BIN-PHYLO-Conservation scores
Calculating Conservation Scores from Phylogenetic Trees
(Quantifying conservation scores in trees)
Abstract:
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Contents
Evaluation
Evaluation: NA
Contents
Coloring a 3D model by conservation
With the superimposed coordinates, you can begin to get a sense whether either or both binding modes could be appropriate for a protein-DNA complex in your Mbp1 orthologue. But these are geometrical criteria only, and the protein in your species may be flexible enough to adopt a different conformation in a complex, and different again from your model. A more powerful way to analyze such hypothetical complexes is to look at conservation patterns. With VMD, you can import a sequence alignment into the MultiSeq extension and color residues by conservation. The protocol below assumes
- You have prealigned the reference Mbp1 proteins with your species' Mbp1 orthologue;
- You have saved the alignment in a CLUSTAL format.
You can use Jalview or any other MSA server to do so. You can even do this by hand - there should be few if any indels and the correct alignment is easy to see.
Task:
- Load the Mbp1 APSES alignment into MultiSeq.
- (A) In the MultiSeq Window, navigate to File → Import Data...; Choose "From Files" and Browse to the location of the alignment you have saved. The File navigation window gives you options which files to enable: choose to Enable
ALNfiles (these are CLUSTAL formatted multiple sequence alignments). - (B) Open the alignment file, click on Ok to import the data, it will take a short while to load. If the data can't be loaded, the file may have the wrong extension: .aln is required.
- (C) find the Mbp1_SACCE sequence in the list, click on it and move it to the top of the Sequences list with your mouse (the list is not static, you can re-order the sequences in any way you like).
You will see that the 1MB1 sequence and the APSES domain sequence do not match: at the N-terminus the sequence that corresponds to the PDB structure has extra residues, and in the middle the APSES sequences may have gaps inserted.
- Bring the 1MB1 sequence in register with the APSES alignment.
- (A)MultiSeq supports typical text-editor selection mechanisms. Clicking on a residue selects it, clicking on a row selects the whole sequence. Dragging with the mouse selects several residues, shift-clicking selects ranges, and option-clicking toggles the selection on or off for individual residues. Using the mouse and/or the shift key as required, select the entire first column of the sequences you have imported.
- (B) Select Edit → Enable Editing... → Gaps only to allow changing indels.
- (C) Pressing the spacebar once should insert a gap character before the selected column in all sequences. Insert as many gaps as you need to align the beginning of sequences with the corresponding residues of 1MB1:
S I M ... - (D) Now insert as many gaps as you need into the structure sequence, to align it completely with the Mbp1_SACCE APSES domain sequence. (Simply select residues in the sequence and use the space bar to insert gaps. (Note: I have noticed a bug that sometimes prevents slider or keyboard input to the MultiSeq window; it fails to regain focus after operations in a different window. I don't know whether this is a Mac related problem or a more general bug in MultiSeq. When this happens I quit VMD and restore the session from a saved state. It is a bit annoying but not mission-critical.)
- (E) When you are done, it may be prudent to save the state of your alignment. Use File → Save Session...
- Color by similarity
- (A) Use the View → Coloring → Sequence similarity → BLOSUM30 option to color the residues in the alignment and structure. This clearly shows you where conserved and variable residues are located and allows to analyze their structural context.
- (B) You can adjust the color scale in the usual way by navigating to VMD main → Graphics → Colors..., choosing the Color Scale tab and adjusting the scale midpoint.
- (C) Navigate to the Representations window and apply the color scheme to your tube-and-sidechain representation: double-click on the NewCartoon representation to hide it and use User coloring of your Tube and Licorice representations to apply the sequence similarity color gradient that MultiSeq has calculated.
- Once you have colored the residues of your model by conservation, create another informative stereo-image and paste it into your assignment.
Self-evaluation
Further reading, links and resources
Notes
About ...
Author:
- Boris Steipe <boris.steipe@utoronto.ca>
Created:
- 2017-08-05
Modified:
- 2017-08-05
Version:
- 0.1
Version history:
- 0.1 First stub
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This page is only a stub; it is here as a placeholder to establish the logical framework of the site but there is no significant content as yet. Do not work with this material until it is updated to "live" status.
