CootTutorial

Adaptation of Paul Emsley's distributed Tutorial (Sept '08) by Michael Chapman, Oct '09

1 Mousing

The following is for quick reference, once you have some objects on the display to view.

Left-mouse Drag	Rotate view
Ctrl Left-Mouse Drag	Translates view
Shift Left-Mouse	Label Atom
Right-Mouse Drag	Zoom in and out
Middle-mouse	Centre on atom
Scroll-wheel Forward	Increase map contour level
Scroll-wheel Backward	Decrease map contour level

2 Introductory Tutorial

In this tutorial, we will learn how to do the following:

Start Coot
Display coordinates
Display a map
Zoom in and out
Recentre on Different Atoms
Change the Clipping (Slab)
Recontour the Map
Change the Map Colour
Display rotamers and reﬁne residue

2.1 Get the ﬁles

Hopefully, we will be making the files available to you in a convenient location. If so, skip the rest of the section 2.1.

If not and you are doing this tutorial independently, then the coordinates and data files are available from several locations.

The easiest might be from the examples sub-directory of the Coot installation, such as: C:\Programs\WinCoot_0.6\examples.

Failing that, they are available from Paul Emsley's web site. You can open your web browser to the http: address below, or use the Unix command wget, as below:

$ wget http://www.ysbl.york.ac.uk/˜emsley/coot/tutorial/tutorialmodern.pdb

$ wget http://www.ysbl.york.ac.uk/˜emsley/coot/tutorial/rnasa-1.8all refmac1.mtz

2.2 StartCoot

To use coot,in a terminal window, type:

Unix (MacOS)

$ coot

Windows

This is a little more complicated. The default installation will provide a desktop icon which you can use considering the caveats below. You should only do this if it is a single-user computer, and probably only if you use Coot only for a single project. The problem is that the started from a Desktop icon, Coot will store "state" and backup files in the installation directories, and will not differentiate those of different people / projects. You really want to run Coot in a specific directory for each directory. The documented way to do this is to copy the link to the directory and edit the "run in" setting in the properties. An easier approach is just to copy the run_wincoot.bat file from the installation directory to each user directory where you want to run coot. Then double-click this run_wincoot.bat.

Unix, MacOS & Windows

From here on, the running will be mostly the same, independent of operating system. When started you should get a blank window like that shown below. In future illustrations, this documentation will use a white background, but yours will continue to be dark.

2.7 Recentre on Different Atoms

Interactively

Click Middle-mouse over an atom in the graphics window [Coot recenters on that atom]
Ctrl Left-mouse & Drag moves the view around. If this is a too slow and jerky:

Experiment!

Using dialog:

Select “Draw” from the Coot menu-bar
Select “Go To Atom...” [Coot displays the Go To Atom window]
Expand the tree for the “A” chain
Select1 ASP in the residue list
Click “Apply” in the Go To Atom window
At your leisure, use “Next Residue” and “Previous Residue” (or “Space” and “Shift” “Space” in the graphics window) to move along the chain.

Figure5: Coot’s Go To Atom Window.

You can display the contacts too, as you do this:
- Select “Measures” from the Coot menu-bar
- Select “Environment Distances. . . ”
- Click on the “Show Residue Environment?” check-button
Also Click “Label Atom?” if you wish the Calpha atoms of the residues to be labeled.
- Click “OK” in the Environment Distances window
- Click “Apply” in the Go To Atom window

[You can’t change the color of the Environment distances]

You can turn off the Environment distances if you like.

Figure6: Coot showing Atom Label and environment distances.

3 Model Building

“So what’s wrong with this structure?” you might ask. There are several ways to analyze structural problems and some of them are available in Coot.

Validate --> Density Fit Analysis --> tutorial-modern.pdb. [Coot displays a bar graph]

Look at the graph. The bigger and redder the bar the worse the geometry. There are 2 areas of outstanding badness in the A chain, around 41A and 89A. Let’s look at 89A ﬁrst -click on the block for 89A. [Coot moves the view so that89ACAis at the centre of the screen]

