P212121

1) Open the desired coordinate files in Coot (click here if you need some help)
(You know you have been looking at structures too long when they start to look like faces.)

2) Under Calculate you have two methods of superimposing:
SSM Superpose (we will go with this option in this example: Calculate -> SSM Superpose…) or LSQ Superpose
Note: SSM Superpose stands for secondary-structure matching and if you need to do it outside of Coot there is a server.

3) Select which PDB you would like to move and apply:

The structures should now be superimposed:

If you need to mutate multiple residues simultaneously there is a great option to use in Coot instead of mutating them individually.

Calculate -> Mutate Residue Range…


1) Make sure you are mutating the correct PDB file
2) Select Chain
3) Enter the residues range by number that you would like mutated
4) Enter desired sequence
5) Option for to autofit the mutated residues (you need to have a map for this)

What are some of the possible uses?

1) Create a poly-alanine chain
2) Mutate a structure that varies between species
3) Mutate hyper variable regions

Note: You cannot add residues with this option.

I still see quite a few people downloading a PDB and then opening it in Coot under File. Coot has the ability to open a PDB directly, if you know the 4 digit access code.


Note: You can also open maps from the Electronic Density Server (EDS) right beneath

The very basics of using Coot for those that have not used this program. I am a big fan of Coot and think if you give it a try then maybe you will be too. This post will cover three basics: 1) opening a PDB file, 2) loading maps, and 3) how to move about using your mouse.

1) Opening a PDB file with Coot

Find your desired PDB file (coordinates) and select it
Tip: Using the Filter option can save a lot of time

2) Loading a 2Fo-Fc map

Simply hit ‘Ok’ and you did it!

Here is how to load a Fo-Fc map:
Select (its below the default option) and open with the ‘DEL’ for the phases and amplitudes

3) Moving the macromolecule with your mouse
Left click (hold) allows you to rotate the coordinates
Right click (hold) allows you to zoom in (move toward you or right) and out (move away or left)
Center click allows you to center (focus) on an atom

You can do this by using Coot and a simple text editor. This can be helpful in preparing figures that discuss biological function. The steps are as follows:

1) Display the symmetry related molecules:
Draw -> Cell & Symmetry…
Note: I changed the master switch to ‘Yes’, adjusted the radius to 50, and then hit ‘Apply’

Below is an overview of the crystal packing:

2) Attain a perspective that will allow you to easily click on your desired symmetry molecule. Below is a zoomed in view of that same packing.

3) File -> Save Symmetry Coordinates
Your pointer will become a small cross shape. Now, click on your desired symmetry molecule.

4) Give the file a name you can remember for at least 15 seconds:

5) Open your original pdb file and then newly generated symmetry pdb files in a simple text editor. I am using gedit on a linux machine in this example.

6) Rename the newly created pdb file from ‘chain A’ to ‘chain B’.
Note: If you do not rename the chains, the atoms may create strange connectivity such as long distance disulphide bonds in cysteines.

Important: I have an ” A ” (there is a space on either side of the A). I always scroll through the pdb file to make sure I’m not replacing the letter A in an undesired place. For example, if you simply replace “A” you will mess up all your CA (carbon alphas) and residues abbreviations such as VAL.

7) Copy and paste ‘chain B’ into your original pdb file. You should copy/paste the ‘ATOM’ section and leave out the header (everything above the ATOM section) and the END (it is at the bottom of your pdb file).

Rename the file and save using ‘Save As’. This way you will have the original monomer available should you need it in the future. If you accidentally overwrite the original, you can always just delete the second chain and re-save the file.

9) Display your newly created pdb file and make sure that your new pdb file is correct.

The program COOT by Paul Emsley and Kevin Cowtan is simply, awesome (pdf paper citation).  I usually read the ‘tips’ that appear when starting the program and wanted to compile a comprehensive list:

Note: if this is difficult to read try pressing ‘ctrl then +’ as needed

1. To centre on a particular atom, click it with middle mouse (if that doesn’t seem to work it may be because the molecule is not active).
2. + and – on the keyboard change the contour level
3. To move just one atom (in Regularize, RS Refine, or Rotate/Translate mode) use Ctrl Left-mouse to pick an atom (you have to be accurate).
4. There is a mailing list for Coot development and discussion at http://www.jiscmail.ac.uk/lists/coot.html
5. Use Ctrl Left-mouse to drag (for example) a blob of density to the the pointer
6. Slow recentering? Try Draw -> Smooth Recentering and reduce the number of steps
7. Want distance of the atoms to the pointer? Use Measures -> Pointer Distances…
8. To label an atom: Shift Left-mouse on it
9. To make atoms be insensitive to clicking, deactivate it by unclicking that molecule’s "Active" button in the Display Control window
10. Can’t label or centre on some symmetry atoms? It’s a known bug. (For now, drag that atom closer to the centre of the screen).
11. Use function key ‘F8′ to make a rendered snapshot.
12. Use the Ctrl to rotate the view when changing Chi angles.
13. Too many cis peptides when using dragged refinement? Use the
    Planar Peptide Restraints’ suggested in the Coot FAQ.
14. Use the Ctrl to rotate the view when using Delete.
15. When in skeleton mode, new skeleton can be displayed around the current point using the ‘S’ key.
16. When in baton mode, the baton can be rotated independently from the Guide Points by using the ‘B’ key (it’s a toggle).
17. When Editing Chi Angles, switch between the angles quickly using the ‘1′, ‘2′, ‘3′, ‘4′ keys.
18. Use "refmac-extra-params" to pass refmac your personal parameters.
19. Use "(poly-ala imol)" to turn molecule number imol into poly-ALA. Use "(poly-ala imol ‘SER)" to turn it into poly-SER."
20. Use "(fit-protein imol)" to rotamer search and real-space refine all residue of molecule number imol."
21. Shift Ctrl right-mouse rotates round screen Z.
22. Ctrl + right-mouse + horizontal (left to right) mouse movement moves the view in screen Z.
23. Ctrl right-mouse up-down drag changes the slab.
24. Use "(set-idle-function-rotate-angle 0.05)" to change the spin speed.
25. (ligand-expert)" enables the GUI editting of some ligand-fitting parameters.
26. Use keyboard + and – to zoom in Ramachandran and Kleywegt Plots
27. The ‘U’ key undoes last nagivation (e.g. re-centering on new pdb file).
28. The ‘D’ and ‘F’ keys change the clipping/slabbing.
29. (view-matrix)" prints the current view matrix, useful for molscript, perhaps.
30. Baton-building low resolution maps is better done with maps that have increased sampling rate (2.0 or 2.5).
31. Coot can read SHELXL .res files. (It can write them too.)
32. Clear All Atom Labels" can be found under Measures -> Distances & Angles. Obviously.
33. Esc and Return are keyboard accelerators for Reject/Accept for Refinement and Regularization.
34. Restraints for alpha helical and beta-strand structure can in the Refinement/Regularization Control Panel
35. To disable coot tips: add "(no-coot-tips)" to your ~/.coot file.
36. It is possible to accidently invert chiral centres. Use Validate -> Chiral Centre to check.