BCB
590
Lab
6
Name _____________________________
Function
Prediction and Annotation
Objectives
- Learn how to infer the function of a protein
on the basis of sequence similarity to proteins of known function
- Predict functional residues on the basis of
conservation using ConSurf
- Learn to use some advanced features of PyMOL
to infer functional residues from structural models
Exercises
Required
questions are in red.
Note: If you were not
able to attend the regularly scheduled lab section, it may help to review the
background lecture slides, which can be downloaded from the course webpage.
Please feel free to ask a TA if you have any questions regarding these slides.
In
the previous lab, we saw that
1)
ConSurf (http://consurf.tau.ac.il/)
One of the simplest ways to predict which residues are functionally important
is on the basis of conservation. One webserver that can be used to predict
functional residues on the basis of sequence alone is ConSeq , which calculates
a sequence conservation score for a user-provided sequence. In today’s
exercise, we will actually be using the ConSurf server designed by the same
group, as this allows us to view conserved residues on a structure we will
provide. The conservation calculations are the same, without utilizing the In
this exercise, we will continue to use the Myb protein from the previous lab.
As we noticed during yesterday’s exercise, the model produced by SWISS-MODEL is
not necessarily useful to us if our end goal is to determine the DNA-binding
residues, because the template it selected (PDB ID: 2aje) is not one of the
structures in which the protein is complexed with DNA. In today’s exercise, we
will resubmit the Myb sequence and specify the particular structure we wish to
use as a template.
>MYB305
MDKKPCNSQDVEVRKGPWTMEEDLILINYIANHGEGVWNSLAKSAGLKRTGKSCRLRWLNYLRPDVRRGNITPEEQLLI
MELHAKWGNRWSKIAKHLPGRTDNEIKNYWRTRIQKHIKQAENMNGQAANSEQNDHQEGSSSHMSSAGPTETYSPTS
YSANIDTTFQGPFLTETNDNIWSMEDIWSMQLLNGD
a.
Submit the sequence
to SWISS-MODEL as in the previous lab but this time type 1h8aC in the “Use a specific template” field.
b.
Download the model
structure and save it in a folder for later use.
c.
Go to the ConSurf
website (http://consurf.tau.ac.il/) and
enter the PDB file you’ve just downloaded under the “Enter your own PDB file”
option. SWISS-MODEL does not provide chain information in their PDB files, so
make sure to type “none” in the Chain Identifier box. Scroll down and click “Submit”.
d.
Click on the link to
download all results
e.
Click on the link
near the bottom of the page for displaying conserved residues in PyMOL near the
bottom of the page, and follow the instructions on the resulting page
f.
If you’ve done this
correctly, you should now have a PyMOL session with a group of selections
corresponding to each of the different conservation classes, with blue colors
representing least conserved residues and magenta representing those that are
most conserved.
g.
Display
this molecule using the spheres representation, then save an image of it and
submit this image with your assignment
2)
Advanced PyMOL
commands 
Now, we want to see if the residues predicted on
the basis of sequence appear to actually be the functionally important
residues.
a.
Download the PDB file
for the template we used: 1h8a
b.
Load this file into your
current PyMOL session by using the Open command under the File menu.
c.
We only want to use
the DNA from this file, so we will create a separate object for the DNA atoms,
and then delete the rest. Use the resn command from the last lab to create a
selection containing only the DNA. Remember that you can use the Boolean OR
operator to select multiple bases at once. The names for the nucleic acid bases
are DA, DT, DG, and DC
d.
Under your DNA
selection, click on the A (Action) button and select “create object”.
e.
Now we can delete the
1h8a object by clicking A next to it and selecting “delete object”.
f.
If our modeled
protein is not already aligned with the appropriate region of the actual
structure, we can align them using the command align <modelname>, 1H8A
g.
You should now have
only the dna molecule and our modeled protein structure displayed. Do the conserved residues appear to coincide well with the
ones in the protein-DNA interface?
h.
In addition to visual
inspection, we can actually use PyMOL to define which residuese are in the
interface, by using the around command
to select those atoms within a specified distance of the dna molecule. One
simple definition of interface is to consider any atoms within 5 angstroms of
each other to be interacting. Use the command select interface, dna around 5
AND (NOT dna) to create a
selection of all of the atoms that fit this definition.
i.
This will only select
the atoms that are within this specified distance, but we want to label the
entire residue for any residue that has any atoms that meet the criteria. We
can do this by using the command select interface, byres interface. Using this definition,
how well does the set of highly conserved residues overlap with the set of
actual interface residues? Why do you think some of the highly conserved
residues may be far from the interface?
j.
We can also draw any
hydrogen bonds between the two molecules by using a particular mode of the distance
command. First, we need to create
a selection called “protein” by defining protein as everything that is NOT DNA
(try to figure out how to do this)
k.
Draw the bonds by
typing dist hbonds, protein, dna, mode=2
l.
Use the appropriate
set of commands to display the dna molecule in the sticks representation, and
the protein as cartoon representation with only interface residues displayed in
the sticks representation. Save a copy of this image
and submit it along with your assignment
3)
Other servers
Each year, the journal Nucleic Acids Research (NAR) produces an issue dedicated solely to new and
updated webservers.
Go to this year’s issue (http://nar.oxfordjournals.org/content/vol36/suppl_2/index.dtl)
and find 2 or 3 servers that appear to be relevant to predicting an aspect of
function relevant to a research question you are interested in. Using one of
the sequences we’ve provided in the previous labs, or a sequence of particular
interest to you, attempt to make predictions on your sequence using the sites
you have chosen.
Briefly explain why you chose the
particular sites and sequences you used.
Attach a copy of your results with your
assignment submission
Please email your completed assignment to Peter.
Email: petez
Domain: iastate.edu