Getting started with COOT

Load coordinates from a PDB file: Use "File" ‑> "Open Coordinates..." and select one or more PDB files to be loaded. PDB files contain the coordinates of atoms and are thus used to store the coordinates of the heavy atoms from the phasing procedure and also for storing the positions of the atom coordinates of the insulin model.

Load electron density from a MTZ file: As soon as phases are available, COOT can either calculate the density map from amplitudes, phases and weights (a) or it can make use of already weighted map coefficients (b).

1. Calculation of electron density from amplitude, phases and weights.

   Go to "File" ‑> "Open MTZ, mmCIF, fcf, phs..." and load the target MTZ file. In the following dialog make sure the correct amplitudes and phases are used. Additionally activate the tick box before "Use weights?") and include FOM into map calculation. Use this option for MTZ files after SHELX phasing.

2. Calculation of electron density from precalculated and weighted map coefficients.

   Go to "File" ‑> "Auto Open MTZ...." and select the MTZ file. COOT will automatically choose the right columns of the MTZ. You can use this after the refinement with REFMAC.

Load electron density from a CCP4 map: Activate "File" ‑> "Open map..." and select a CCP4 map. Typical file extensions are .map or .ccp4. Use this option for displaying anomalous maps.

To switch maps and molecules on and off, or even to delete molecules and maps, use "Draw" ‑> "Display Manager". There is also a button "Display Manager" at the top menu of COOT and you might use the F7 button.

You will recognize, that each map and each molecule has its own unique number. This is very handy, when you are dealing with more than one molecule and while model building to keep track of the different built parts. Sometimes the names of the molecules are very similar and when it comes to save one of the molecules, it is important to have the number of the molecule in mind, which should be saved.

Maps

The scroll wheel is used for changing the contour level of an electron density map. Which map is linked to the scroll wheel in the main window is set up in the Display Manager by clicking on the respective "Scroll" button.

To edit the color of a map push "Properties" next to the map name.

COOT does not calculate the complete electron map at once, but it will display a specific region of density around the "coot cursor". The size (map radius) of the the displayed maps can be changed by using "Edit" ‑> "Map Parameters".

Molecules

With the Display manager you can select which molecules you want to display. Note, that the term "molecule" in this sense could also include single atoms or your C-alpha trace. It can thus be understood as some kind of "molecular object".

Next to the name of your molecule, there are two buttons ("Display" and "Active"). The former will allow you to hide/show the specific molecular object, while the latter allows you to "protect" it against accidental manipulation.

With the next button, you can change the display mode. The most important styles are:

There are three different options to center on a residue:

• Click with the middle mouse button onto one atom of the residue.
• Use "Draw" ‑> "Goto Atom" or hit "F6" or choose the "Go To Atom" symbol (green arrow pointing towards a yellow circle) in the top menu bar of COOT.
• Use the sequence view: "Draw" ‑> "Sequence view"

If you click with SHIFT-left_mouse_button or if you double click on an atom, it will be labeled. If you need further information on a residue or atom use the option "Edit" ‑> "Residue Info".

Go to option "Draw" ‑> "Cell & Symmetry" and activate the "Master Switch: Show Symmetry Atoms?".

Useful hints:

• To analyze a crystal contact, use a small radius of 13 Ang. and display a sphere (Button "symmetry by molecule")
• To analyze a crystal packing, use a large radius and display as CAs (Button "symmetry by molecule")

"Draw" → "Screenshot" → "Simple"

During all model building, it is a good idea to save the results from time to time in order to avoid a loss of the model due to a program crash or user errors. Furthermore, saving a model with a meaningful name will change its name in the Display Manager.

• Make a backup with "File" ‑> "Save State...". This will also save the position and the current view. From time to time also choose a new filename instead of overwriting the previous backup file, e.g. backup_01.scm, backup_02.scm, etc. You can later read these backup files with "Calculate" ‑> "Run Script...".
• Once you finished essential parts of your molecule, you should also save the molecule in form of a PDB file. Do it with "File" ‑> "Save Coordinates...". Make sure to choose the right molecule, thus check its number in the Display Manager.

There are two main menus with model building commands.

The first menu is activated via "Calculate" ‑> "Model/Fit/Refine". Small buttons with the graphical symbols are also accessible at the right side of the graphics window. This menu contains mainly commands needed when a great part of the main chain has already been built.

Many of the following command act on a region of residues which after activating the option is specified by clicking on the first and last residue of the region. By pressing the CTRL button, one can rotate the molecule to find the first and last residue. The region can also be a single amino acid or a ligand.

Automatically fits and refines a region based on an electron density map.

Optimizes the stereochemistry of a region (bond length and bond angles). Before accepting you may manually change the zone.

A submenu opens to fix or unfix atoms, which should not be moved when applying automatic building commands.

Shifts and rotates a region as a rigid body (no conformational changes) for optimal fit to the electron density map.

After specifying the region a menu with dial buttons opens to move and rotate the region manually.

Based on a library of allowed side chain conformations ("rotamers") the one which best fits to the electron density is chosen. Will only work if the main chain is built correctly. Otherwise choose "real space refine zone".

After clicking on a residue a list of allowed side chain conformations will show up. Click through all rotamers until one is found which best fits the electron density. The main chain conformation should be correct.

Modify the side chain conformation by manually changing all torsion angles (named chi angles). With this option also high energy rotamers may be chosen, if necessary.

By clicking on four atoms a torsion angle is defined which can be manually changed. This option is usually only used for ligand, for which no rotamer library is available.

Flips both the carbonyl group and the NH group of the peptide bond by 180°.

Flips the side chain conformation of His, Asn or Gln.

The phi and psi angles of the peptide bond can be changed manually.

Mutate the residue and automatically determine the best side chain conformation based on the electron density map.

Mutate the residue.

Add an alanine residue to the N-terminus or C-terminus of the current chain. After activating the option you click on a terminal residue. Before accepting the new residue you may manually change the residue.

If the electron density indicates that the residue is present in two or more conformations, you can add the alternate conformations here.

Place an atom at the current center of the view. Before activating the option you should move the density feature, usually a water molecule or ion, to the center by moving the structure with CTRL-left_mouse_button. Rotate with the left mouse button only and again center the density until the density is completely centered. After activating the option you can choose the type of atom to add.

If you want to cancel an option like "Regularize Zone" when picking atoms, choose this option.

With this option you can delete atoms, side chains or regions.

Undo an operation. Does not always work.

Redo an operation that was undone.

The second menu is activated via "Calculate" ‑> "Other Modelling Tools...". It mainly contains commands to start manual model building from scratch. Mentioned are the option you might need in the practical.