Find the most sequence-similar structure to a protein of unknown structure
Build a VAST alignment to this structure
Use the VAST alignment as a structural template for the query protein
Align the query sequence to the VAST alignment using Cn3D
Introduction
In this exercise we will attempt to find a structural template for
a DNA photolyase from D. melanogaster, accession AAB32328, using a well-
studied group of bacterial photolyases. This
exercise has no real end, but rather is an example of how one might begin a modeling
project!
Steps
Retrieve the protein sequence using Entrez
Use Entrez Protein to retrieve AAB32328.
Click on the accession and review the record.
Locate conserved domains in the protein
Click the Conserved Domains link to view the matching CDs, and then click the Full Report button.
What two major functional domains does the protein contain (look for single-domain CDs, not COGs)?
From what database were these CDs derived?
Find a sequence-similar structure to the query
Click on the link containing the protein accession to the right of "Query sequence" at the top of the page.
Click 'Related Structure' in the Links menu.
Change the menu from 'All MMDB' to 'Non-identical', and click List.
View the BLAST results
Change the display menu from 'Graphic' to 'Table' and click List.
Based on their names, do the top few hits seem to be reasonable candidates for homologs?
Analyze the BLAST results
Make a note of the most similar structure. What is its e-value?
Make a note of the next three most sequence-similar structures. What are their e-values?
Analyze the locations of matching CDs
Change 'Table' back to 'Graphic' and click List.
Which CD does the top structure match best?
Do any of the structures fully match both CDs?
Retrieve VAST neighbors to the target structure
Since the two single-domain CDs in AAB32328 are not curated records, we will build a VAST multiple alignment as a potential template, using the most similar structure as the master.
Click on the accession of the most similar structure.
Click on the grey chain A bar to load the VAST neighbors.
Analyze the VAST neighbors
Change the List subset menu to "High redundancy" and click List.
Compare the three best VAST neighbors to the sequences found by BLAST.
How do the VAST alignments compare to the BLAST alignments? Which
are better?
Import a VAST alignment into Cn3D
Change the menu from 'Graphics' to 'Table' and click List.
Check the boxes to the left of the three best VAST hits that are photolyases and that are not identical to
the query (check the %id column).
Click "View 3D Alignment".
Clean up the VAST alignment
In the sequence window, select Edit / Enable Editor.
In the structure window, select Style / Coloring Shortcuts / Secondary Structure.
Using the scroll bar in the sequence window, carefully scan the alignment for blocks of fewer than 4 residues.
Delete these blocks by selecting Edit / Delete Block, and then clicking on the block to delete.
What kind of secondary structure elements were these little blocks in?
Import AAB32328 into Cn3D
Select Imports / Show Imports.
In the Imports window, select Edit / Import Sequences.
Select Network and enter AAB32328.
You should see AAB32328 (gi 688103) paired, but unaligned, to the master structure.
Alignment trial 1: PSI-BLAST
Select Algorithms / BLAST/PSSM Single.
Position the pointer over the sequence pair, and click.
Position the pointer over the aligned pair, and find the e-value of the
alignment shown in the bottom of the window.
Alignment trial 1: PSI-BLAST
Look for red shaded areas that indicate portions of the PSI-BLAST alignment that do not match the VAST alignment), and also for green shaded areas that indicate geometry violations.
What portion(s) of the sequence did not align?
Alignment trial 2: BLOCK Aligner
Select Algorithms / Block Align Single.
Click on the paired sequences.
In the options window, accept all defaults except uncheck "Global Alignment."
Fixing a geometry violation
Find the two green-shaded residues in the bottom row. The shading indicates a
geometry violation, usually meaning that the blocks are too tight and need to be relaxed (shortened).
In the alignment window, choose Mouse Mode / Horizontal drag
Fixing a geometry violation
Find the block on the left side of the geometry violation. Note that this block ends with random coil.
Click on the block end (->) and drag it one residue to the left.
Repeat the BLOCK Aligner. The green should disappear!
Now for the rest of the sequence...
What portion of the sequence is still not matching?
At this point we will focus mainly on this problematic region.
Alignment trial 3: Threader
Choose Algorithms / Thread Single.
Click on the paired sequences.
Accept all defaults, but uncheck "Merge results after each
row is threaded".
Click OK to begin threading. The Threader will now attempt to align
only the portions of the sequence not already in blocks. It may take a few minutes.
Merge the trial alignment
When you see blocks appear, the threader is finished.
Position the pointer on the aligned sequences to see the score in the bottom
of the window.
Select Alignments / Merge All to merge the new alignment
into the VAST alignment. AAB32328 now appears as the bottom row.
Analyze the new alignment
You can now evalute the alignment by coloring
by sequence conservation, hydrophobicity (looking for conserved
hydrophobic cores), or by finding the residues that contact the FAD
cofactor.
This is a very preliminary example of using the threader, which in general should be run several times using different block alignment structures and combinations of frozen blocks, and then analyzing the threading scores and resulting alignments. This preliminary alignment is only the beginning!
The Alignment Challenge: An enzyme of unknown structure