In this example, we will make a model for the part of the
mouse FAS antigen ligand (SWISSPROT entry P41047), which is known to be
similar to the tumor necrosis family.
The concept of modelling is to find one or more suitable template(s)
(at least 20-25% aa similarity) with known structures, align the
templates, align the sequences of interest with the templates and thread
it onto the template structure. The last step is energy minimisation which
is done by a computer program.
In this exercise we'll use the Swiss PDB viewer (Deep View) as it's
available for free and also in a version for Microsoft Windows (which most
of you probably use on your own machines).
- Click on the "visible" check box of the Control Panel, to show the
sequence we want to model, and choose the "Update threading now" item
of the "Swiss-model" menu (this item is not accessible if the "Update
Threading Display automatically" item is enabled; which is the case
by default). While you are here, enable the "Update threading display automatically"
item of the "Swiss-model" menu if it is not already done.
FASL should now be "wrapped" around 1TNRA.
- Now, colour the 1TNRA protein in yellow, and the 2TUNA protein in
green (this is done with the control panel, by shift-clicking on any
colour box located at the right of the panel; be sure that the protein
you want to colour is the one displayed in the control panel.
Alternatively, use "Color" - "Layer" as before and then CPK colour FASL.).
Threading FASL onto 1TNRA
- Scroll the align window up to the first big gap and select the region
starting from GQSCN and finishing with LNHK. To select also corresponding
amino acids of 1TNRA and 2TUNA, hold the shift key while you do so.
- Hit the "Enter" key, and hit the "=" key
to recenter the view.
You can see that there is a long "false" bond, corresponding
to the gap.
- Should you want to modify the location of the gap, all you need to
do is to make sure the align window is active and select some amino-acids
flanking the gap (for example SCN) and hit the left or right arrows
keys to "slide" the selected residues at the other part of the gap.
You can also select a residue and use the "space" or "backspace" keys
to add or remove gaps at the selected residue.
Note: To insert a gap in the template sequences, click on the
first template and hit the space key. A gap will be added in all the
templates. To add a gap or modify the structural alignment, you have
to hold down the "Control" key while hitting the space key.
- The alignment was correct. Put everything back as it was.
Now we will demonstrate the use that can be done
of mean force potential to help you thread correctly a protein. Note
that this tool should be used with caution.
- Make sure the current layer is FASL, and click on the little white
arrow located in the left side of the Align Window.
The window expands, and displays a curve depicting how each residue
likes its surrounding. If a residue is "happy", its energy is below
zero, whereas an "unhappy" residue will have an energy above the zero
axis. This is the mean force potential energy, computed according
to a "Sippl-like" method.
[Sippl, J.M. (1990)
Calculation of Conformational Ensembles from Potentials of Mean Force:
an approach to the knowledge based prediction of local structures in
J. Mol. Biol. 213,859-883]
- Click on the "smooth" text, and set a smoothing factor of 1. It means
that the energy off each residue will be the average of itself plus
the energy of 1 flanking residue on each side.
- Enable the "Auto Color by Threading Energy" item of the "Swiss-Model"
- Click on the "E= " text, this will recompute the energy for the
- Select some residues (5-8), and move them with the arrow keys. As you can
see, the curve changes, and the E value changes.
- When you're done playing, put everything back the way it was.
Note: this tool provides hints and should be used in conjunction
with the analysis of the hydrophobicity of residues, the overall structure,
and common sense! It works better for displacement of large fragments
than for just one or two residues, as it analyses the mean force potential
for the whole protein.
Validating your alignment and submitting a modelling
- You can evaluate how good your threading is by using the "aa
Making Clashes" and "aa Making Clashes with Backbone" items of the "Select" menu. This will allow you to quickly
focus on potentially problematic regions. You can then choose the "Fix Selected Sidechains"
item of the "Tools" menu, which will browse the rotamer library to choose
the best rotamer, exactly as during a mutation process. By repeating
the "Select aa making clashes" process, you should see that far less
amino-acids are making problems. If not, this is probably a good clue
that your threading is incorrect.
Important Note: Actually fixing the sidechains is just for you
to evaluate prior to submitting the request whether the model will really
be very bad or not. It will have absolutely no incidence onto the model
building, as sidechains are reconstructed anyway. This reconstruction
process is useful if - and only if - you intend to directly use an external
- Another handy tool for locating areas causing trouble is the
"Ramachandran Plot" in the "Wind" menu. A Ramachandran plot visualises
the torsion angles of the peptide backbone and almost all
residues of natural proteins are found within the areas denoted on the plot. An exception is
glycine and alanine residues. Problems with the peptide backbone are
quickly spotted with this tool.
Click here for more information on accuracy determination.
- When everything seems okay, you can submit a modelling request to
Swiss-Model simply by choosing the "Submit modelling request" of the
"Swiss-model" menu. By default, you will get a Swiss-PdbViewer project
file, with your model aligned onto the templates you used, and ready
for comparison. The project comes back at the e-mail address you have
- If there's time left you can always experiment with your own
favourite protein ....