Protein Sequence, Structure, and Functio n Lab

1. Protein Sequence, Structure, and Function Lab Gustavo Caetano - Anolles Powerpointby Casey Hanson Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

2. Exercise In this exercise we will be doing the following: • Visualize the structure of various proteins in the Protein Data Bank. • Use the Superfamily HMM tool to uncover common protein domains in aligned sequences. • Reconstruct Phylogenies of Structurally Related Proteins Using Mr. Bayes. Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

3. Step 0: Shared Desktop Directory For viewing and manipulating files on the classroom computers, we provide a shared directory in the following folderon the desktop: classes/mayo In today’s lab, we will be using the following folder in the shared directory: classes/mayo/caetano Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

4. Protein Visualization in PDB In this exercise, we will become familiar with the Protein Data Bank, a database that provides various information on the structure and function of proteins. We will concentrate on Acyl Phosphotase (2ACY) in our exercises. We will primarily be using this tool to visualize the 3D structure of proteins in the browser, and then making predictions on their secondary structure from this view. We will validate our predictions using a PDBsum Hera Diagram. Additionally, we will use CATH (a tool that imposes a hierarchical structure to PDB) to look at the folds (hierarchy) for 2ACY. Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

5. Step 1A: Accessing PDB Open a browser and go to the following web address: http://www.rcsb.org/pdb/ In the search box, type the PDB ID of Acyl Phosphotase: 2ACY Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

6. Step 1B: Accessing PDB On the right side of the screen, under biological assembly, click View in 3D. On the next page you may get warnings regarding Java. If so follow the directions on the next slide. • If Java™ needs your permission to run, click Run This Time • If a Security Warning pops up, select the checkbox and click Run. • If a Block Window pops up, select Don’t Block Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

7. Step 2: Visualization of 2ACY The visualization window should look something like the this: Holding Left Click down and moving the mouse should enable you to rotate the protein in 3D space! Look at the protein. Can you detect what its secondary structure is from this 3D diagram? Write down your prediction in Notepad and we will test it next. Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

8. Step 3A: Verification of 2ACY Secondary Structure Though we could do this in PDB, we will consult a secondary resource to verify our prediction. Go to the following web address: http://www.ebi.ac.uk/pdbsum/ In the search box PDB code box, type 2ACY and click Find. Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

9. Step 3B: Verification of 2ACY Secondary Structure Under the Protein Chain header click the The Protein Chain page should look like the following: Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

10. Step 3C:Verification of 2ACY Secondary Structure How does your prediction compare with this domain? Click on the domain icon on the right side of the screen for a nice diagram of the domain. N terminus Helix sheet C terminus Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

11. Step 4A: Analysis of Folds for 2ACY Now, we will look at 2ACY’s fold in the CATH heirarachy. CATH (Class, Architecture, Topology, and Homologous Superfamily) is a novel hierarchical clustering of proteins according to these 4 attributes. To view the CATH hierarchy from our 2ACY Domain page, click on the CATH button . Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

12. Step 4B: Analysis of Folds for 2ACY The resulting page for Domain 2ACYA00 (our one domain in 2ACY) should include the following CATH Classification. The decomposition of 2ACY according to CATH follows the following path: Alpha Beta 2-Layer Sandwich Alpha-Beta Plaits Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

13. Using blastPFor Finding Sequence Matches to GATA-1 In this exercise, we will utilize a different BLAST tool called blastP to find all protein sequence matches to GATA-1 (the erythroid transcription factor from an earlier lab). Using SUPERFAM HMM we will analyze which protein domains these homologous sequences have. Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

14. Step 5A: BLASTing GATA-1 Go to the following web address to access BLAST: http://blast.ncbi.nlm.nih.gov/Blast.cgi The program we want to run is protein blast.. Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

15. Step 5B: BLASTing GATA-1 The protein FASTA sequence is available in our shared-directory: classes/mayo/caetano/gata1.fasta Click the Choose File button and upload our gata1.fasta file. Under Database choose Protein Data Bank(pdb). Ensure that for Algorithm, blastp is selected. Click BLAST. Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

16. Step 5C: BLASTingGATA-1 The screenshot below details the correct configuration. Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

17. Step 5D: BLASTing GATA-1 The distribution of hits should look similar to below: Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

18. Step 5E: BLASTing GATA-1 In this step, we will download all of the significant alignments in this plot. Scroll down the window to the Sequences producing significant alignments box: Click Select All. Click Download Select FASTA (complete sequence) Click Continue Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

