BIO Assignment Week 12

Assignment for Week 12
Structure and Function

Note! This assignment is currently active. All significant changes will be announced on the mailing list.

 
 

Concepts and activities (and reading, if applicable) for this assignment will be topics on next week's quiz.

 

Introduction

Integrating evolutionary information with structural information allows us to establish which residues are invariant in a family–these are presumably structurally important sites–and which residues are functionally important, since they are invariant within, but changeable between subfamilies.

To visualize these relationships, we will load an MSA of APSES domains with VMD and color it by conservation.

Coloring by conservation

With VMD, you can import a sequence alignment into the MultiSeq extension and color residues by conservation. The protocol below assumes that an MSA exists - you could have produced it in many different ways, for convenience, I have precalculated one for you. This may not contain the sequences from YFO, if you are curious about these you are welcome to add them and realign.

Task:

Load the Mbp1 APSES alignment into MultiSeq.

1. Access the set of MUSCLE aligned and edited fungal APSES domains.
2. Copy the alignment and save it into a convenient directory on your computer as a plain text file. Give the extension .aln .
3. Open VMD and load the 1BM8 structure.
4. As usual, turn the axes off and display your structure in side-by-side stereo.
5. Visualize the structure as New Cartoon with Index coloring to re-orient yourself. Identify the recognition helix and the "wing".
6. Open Extensions → Analysis → Multiseq.
7. You can answer No to download metadata databases, we won't need them here.
8. 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 ALN files (these are CLUSTAL formatted multiple sequence alignments).
9. Open the alignment file, click on Ok to import the data. If the data can't be loaded, the file may have the wrong extension: .aln is required.
10. 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 1BM8 sequence and the Mbp1_SACCA 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.

Task:

Bring the 1MB1 sequence in register with the APSES alignment.

1. 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. Note: don't include the 1BM8 sequence - this is just for the aligned sequences.
2. Select Edit → Enable Editing... → Gaps only to allow changing indels.
3. 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 1BM8: S I M ... . Note: Have patience - the program's response can be a bit sluggish.
4. Now insert as many gaps as you need into the 1BM8 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 a saved session. It is a bit annoying but not mission-critical. But to be able to do that, you might want to save your session every now and then.)
5. When you are done, it may be prudent to save the state of your alignment. Use File → Save Session...

Task:

Color by similarity

1. 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.
2. Navigate to the Representations window and create a Tube representation of the structure's backbone. Use User coloring to color it according to the conservation score that the Multiseq extension has calculated.
3. Create a new representation, choose Licorice as the drawing method, User as the coloring method and select (sidechain or name CA) and not element H (note: CA, the C-alpha atom must be capitalized.)
4. Double-click on the NewCartoon representation to hide it.
5. 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.

Study this structure in some detail. If you wish, you could load and superimpose the DNA complexes to determine which conserved residues are in the vicinity of the double helix strands and potentially able to interact with backbone or bases. Note that the most highly conserved residues in the family alignment are all structurally conserved elements of the core. Solvent exposed residues that comprise the surface of the recognition helix are quite variable, especially at the binding site. You may also find - if you load the DNA molecules, that residues that contact the phosphate backbone in general tend to be more highly conserved than residues that contact bases.

That is all.

 

Links and resources

 

Footnotes and references

 

Ask, if things don't work for you!

If anything about the assignment is not clear to you, please ask on the mailing list. You can be certain that others will have had similar problems. Success comes from joining the conversation.

• Do consider how to ask your questions so that a meaningful answer is possible:
  • How to create a Minimal, Complete, and Verifiable example on stackoverflow and ...
  • How to make a great R reproducible example are required reading.