3.1 Rotamers

Examine the situation. . .[ The side chain is pointing the wrong way. Let’s Fix it... Note that the incorrect rotamer has been squeezed into the structure by rotation of the carbonyl out of the density. It is very common for one error to lead to other "compensating" errors in the neighborhood. Thus, if one had been looking at outliers in backbone torsion angles and residues 88-89 had been highlighted, one would look around to see if there is a problem in neighboring atoms that could be causing the strange backbone torsion angles.]
Select “Rotamers...” from the toolbar.
In the graphics window,(left-mouse) click on an atom of residue 89A (the Calpha, say) [Coot displays the “Select Rotamer” window. Listed are rotamer configurations that have been mined from a large database of high quality structures. Listed also are the frequencies that such rotamers are found. Usually, you should be trying to choose a common one. Sometimes there will be steric conflicts that mean that it is one of the rare ones that is appropriate.]
Choose the Rotamer that most closely puts the atoms into the side-chain density
Click “Accept” in the “SelectRotamer” window [Coot updates the coordinates to the selected rotamer]
Click “Real Space Reﬁne Zone” in theModel/Fit/Reﬁne window.
In the graphics window, click on an atom of residue 88A and one in residue 90A. [Coot displays the reﬁned coordinates in white in the graphics and either a new “Accept Reﬁnement” dialog or ribbon above the display area. Notice that the Phe ring has been rotated about 30 deg to better fit the density. Note also how the refinement is now able to move the carbonly oxygen into the density even while maintaining good stereochemistry.]
Click “Accept” in the “Accept Reﬁnement” window/ribbon. [Coot updates the coordinates to the reﬁned coordinates. 89A now ﬁts the density nicely.]

3.2 More Real Space Reﬁnement

Now let’s have a look at the other region of outstanding badness:

Click on the graph block for 41A (if you have kept the fit-density dialog, or else use the "Go To Atom" dialog). [Coot moves the view so that 41A CA is at the centre of the screen]
Examine the situation. . .
Residue 41is in a mess and not ﬁtting the density. Can you ﬁx it? [Yes, you can]
The trick is Real Space Reﬁne that zone. So. . .
You might want to try this two different ways:
- First try refining just residue 41.
  - Click on "Real Space Refine Zone" in the toolbar.
  - Click residue 41 A twice.

[As the backbone is far from the correct location, you will likely have to drag the residue into approximately the right place for refinement to converge properly. This is an example of non-linear refinement being local. It finds a local optimum, not necessarily a global optimum. Move residue 41A by dragging the white intermediate atoms with the left mouse button. When you release the button, refinement will start automatically.]

At this point you would normally accept the structure if you think it is an improvement. However, let's reject it, so that you can see an alternative approach.

Now try repeating the real space refinement, but this time refining the zone 40 to 42:
- Click on “Real Space Reﬁne Zone” in the Model/Fit/Reﬁne window
- Select the range 40 - 42 by clicking on atoms in the graphics window [Coot displays intermediate (white) atoms]
- Move the single carbonyl oxygen atom of residue 41 using Ctrl Left-mouse to pick and move(just) that intermediate atom. (If you do this without the Ctrl, the entire zone will move, which is not too useful.)

[When you let go, you may be surprised how well refinement takes care of not just the carbonyl, but it now places the entire side chain in the density.]

[Before you now click "Accept", compare the backbone structures of the intermediate atoms to the old ones. Perhaps you can recognize why the model was no originally built in the correct configuration. Look for something unusual (but valid) in the new intermediate structure. Click accept.]

[Near the end of the side chain of residue 41 you will see a large blob of density. It is seen in both the blue (regular) map and green (a difference map). The difference map shows regions where model is missing (green) or is present where there is no data (red). This large blob suggest that there might be a ligand that needs to be modeled. What? - not an easy answer.]

4 Blobology

4.1 Find Blobs

To be found under Validate (called “Unmodelled Blobs”). You can use the defaults in the subsequent pop-up. Press “Find Blobs” and wait a short while. You will get a new window that tell you that it has found unexplained blobs. Time to ﬁnd out what they are.

4.1.1 Blob3 - in the interests of time, skip to 4.1.2 Blob2 (or 4.1.3 Blob 1)

Let’s start from Blob3 (the blobs are ordered biggest to smallest -Blobs3 and 4 (if you have it) are the smallest).

Click on “Blob3” [Coot centers the screen on a blob]
Examine the situation. . . [We need something tetrahedral there. . . ]
“Place Atom At Pointer” on the Model/Fit/Reﬁne toolbar [Coot shows a Pointer Atom Type window]
“SO4” in the new window. . .
In the “Pointer Atom Added to Molecule:” frame, change “New Molecule” to ‘‘tutorial-modern.pdb”
Click OK.
Examine the situation. . . the orientation is not quite right. Let’s Real Space Reﬁne it (you should know what to do by now. . .)
(“Real Space Reﬁne Zone” then click an atom in theSO₄ twice. Accept) [TheSO₄ ﬁts better now. Blob4 is like Blob3 (isn’t it?).]

