If you have access to Alwyn Jones' crystallographic modelling program O, you may want to use it to inspect electron-density maps that you get from EDS, the Uppsala Electron-Density Server. For every entry, EDS provides pre-cooked files for use with O that provide you with a lot of useful information about the model, the density, and the fit of the model to the density.

Important notice: this guide describes the EDS files as they were created prior to 1 December, 2006. If the EDS entry you are interested in was processed after this date, you should also read the update notes at the end of this guide.

In case you have never used O before, this page provides a brief introduction to some of the basics of O. It is assumed that someone has installed and set up O for you, so that all you need to do to start the program is to type a single command (often, this will be the command "ono"). This example uses the EDS entry 1CBS - go to this EDS entry now.

On your current computer, go to or create a directory that you own and in which you can (temporarily) store the files for this exercise (e.g., do something like: "cd ; mkdir eds_test ; cd eds_test")

On the EDS page for entry 1CBS, find the "Download" section in the menu on the left, and click on the link to download "All files (.tar.gz)".

On the next page, either click on the link for "1cbs.tar.gz", or click on the link with your mouse (which button you need to use depends on the type of computer you are using) and select the "Save link as ..." option from the pop-up menu. Save the file in your own directory (e.g., in "~/eds_test").

Open a shell window, go to the directory where you saved the file, and type the following command: "tar xovpfz 1cbs.tar.gz". This creates a new directory (called "1cbs"). Go to this directory ("cd 1cbs").

Now you are ready to start the program O, for instance by typing "ono". The program will ask you a number of questions, but (provided O has been set up correctly) you can simply hit the "RETURN" key to accept the default values offered by O. (Note: it is recommended that you use the latest version of O. However, most features will work with O version 9 or later.)

O will now start doing some work for you. In fact, it executes a macro (a set of O commands set up for this entry by EDS) that reads in the model and the maps and generates some graphics objects of them. When it is done (and ready to accept commands from you), it will show you the O graphics window.

There are a few floating windows that are important. The first is the "Object_menu" that contains a list of all graphics objects that O has created for your EDS entry:

These objects are:

1. 1CBSCA = a C-alpha trace of the protein in 1CBS (all carbons are yellow)
2. 1CBS = all atoms of entry 1CBS (including ligand and waters; carbons yellow, nitrogens blue, oxygens red)
3. 1CBS_1 and 1CBS_2 = the 2mF_o-DF_c map of 1CBS, contoured at a level of 1.0 times sigma (dark blue) and 4.0 times sigma (cyan)
4. D1CBS_1 and D1CBS_2 = the mF_o-DF_c map of 1CBS, contoured at a level of 4.0 times sigma (green) and -4.0 times sigma (red)
5. RSCC = a C-alpha trace, colour-ramped according to the value of the real-space correlation coefficient for every residue (from 0.5 = red to 1.0 = dark blue)
6. RSR = a C-alpha trace, colour-ramped according to the value of the real-space R-factor for every residue (from 0.0 = dark blue to 0.5 = red)
7. OWAB = a C-alpha trace, colour-ramped according to the value of the average temperature factor of every residue (from 0.0 = dark blue to 80.0 = red)
8. OCC = a C-alpha trace, colour-ramped according to the value of the average occupancy of every residue (from 0.0 = red to 1.0 = dark blue)
9. HETERO = an object that only contains the hetero-entities of the entry (ligands, co-factors, etc.)

With the left mouse button, click on one of the atoms. The residue number and atom name will be displayed next to it. At the top of the display you will also get some more information about the atom and the residue. Congratulations! You have "picked" (i.e., identified) your first atom in O. What is the B-factor of your atom? What is the real-space R-factor of the residue?

Apart from picking atoms, you will also want to learn how to change the view by rotating, translating, zooming and slabbing (or clipping). There are several ways to do this in O, but usually people use either the mouse or the arrow keys. Let's use the arrow keys first, because they allow for precise and reproducible (and manually undo-able) manipulations of the display. In the lower left corner of O's graphics window, you see 8 commands, of which one is marked with an arrow:

By default, the arrow points at "Rot X", i.e., rotation around the X-axis. Press the left and/or right arrow keys and observe what happens - you are rotating around the X-axis! Now use the up arrow key to point the arrow at "Rot Y" and use the left and right arrow keys again. Simple, eh?

Use the up arrow key to select "Zoom". Which key zooms in and which one zooms out? Notice what happens if you keep the left arrow key pressed for a few seconds. Change the zoom until the protein fits entirely inside the graphics window. Now select "Slab" and press the left arrow key a couple of times. What happens? Now figure out how to translate the view yourself.

All these manipulations can also be carried out with the mouse. The right mouse button, when pressed, controls the view. Moving the mouse rotates the picture in the direction of the motion. To rotate the view around a vertical axis, slide the mouse to the right or left. To rotate around a horizontal axis, slide the mouse up or down.

Pressing the right and middle buttons at the same time, rotates the picture around the third axis, perpendicular to the screen. Moving the mouse down rotates the picture clockwise, and moving it up rotates it anti-clockwise. Pressing the "CTRL" key at the same time enables you to translate.

The middle button when depressed by itself, controls both zoom and slabbing: up-down for zoom, and right-left for slabbing.

The left key is used for identifying atoms and activating the windowing system.

Experiment with these mouse controls. Try to find the ligand and zoom in on it.

Often you will want to centre on a particular atom so as to make it the pivot for rotations in O. There are several ways to do this. The first is to use the "Centre_ID" command that is shown in the "User Menu". Click on this command with the left mouse button and then click on the atom you want to centre on. Now rotate the view and note how the atom has become the pivot point.

