Launch guide window

Launch NCBI Home Page

Goals

• Locate a disordered region in the structure of RNase H from enterobacteria phage T4.
• Use VAST to find a structural template for this region.
• Use a curated CD to predict the function of the disordered region.

Steps

1. Retrieve the structure in Entrez
   
   • Retrieve accession 1TFR from Entrez Structure.

   • Click on the accession to load the structure summary page.

2. Locate the disordered regions using Cn3D
   
   • Click the structure image to launch Cn3D.
   • Make a note of the positions of the three disordered regions (shown as gray letters).

3. Retrieve the VAST neighbors for 1TFR
   
   • Quit Cn3D and return to the structure summary page for 1TFR.
   • Click the gray Chain bar to retrieve the VAST neighbors for the entire chain.

4. Analyze the VAST neighbors for 1TFR
   
   • Change the List setting from "Graphics" to "Table" (rightmost menu in the List row).
   • Click List to update the view.
   • What is the most similar structure?
   • What is the sequence identity and RMSD between this protein and 1TFR?

5. View a VAST alignment in Cn3D
   
   • Check the box to the left of the most similar VAST neighbor to 1TFR.
   • Click "View 3D Alignment."

   • Color the structures by Object (use Style / Coloring shortcuts).
6. Analyze the VAST alignment
   
   • Compare the disordered regions in 1TFR to the corresponding regions in the VAST neighbor using the list of their locations you prepared earlier.
   • Are there regions that have coordinate data in one structure but not the other? (Hint: Use the sequence window. Disordered residues will be gray, and residues with coordinates will be colored.) Make a note of these regions.
7. Assess the uncertainty in the structural data
   
   • Color the structures by temperature (use Style / Coloring shortcuts).
   • How well determined are the regions you identified in the previous step? (more red = more error)

8. Find conserved domains in the structure
   
   • Quit Cn3D. Return to the structure summary by clicking on the MMDB ID 5678 at the very top of the VAST neighbor page.
   • Click on the aligned CD record to view that multiple alignment.

9. Analyze the conserved domains in the structure
   
   • What is this functional domain? What kind of CD record is this (what source database)?
   • Do any of the other aligned structures look familiar?
   • Find the features menu in the "Show Alignment" row. What functional features are annotated on this domain?

10. View the CD in Cn3D
    
    • Launch Cn3D by clicking "Structure View."
    • Click "Show Annotations Panel" in the CDD Descriptions panel, and highlight each functional feature.
    • Do any of them occur near the long, internal disordered region in 1TFR? What might this region do?

11. Locate structural features unique to 1TFR
    
    • Select Style/Coloring Shortcuts/Object.
    • Can you find a region close in space to the disordered region analyzed in the previous step that is unique to 1TFR?
    • Can you design a deletion mutant of 1TFR that may reveal specific interactions between RNase H and other proteins?

12. EXTRA CREDIT (optional): Use BLAST to find sequence-similar structures to 1TFR
    
    • Keep Cn3D open, and open a new browser window.
    • From the NCBI home page, retrieve 1TFR in Entrez Protein.
    • Click on "Related Structure" in the Links menu to the right of the accession.

13. EXTRA CREDIT (optional): View the BLAST results
    
    • Do you find any structures? Do any of the structures look familiar from your VAST search?
    • Compare the BLAST alignment to the VAST alignment between these structures and 1TFR. How did BLAST do?

Revised December 3, 2007