Using PROTEIN EXPLORER to Visualize Mutation vs. Conservation in 3D from a Multiple Protein Sequence Alignment


Garry Duncan and Eric Martz
(July 16, 2000 for PE version 1.47-beta)

Amino acid sequences retrieved from databases can be aligned using software such as the Biology Workbench (NCSA), and the alignments imported into Protein Explorer (PE) where mutations or conservation may be visualized in 3D.  Below is an example illustrating how to do this.


The following procedures allow you view in 3D an aligned protein sequence of enolase, an enzyme found in all living organisms because of its role in glycolysis.  Using the Biology Workbench, the amino acid sequences for enolase were retrieved for the following six species: Homo sapiens (human),  Drosophila melanogaster (fruit fly), Saccharomyces cerevisa (yeastóunicellular eukaryote), Methanococcus jannaschii (an Archaea), Escherichia coli (gram negative bacterium) and Bacillus subtilis (gram positive bacterium).  The sequences were aligned, converted to FASTA format, copied to the clipboard and pasted into PE.
 

Enolase:  Sample for which you can view the alignment differences in a 3D rotating molecule:

  1. Using Netscape 4.04 or later (sorry, Internet Explorer doesnít work and this is beyond our control), launch the Protein Explorer to view enolase (structure file 4enl): http://www.umass.edu/microbio/chime/beta/pe_alpha/protexpl/pe.htm?id=4enl   (note that the word pe_alpha contains an underscore character, not a space).

    If you do not have the 2.0 or greater version of Chime, you will be given a link to download it and add it as a plug-in to Netscape.  After you have downloaded it and installed it as a plug-in, you will need to exit from Netscape, and repeat step 

  2. If you are using Netscape 4.04 or later, and you have Chime 2 installed, a page will appear about starting a PE session. Resize this window if desired, then click ëStart Explorer session

  3. In the right-hand frame, you should see a protein molecule  rotating (enolase). Click the button Hide/Show Water until the red balls (water oxygens) are hidden, so you can see the backbone trace of the protein clearly.

  4. Click on Explore More, then at the bottom of the QuickViews page, click on Advanced Explorer.

  5. Now you are ready to do an alignment.  In the upper left frame, click on the hyperlink ëMSA3D Multiple Sequence Alignment Coloring."

  6. Click on either ëTry a ready-made demonstration exampleí or on ëMSA3D ALIGNMENT FORM.í  (MSA3D = multiple sequence alignment in 3D.)

  7. You are now in the MSA3D Alignment Form window where you would normally copy and paste the amino acid sequences in FASTA format.  Disregard the two blank boxes (into which sequences are copy/pasted) for a moment.  Instead, we are going to click on a ready-made example of some real data.  If you donít already see it, scroll down until you see the heading Ready-Made Examples and click on the Enolase example.  Sequentially, two dialog boxes will pop up.  Cancel the first (since 4enl is already loaded), and OK the second.  Note that the aligned sequences have now been placed into the Alignment Box (all 6 sequences, including yeast) and the 3D Sequence Box which contains the known PDB 3D structure for enolase from yeast.

  8. Click the SUBMIT button just above the Ready-Made Examples heading.  In a moment, a new Netscape page will open, showing the alignments for the six species.  (This process may take several moments, so be patient.)  The color codings are indicated at the top of the page.  For example, medium green indicates that an amino acid at a specific position is identical for all 6 species.  If your screen is large enough, you will see the 3D structure rotating on another Netscape web page.  Click on that web page to bring it to the front.

  9. The backbone trace of enolase has been colored as indicated. The results are more easily appreciated when the full structure including sidechains is shown with all atoms "spacefilled" (to van der Waals radii). Click on the links Identical, Similar, Different to spacefill all categories.

  10. Point to the 3D model, click and hold down on the mouse button and move the mouse.  This action allows you to rotate the molecule.  The catalytic site is marked by a brown Zn ion (nearly buried) and an easily spotted red-and-yellow sulfate ion which happens to be bound there. Note that the active site is entirely dark green (complete identity), showing billions of years of evolutionary conservation, while the peripheral region of the molecule is yellow.

  11. If you have time, make your own alignment for any molecule of your choice in Biology Workbench. The alignment must include at least one sequence for which a 3D structure is available. Follow the MSA3D instructions in PE to paste the alignment and color the molecule accordingly.