This journal entry is due on Wednesday, October 22 at midnight PDT (Tuesday night/Wednesday morning). NOTE that the server records the time as Eastern Daylight Time (EDT). Therefore, midnight will register as 03:00.
Background for HIV Structure Project
- Huang, C. C., Tang, M., Zhang, M. Y., Majeed, S., Montabana, E., Stanfield, R. L., Dimitrov, D. S., Korber, B., Sodroski, J., Wilson, I. A., Wyatt, R., & Kwong, P. D. (2005). Structure of a V3-containing HIV-1 gp120 core. Science, 310(5750), 1025-1028. DOI: 10.1126/science.1118398
- Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J., & Hendrickson, W. A. (1998). Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature, 393(6686), 648-659. doi:10.1038/31405
- Merk, A., & Subramaniam, S. (2013). HIV-1 envelope glycoprotein structure. Current opinion in structural biology, 23(2), 268-276. DOI: 10.1016/j.sbi.2013.03.007
- Stanfield, R. L., Cabezas, E., Satterthwait, A. C., Stura, E. A., Profy, A. T., & Wilson, I. A. (1999). Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing fabs. Structure, 7(2), 131-142. DOI: 10.1016/S0969-2126(99)80020-3
- Turner, B. G., & Summers, M. F. (1999). Structural biology of HIV. Journal of molecular biology, 285(1), 1-32. DOI: 10.1006/jmbi.1998.2354
Individual Journal Assignment
- Store this journal entry as "username Week 8" (i.e., this is the text to place between the square brackets when you link to this page).
- Create the following set of links. These links should all be in your personal template; then use the template on your journal entry.
- Link to your journal entry from your user page.
- Link back from your journal entry to your user page.
- Link to this assignment from your journal entry.
- Don't forget to add the "BIOL368/F14" category to the end of your wiki page.
Defining Your HIV Structure Research Project
For this project, you can choose to work with the same sequences you used for the HIV Evolution Project, or you may choose different sequences. You will reframe your question from the HIV Evolution Project to make it a structure→function question. Instead of looking at how the evolution of variation of the viral DNA sequence affects the different patient groups, you will look at how variations in the viral sequence affect the structure and, therefore, function of the virus.
For this week's journal assignment, your electronic lab notebook entry should contain the answers to the following:
- What is your question?
- Make a prediction (hypothesis) about the answer to your question before you begin your analysis.
- Which subjects, visits, and clones will you use to answer your question?
- You should choose a combination of subjects, visits, and clones that will add up to approximately 50 sequences. You will need about that many sequences to answer a reasonably complex question. However, you cannot use more because the multiple sequence alignment tool cannot handle more than that many sequences.
- Justify why you chose the subjects, visits, and clones you did.
Once you have your question, hypothesis, and data you will use, you should then move on to answering your question (see the Week 9 Assignment). Although the project is not due until October 29, you should work ahead. Your electronic notebook should contain your notes, methods, results, and interpretations as you carry out your project. You should document as you work, taking your notes on the wiki as much as possible. Post data, figures, screenshots, to support your project. You can post files that are in progress; remember, you can upload a new version of the file and the wiki will automatically link to the new version (while keeping the old).
Working with Protein Sequences In-class Activity
- This week we will begin to learn how to analyze protein structures. For today, we will be using the Bioinformatics for Dummies book extensively, so be sure to bring it to class. We will be using some bioinformatics tools to analyze the structure of the gp120 envelope protein.
- Chapter 4: Reading a SWISS-PROT entry (pp. 110-123 in the second edition). The example worked through in the book is the epidermal growth factor receptor. Work through this example using the HIV gp120 envelope protein instead.
- Swiss-Prot is now part of what is known as 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. The user interface to this protein database has undergone many revisions since this book was published, but all of the same information can still be found.
- If you search on the keywords "HIV" and "gp120", how many results do you get?
- Use the entry with accession number "Q75760" which corresponds to the HIV gp120 sequence that was used for the crystal structure for the Huang et al. (2005) paper.
- Chapter 5: ORFing your DNA sequence (pp. 146-147 in second edition). In the previous section of the course, we were working with DNA sequences from the HIV gp120 envelope protein. Take one of your DNA sequences and follow the instructions to find the open reading frames in the sequence. Since you were working with just a portion of the entire envelope protein, you may get some strange results. Compare your results with the UniProt entry for the protein above to decipher what the output means.
- Chapter 6: Working with a single protein sequence (pp. 159-195 in second edition). Apply the following tools to the entire HIV gp120 envelop protein sequence that you obtained from UniProt above. We will then compare the results of these analyses with the actual structure of the gp120 protein obtained by X-ray crystallography.
- The ExPASy tools page which lists many available tools for protein analysis
- ScanProsite (NOTE: uncheck the box for "Exclude motifs with a high probability of occurrence from the scan")
- Chapter 11: Predicting the secondary structure of a protein sequence and additional structural features (pp. 330-336 in second edition). Predict the secondary structures and other structural features that occur in your HIV gp120 sequence and compare it to the published crystal structure from Huang et al. (2005).
- To compare your analyses with the actual crystal structure of gp120, download the structure file for the paper we read in journal club from the NCBI Structure Database.
- Store your journal entry in the shared BIOL368/F14:Class Journal Week 8 page. If this page does not exist yet, go ahead and create it.
- Link to the shared journal entry from your user page; this should be part of your template.
- Link the shared journal page to this assignment page.
- Sign your portion of the journal with the standard wiki signature shortcut (
- Add the "BIOL368/F14" category to the end of the wiki page (if someone has not already done so).
- After working with the protein tools on today's assignment, compare your experience with working with the nucleic acid tools versus the protein tools. Which do you like better, and why?