CHM 576
Problem Set 3
In this problem set, you will complete the solution of a protein structure. It is not
uncommon to find relatively mobile loops on the surface of a protein that require building
as the last step in completing a protein structure. I have simulated such a scenario here by
deleting a loop of six amino acids from a nearly complete structure. You will recognize
the protein as the same structure included with Coot in the examples folder.
I have modified Chain B by deleting amino acids 60 – 65. Your job will be to recreate
this loop with the amino acid sequence Pro Gly Ala Arg Thr Arg.
1.1 Download the files
Open your browser to the course web site and download two files to the same folder on
your computer. You will need a coordinate file (problem3.pdb) and a reflection/map file
(problem3.mtz).
1.2 Start Coot
Double-click the icon on your desktop or select through the Start menu.
1.3 Open the coordinate file and reflection file
 Select “File” from the Coot menu bar.
 Select the “Open Coordinates…” menu item
 Find the folder where you saved the files you downloaded previously and select
“problem3.pdb.” Click the “Open” button.




Select “File” from the Coot menu bar.
Select the “Open MTZ, mmCIF, fcf or phs…” menu item
Select “problem3.mtz” and click the “Open” button.
A new window like that shown below will open. Accept the default values for all
options and click “OK.” After a short delay, electron density will display in the
Coot window.
1.4 Beginning of the loop
The last amino acid included prior to the missing loop is number 59 in Chain B. To start,
you need to position the viewer at that location.



Select “Draw” from the Coot menu-bar.
Select the “Go To Atom…” menu item.
Select Chain B and Residue Number 59, the click Apply.
Not ice that clicking Next Residue in the Go To Atom… window takes you to Residue
Number 66. There is a big gap in the protein between amino acids 59 and 66, but there is
a tube of electron density between these amino acids. You will build the correct amino
acids into this loop in the protein. I also prefer not to close this window as it will be
useful for navigating the loop as you rebuild the missing parts of the protein.
1.5 Where am I? In the crosshairs…
When rebuilding a loop of amino acids, it is easy to lose track of the center of the screen
and the amino acid on which you are currently operating. A preference I have is to turn
on a crosshair in the middle of the screen to always indicate



Select “Draw” from the Coot menu-bar
Select the “Crosshairs…” menu item
Click to circle by “On,” then click “OK” to close the “Crosshairs…” window.
2. Rebuilding the loop
Version A
The most obvious way to rebuild missing amino acids is to do so one at a time. A
function in Coot allows you to add an amino acid to the terminus of a chain, and it
frequently places the new amino acid in the correct location and orientation (rather
handy). Let’s start by adding amino acid 60 to the B chain of our protein.

Select “Add Residue…” on right side of the Coot window
OR



Select the Calculate menu-item
Select the Model/Fit/Refine… menu item
Click Add Terminal Residue…
At this point, nothing much happens. Coot is waiting for you to select an amino acid after
which to append the new residue.


Click anywhere on amino acid 59
A new window will open asking you to Accept or Reject the placement of the
new Terminal Residue
If the new amino acid is generally located within the electron density, click Accept. If the
placement just looks wrong, Reject the choice and try again, maybe using another
method.
After Accepting the new Terminal Amino Acid, a peptide bond will be made to amino
acid 59. Now you can more closely inspect the results of your “peptide synthesis.” You
will notice that the new amino acid is an Alanine. This is most handy if you needed
Alanine, but is a good start if another amino acid is required.
The 60th amino acid should actually be Pro rather than Ala. This is easy to fix with the
Simple Mutate function available on the right of the Coot window or in the
Model/Fit/Refine window which you likely still have open:
Use the Go To Atom window to center the crosshairs on Ala 60
Select Simple Mutate
Click Ala 60
A window with the three letter code for each amino acid opens
Click on Pro and Ala will be replaced with Pro
Inspect your new Proline. It fits pretty well into the electron density, but likely needs to
be tweaked a bit.



Select Real Space Refine Zone from the Model/Fit/Refine window
Double click on Pro 60
Accept the new position of Pro in the electron density
Completion of the loop is simply an iteration of the above process for amino acids 61 to
64. You may have noticed that another option for rebuilding, Mutate and Auto Fit is
available in Coot. After adding any amino acid to the terminus of a chain, your next
likely operation is to refine its position. These two operations are combined into one with
Mutate and Auto Fit. You lose the opportunity to Reject the fit of the mutated amino acid,
but you can always start over with Delete to remove the new amino acid completely.
The fourth amino acid in the loop you are rebuilding is Arg, and I’ve found repeatedly
that this amino acid is not placed very well by Mutate and Auto Fit… You can use Real
Space Refine Zone to fix this manually. Sometimes you may find it useful to move one
atom at a time rather than an entire amino acid. Hold down the control key while
moving (left mouse button down) to move just one amino acid at a time.
You will find that Coot thinks it knows better than you and will frequently ignore your
gentle suggestions to move one atom. Be insistent and you can get Coot to eventually see
your way if you are clever. Also use the other tools at your disposal. Finding the correct
Rotamer first will make fitting the entire amino acid into the electron density easier.
If all goes well, your final amino acid will join properly with the remainder of the protein
and you can publish your results, obtain more funding, win a Nobel Prize, and have time
to drink beer. Typically you would need to refine the structure further using methods we
won’t discuss here, but the majority of the structure solution proceeds according to the
methods you just employed.
As a side note, it would be perfectly acceptable to place Ala for each missing amino acids
in the loop, then go through and mutate them to the correct amino acids. It is a matter of
preference frequently, although it is sometime easier to use this method than to mutate
each amino acid as you go.


After rebuilding this loop, be sure to address the other structural errors
indicated in the tutorial.
Save the completed structure and email your results to me.
Version B
Loop rebuilding is a sufficiently common exercise that methods have been developed to
(semi-) automate the process. In this half of the problem set, we will use these tools to
repeat the exercise accomplished above.
First, you need to close the molecule (be sure you saved a copy first!) and map used
above. This can be done most easily using Close Molecule/Map from the File menu-item
in Coot.
Reload the protein and map file in Coot.

Select the Calculate menu-item in Coot

Select Fit Loop…
A new window will open to automagically build your missing loop.



Be sure to select Chain B!
Residue Numbers 60 to 65 inclusive
Enter the proper one letter code for the sequence of the amino acids in the loop.
Sometimes more than one possible structure for the loop region is found. Choose the one
that is most appealing to you and Accept or Reject the solutions if none looks good to
you.
Even a well-built loop will need a little tweaking by hand. Use the tools discussed above
and in the Tutorial to fix these problems.


After rebuilding this loop, be sure to address the other structural errors indicated
in the tutorial.
Save the completed structure and email your results to me!