment
- You will be expected to consult with your partner, in order to complete the assignment.
- Each partner must submit his or her own work as the individual journal entry (direct copies of each other's work is not allowed).
- You must give the details of the interaction with your partner in the Acknowledgments section of your journal assignment.
- Homework partners for this week are the same as last week:
- Fatimah & Kam
- Nathan & Macie
- Aiden & Anna
- JT & Yaniv
- Owen & Ian
- Taylor & Nida
Format and Content Checklist
- Store this journal entry as "username Week 5" (i.e., this is the text to place between the square brackets when you link to this page).
- Write something in the summary field each time you save an edit. You are aiming for 100%.
- Invoke the template that you made as part of the Week 1 assignment on your individual page.
- Purpose: a statement of the scientific purpose of the assignment. Note that this is different than the learning objective stated on the assignment page. What science will be discovered by completing this assignment?
- Combined Methods/Results (Electronic Lab Notebook): documentation of your workflow for this exercise. It should include:
- The protocol you followed in enough detail for someone else to be able to conduct the same investigation. There should be enough detail provided so that you or another person could re-do it based solely on your notebook. You may copy protocol instructions on your page and modify them as to what you actually did, as long as you provide appropriate attribution.
- Answers to any specific questions posed in the exercise.
- Screenshots and images to document your answers.
- Data and files: links to all data and files used and generated.
- It is a critical skill for data and computer literacy to back-up your data and files in at least two ways:
- References to data and files should be made within the methods and results section. In addition to these inline links, create a "Data and Files" section of your notebook to make a list of the files generated in this exercise.
- Scientific Conclusion: a summary statement of the main result of exercise/research. It should mirror the purpose. Length should be 2-3 sentences, up to a paragraph.
- Acknowledgments section (see Week 1 assignment for more details.)
- You must acknowledge your homework partner with whom you worked, giving details of the nature of the collaboration. You should include when and how you met and what content you worked on together.
- Acknowledge anyone else you worked with who was not your assigned partner. This could be the instructor, the TA, other students in the class, or even other students or faculty outside of the class.
- If you copied
wiki syntax or a particular style from another wiki page, acknowledge that here. Provide the user name of the original page, if possible, and provide a link to the page from which you copied the syntax or style.
- If you copied any part of the assignment or protocol and then modified it, acknowledge that here and also include a formal citation in the Reference section.
- You must also include this statement:
- "Except for what is noted above, this individual journal entry was completed by me and not copied from another source."
- Sign your Acknowledgments section with your wiki signature (four tildes,
- References section (see Week 1 assignment for more details.)
- Use the APA format.
- Cite this assignment page.
- Cite any protocols that you copied and modified (this must also be noted in the Acknowledgments section).
- Cite any other methods, software, websites, data, facts, images, documents (including the scientific literature) that was used to generate content on your page.
- Do not include extraneous references that you do not cite or use on your page.
- Wan, Y., Shang, J., Graham, R., Baric, R. S., & Li, F. (2020). Receptor recognition by the novel coronavirus from Wuhan: an analysis based on decade-long structural studies of SARS coronavirus. Journal of virology, 94(7). DOI: 10.1128/JVI.00127-20
- For a refresher you can read the chapter on the Shape and Structure of Proteins from the text book Molecular Biology of the Cell, 4th edition, by Alberts et al. for free on the NCBI Bookshelf here: https://www.ncbi.nlm.nih.gov/books/NBK26830/
- Recorded video lectures and slides from Cell Function are also available to view on Brightspace
Databases & Tools
Exploring the Spike protein Structure
- Find 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 was only added to the database with the April 22 release.
- Search using the keyword "SARS-CoV-2" in the main UniProt search field. How many results do you get? Are all the results viral proteins?
- Use the entry with accession number "P0DTC2" which corresponds to the reference entry for the SARS-CoV-2 spike protein.
- What types of information are provided about this protein in this database entry?
- View one of the structures of the SARS-CoV spike protein from Wan et al. (2020) in the NCBI Structure Database using the web-based iCn3D viewer. (If you would prefer to work offline, you can install the stand-alone Cn3D viewer on your own computer.) Choose one of the three structures below to focus on initially.
- Figure 1A SARS-CoV RBD (year 2002) complexed with human ACE2: 2AJF
- Figure 1C SARS-CoV RBD (optimized for human ACE2 recognition) and human ACE2: 3SCI
- Figure 4B SARS-CoV RBD (optimized for civet ACE2 recognition) and civet ACE2: 3SCK
- When answering the following questions, provide a screenshot pasted into your wiki:
- Create a view of the protein you have selected that recreates Figure 2A from the Wan et al. (2020) 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.
