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:
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
Enolase: Sample for which you can view the alignment differences
in a 3D rotating molecule:
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
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.í
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
Click on Explore More, then at the bottom of the QuickViews page,
click on Advanced Explorer.
Now you are ready to do an alignment. In the upper left frame, click
on the hyperlink ëMSA3D Multiple Sequence Alignment Coloring."
Click on either ëTry a ready-made demonstration exampleí or on ëMSA3D
ALIGNMENT FORM.í (MSA3D = multiple sequence alignment in 3D.)
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.
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.
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
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.
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.