19. Step 6A: Running Superfamily HMM The file from the previous step is available in our shared directory as: classes/mayo/caetano/gata1_homologs.fasta To run SUPERFAMILY HMM go to the following web address: http://supfam.cs.bris.ac.uk/SUPERFAMILY/hmm.html Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

20. Step 6B: Running Superfamily HMM On the next screen, next to Multiple Sequence FASTA File, click Choose File. Select our homolog file we just downloaded or the file in the shared directory: gata1_homologs.fasta. Ensure that Amino Acid sequence is selected from the dropdown menu at the top. Ensure Notification is Browser. Click Submit. Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

21. Step 6C: Running Superfamily HMM Ignore the short sequence warnings and click the View the domain assignment results link at the bottom of the page. The results are shown in pictorial and tabular form (scroll down on the page) and are sorted according to e-value of whether or not the sequence belongs to a given superfamily. The picture to the right shows a diverse set of domains showing up. Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

22. Step 6D: Running Superfamily HMM Many homologs though show the same domain family as 1GAT. In the tabular view, you can see the e-valuesfor superfamily assignments and family assignments for each on of these homologs. In general, the superfamily assignment must not exceed 0.00001 to be considered significant, while the family assignment can not exceed 0.001. Those sequences that violate these constraints have their e-values grayed in the tabular view. Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

23. Finding Structural Neighbors and Rebuilding Phylogenetic Trees In this section, we will search a database for sequences with a similar structure to a protein of interest, 3GE4 – a DNA STARVATION PROTEIN. In particular, we will look at Chain A. Then, utilizing Mr. Bayes we will reconstruct a Phylogenetic Tree utilizing the alignment data we get from DALI, our structural alignment program. Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

24. Step 7A: DALI NOTE: DO NOT CLICK SUBMIT. WE HAVE PRECOMPUTED THE RESULTS. There is a nice web interface for using DALI at the following link: http://ekhidna.biocenter.helsinki.fi/dali_server/start To run our query against the database we need to just specify two things. In PDB identifier type 3GE4 In Chain type A. Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

25. Step 7B: DALI The DALI results for this protein-chain have already been computed and are available in the HTML file in our shared directory. In the browser, it should look similar to the following: a ranked list of sequences to the query (3GE4) decreasing in similarity. Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

26. Step 7C: DALI Select the following hits: (Ctrl-F to search for something in the web page) 3ge4-A 1tjo-C 1ji4-L 1bcf-H 3uoi-j 1eum-A Click on Structural Alignment Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

27. Step 7D: DALI The structural alignment is shown below where the TOP figure shows the alignment of the residues while the BOTTOM figure shows the secondary structure identifier for the residue (L = coil, H= Helix, E = Strand). Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

28. Step 8A: Reconstructing Phylogenies Using Mr. Bayes A nexus file for the tracks we selected in the previous stage is provided in the shared directory: classes/mayo/caetano/alignment.nex We will run a program called Mr. Bayes that will reconstruct the phylogenies from these structural alignments. Its icon is located on the desktop. Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

29. Step 8B: Reconstructing Phylogenies Using Mr. Bayes Unfortunately, Mr. Bayes does not handle paths well. In order to use our alignment.nex file, we have to copy it into the directory where Mr. Bayes is installed. To navigate to this directory, Right Click on the Mr. Bayes icon on the Desktop. Click Find Target… Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

30. Step 8C: Reconstructing Phylogenies Using Mr. Bayes Open up our shared directory in a window side by side with our Mr. Bayes directory. Drag our alignment.nex file to the Mr. Bayes directory. Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

31. Step 8D: Reconstructing Phylogenies Using Mr. Bayes Run the following commands in Mr. Bayes to reconstruct the phylogeny. $ execute alignment.nex $ showmodel $ set autoclose=yes; # close chains and go to next statement $ mcmcpngen=10000 printfreq=100 samplefreq=100 nchain=4 savebrlens=yes filename=alignment; # define parameters of the run $ mcmc; # Run Markov Chain Monte Carlo $ sump # Summarize your mcmc results $ sumt# Output Trees Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles

32. Step 9: Analyzing the Phylogenies The phylogeny is shown in the output of Mr. Bayes. A screenshot is shown below. Protein Sequence, Structure, and Function Lab v1 | Gustavo Caetano - Anolles