4.1.2 Blob2

Click on the button “Blob 2” and examine the density. Something is missing from the model. What? This protein has been co-crystallized with its ligand substrate. That’s what missing: 3’GMP. So let’s add it. . .

On a Mac or Unix computer with RefMac and CCP4 software installed

File --> Get Monomer. . .
Type 3GP in the box (UseUpperCase). Press return. . . [Coot pauses for a few seconds while LIBCHECK and REFMAC run] [3GPappears]

[If you don't have RefMac and CCP4 available, a non-fatal error message should appear by now. Proceed with the WinCoot directions. If no error message occurs, skip to "All computers".]

WinCoot on a PC (RefMac / CCP4 generally not available):

Open the ﬁle monomer-3GP.pdb.

All computers...

It is generally easier to work without hydrogens, so let’s delete them
- “Delete. . . ” from the Model/Fit/Reﬁne window
- “Hydrogens in Residue”
  click on an atom in 3GP [Hydrogens disappear] The 3GP is displaced from where we want it to be.
“Rotate/Translate Zone” from the Model/Fit/Reﬁne window
Click on an atom in 3GP twice [Intermediate(white) atoms appear]
Drag it or use the sliders to move the intermediate fragment to approximately the right place.
Click “OK” in the slider window
Now optimize the ﬁt using “Real Space Reﬁne Zone” like we did before.
Now merge this ligand into the protein:
- Calculate --> Merge Molecules. . .
- Click (activate) “monomer-3GP.pdb”
- into Molecule: tutorial-modern.pdb
- Merge

4.1.3 Blob1

Click on “Blob 1”
Examine the situation. . .What is this density? [We need to add residues to the C-terminus of the A chain.] The missing residues are GLN, THR, CYS (QTC).
Calculate --> Fit Loop. . . [Coot pops-ups a Fit Loop dialog] Add residue number 94 to 96 in the A chain. The single letter codes of the extra residues are QTC
Make sure that you can seethe unmodelled density well, then click “Fit Loop” -- Watch. . .(fun eh?)
Examine the density ﬁt. [It should be ﬁne, except at the C-terminus. We need to add an OXT atom to the residue]
Calculate --> Other Modelling Tools. . . --> Add OXT to Residue. . . --> Add it

[Looks to me (Michael) that this auto-building has done a great job of residues 94 & 95, but a lousy one for 96. There is density extending to Cys 7 A, and I suspect that residue 96 should be part of a disulfide with its side chain pointing in the opposite direction. If you are ahead of everyone else at this point, you might want to use the skills you have used to model the Cysteine to point in the other direction.]

Ending Coot

This is where we will leave the distributed tutorial. Before exiting, one would normally save coordinates and the state. Decide whether you want to do this (using menu File) or whether you would want the files to be ready to start the tutorial from scratch. Then File --> Exit.

More Challenging Maps

The examples used for the Coot tutorial are chosen to put Coot in its best light, and to be easy on the student learning Coot. i.e. they are examples where the density is clear and at high resolution making it relatively easy to see where the model atoms should be placed. Here is an example from the other end of the spectrum, one of the poorer parts of a structure determined at 2.4 A resolution. While these regions might represent only a few percent of the structure, they take a big proportion of the time remodeling. The files will only be available locally in tutorials mentored by Michael Chapman. In the future they might be available from the PDB database, but not now.

Start up Coot again, rejecting the option to use a previously saved state. See if you can use the skills that you have learned to examine the differences between the original model (1M80.pdb) and one (1M80-coot-10.pdb) that was remodeled using an improved map (calculated from ak_apo_091022d_002_map_coeffs.mtz). The region near chain A residue 294 is the most illustrative. A few residues to each side, the density is reasonably good. However, here it may appear missing, until you lower the contor level. Perhaps you can see that one of the models is a better fit, but the density through both models is weak, and it is not difficult to see how the worse model might have been built into an inferior map. If you look carefully, you can see that the error created a frame-shift of one residue. Thus everything is off in terms of sequence until there is another error a few residues away. If you have a bunch of time, you could hide the corrected structure, and try to remodel 1M80.pdb into the new path of the density. When you are done, compare to 1M80-coot-10.pdb to see how well you have done.

Colophon

This document was originall written using XEmacs 21.5 in LATEX using AUCTEX and is distributed with theCoot source code. It was converted to html and edited in SharePoint Designer.