- Is this a primary, secondary, tertiary, or quaternary structure? Explain your reasoning.
- How many domains are in this structure? Explain your reasoning.
- Click on the Style > Proteins menu and show screenshots of the following styles:
- Cylinder and Plate
- C Alpha Trace
- Ball and Stick
- What can you see in each of these styles that is unique to the style?
- In the “Spheres” view, click on the Color menu and show screenshots of the following color schemes:
- What can you see in each of these color schemes that is unique to the color scheme?
- Find the N-terminus and C-terminus of each polypeptide.
- What secondary structures are found in the ACE2 protein? How did you figure this out?
- What secondary structures are found in the Spike protein? How did you figure this out?
- Now we are going to look at side chain-side chain interactions in the civet ACE2-spike protein structure shown in Figure 4B.
- Click on the link to interact with the structure in iCn3D
- Click on the Windows menu to “View Sequences & Annotations”
- In the new window that appears to the right, click on the “Details” tab to show the actual amino acid sequences
- There are 2 sets of ACE2-spike proteins because of the way the proteins crystallized.
- Focus on the pink and tan chains and orient them like is shown in Figure 4B
- We are going to make the amino acid side chains shown in the figure visible.
- In the sequence window go to sequence “Protein 3SCK_A” (in pink) and select the following amino acids
- The part of the ribbon that represents these amino acids should be highlighted in yellow in the structure
- Go to the Styles menu and select Proteins > Ball and Stick
- Go to the Color menu and select Atom
- You should see the side chains shown in the figure.
- Repeat this process for the tan spike protein sequence 3SCK_E for the following amino acids:
- After you have done this, take a screenshot oriented like in Figure 4B.
- E35 on ACE2 makes an ionic bond with R479 on spike protein. Which of these is acidic and which is basic? How can you tell that from the image you created?
- T31 on ACE2 makes a hydrogen bond with Y442 on spike protein. Which side chain classification do these two amino acids belong to (acidic, basic, uncharged polar, nonpolar)? How can you tell that from the image you created?
- You can make the dashed lines for these bonds in iCn3D as follows:
- Go to the View menu and select H bonds & Interactions
- In part 1 of the window that appears, uncheck “Contacts/Interactions” leaving Hydrogen Bonds and Ionic Interaction checked
- In part 2 of the window, select the first set “3SCK_A” (pink)
- In part 3 of the window, select the second set “3SCK_E” (tan)
- In part 4 of the window, click the button “3D Display”
- You should now see the dashed lines representing the ionic bonds and H-bonds between the two polypeptide chains and amino acids we described above. Take a screenshot when you are done.
Beginning your research project
- Consulting with your partner, answer the following:
- What question will you answer about sequence-->structure-->function relationships in the spike and/or ACE2 protein?
- What sequences will you use?
- You will be able to consult with Dr. Dahlquist to locate the sequence information you need.
- You will need to make a multiple sequence alignment and phylogenetic tree. You will also need to visualize protein structures as part of your project.
- I will approve all project questions. If you want me to approve your project question in advance of next week's class so that you can start work, please schedule an office hour appointment with me to discuss it. Links to my drop-in office hours and Calendly are on Brightspace.
- Don't forget that you have been asking questions for each weekly assignment, you may just need to refine one of your questions for this project.
- This section of your individual journal entry may be copied between homework partners. Remember to acknowledge this in your Acknowledgments section.
- Compose your journal entry in the shared Class Journal Week 5 page. If this page does not exist yet, go ahead and create it (congratulations on getting in first :) )
- Create a header with your name, and then answer the questions in your own section of the page.
- You do not need to invoke your template on the class journal page.
- Any Acknowledgments and References you need to make should go in the appropriate sections on your individual journal page.
- Sign your portion of the journal with the standard wiki signature shortcut (
- Add the category "BIOL368/F20" to the end of the wiki page (if someone has not already done so).
- After manipulating the protein structure yourself, what new insights to you have into the article we read for journal club?
- The "protein folding problem", i.e., the difficulty of predicting protein structure from sequence information alone, has prompted a group to create Foldit, a protein-folding game that anyone can play on the web. Scientific papers have been published based on the crowd-sourced work of gamers playing Foldit, with the gamers as co-authors. Read the blog post (scroll to the middle of the page where it says "Experiment results for coronavirus spike binders") about how Foldit is contributing to coronavirus research. I'm not asking you to play Foldit, but please comment on whether this game is something that you would like to play and why (it might be a good "safer at home" distraction once the semester ends!).