You can also centre on atoms or residues that you don't see or can't identify. Click on the shell window from which you launched O. To centre on an atom, type something like: "cen_at 121 cb". Go back to the display and rotate the view - you will notice that you have centred on the C-beta atom of Leu 121. If you just want to centre on the centre-of-mass of a residue (or if you don't know any atom names in it), type something like this: "cen_zo 200". Go back to the display window and check that you are now centred on the ligand. You can even centre on a specific position in space (using the "cen_xyz" command). You can also use the "cen_zo" command to centre on a region ("zone", in O-speak) of residues, e.g.: "cen_zo 5 8" will centre on the centre-of-gravity of the atoms in residues 5, 6, 7 and 8.

In fact, you don't have to type these centring commands in the shell window - try typing one into the display window (e.g., "cen_at 43 cd1"). The actual O commands you have used are "Centre_atom" and "Centre_zone", but you only need to type an abbreviation that is unique. If it is not unique (for instance, type "cen" in the shell window), O will list all commands that begin with that string. Furthermore, O tolerates dashes instead of underscores in command names, so typing "cen-zo 100 106" will work just fine.

Now you know how to get around in the model: how to rotate, zoom and slab, and how to centre on specific atoms or residues. All you need to know now to use O to inspect EDS maps is how to draw the maps. First, click on both maps in the "Object_menu". Adjust the zoom and slab until you have a good view of the ligand and its density. You will notice that the density doesn't cover all of the ligand. To update the map drawing, click on the commands "Fm_dr 1cbs" and "Fm_dr d1cbs" in the "User_menu". Explain what these two commands do.

Now we can manipulate the map drawing. The map contouring parameters are controlled through the two windows called "D1CBS" and "1CBS". The slider labelled "Rad." controls the contouring radius (in Å). Pick up the slider with the left mouse button and while you keep the button down, move it to the right to increase the contouring radius. The map has been contoured at two different levels and for each of these you can change both the contouring level (in units of sigma of the map, which is not the same as units of sigma in the unit cell, unfortunately) and the colour (by changing the red, green and blue components). Experiment with changing all these parameters for the "1CBS" map.

The "D1CBS" is contoured at a high positive (green) and a high negative level (red). Roughly, the former shows places where "there should be some more electrons in the model" (especially when similar features occur in the "1CBS" map), and the latter places where "there are too many electrons in the model". Hopefully, there will be very little signal in this map (which is a sign that the refinement of the model was rightfully terminated).

So - now you should be ready to inspect models and maps from EDS inside O. Use the objects "RSR" or "OWAB" to see if you can find places in the model that have poor density. Use the "HETERO" object to quickly locate the ligand (first switch off the "1CBS" object, of course). What is your impression of this model based on the quality of the electron density (and the fit of the model to the density) ?

In case you're wondering, to stop O without saving your work, type the command "Quit". If you do want to save your work, use the "Save_DB" command. You can later resume your work by providing the name of the saved file as an argument to the "ono" command.

Finally, a few useful resources if you want more information about O or EDS:

• O essentials - a more elaborate introduction to O
• A to Z of O - a full description of all O commands
• EDS help - an extensive description of EDS

EDS entries processed from 1 December, 2006 onward come with improved O-related files. The major changes are discussed below.

1. Automatic display of symmetry-related molecules. One of the most important changes is that the O files now also generate graphics objects for symmetry-related molecules that are close to the location in space where you are inspecting the maps and models. When inspecting crystal structures, one should always take crystal symmetry into account. Sometimes structural features or changes can be explained by crystal-packing constraints. In other cases, a physiological dimer may be formed by two symmetry-related monomers, etc. You will recognise any symmetry-related molecules by the fact that their carbon atoms are coloured green. Their graphics objects will be called "SYM1", "SYM2", etc.
   

2. Automatic updating of map and symmetry objects when centring. There was a bug in the previous macros that made that the maps were not (always) updated automatically when you centred somewhere. This has been fixed. Whenever you use one of the centring commands (e.g., "Centre_ID") both maps will be updated and so will the display of any nearby symmetry-related molecules.

3. Display of all hetero-entities on start-up. Previously, the first thing you saw was a display of the C-alpha trace of your molecule, coloured by RSR values. Now, the "HETERO" object is also displayed. This makes it very easy to quickly find the ligands, inhibitors, metal ions, etc. that often make a structure particularly interesting.

4. New "User Menu". A completely new "User Menu" is provided, which will hopefully make it easier to tune the behaviour of O to your needs. It contains the following commands:
   • "Centre_ID" - as before, you can use this command to centre on any atom currently displayed.
   • "@map_sym" - if you have switched off the automatic updating of map and symmetry objects (see below), you can tell O to update these objects at any time by clicking on this command.
   • "@reset_view" - sometimes one gets completely lost inside a structure or perhaps the slab has become infinitely small or you have zoomed out a couple of miles. Don't panic! Just press this command, and you will get back to the same view that greeted you initially (i.e., only the "RSR" and "HETERO" objects are displayed, and zoom and slab have reasonable values).
   • "@all_obj_off" - this is another command in the "don't panic" category. If you click on it, all graphics objects will be switched off. (Remember, you can switch objects on and off one by one by clicking on their names in the "Object_menu").
   • "@auto_map_sym" and "@man_map_sym" - by default, O will update the map and symmetry objects whenever you centre on a new position. In some cases, this may be undesirable (e.g., when the molecule is very big so that the evaluation of the symmetry takes a lot of time). In such cases, you can switch off the automatic updating by clicking on the command "@man_map_sym" ("MANual MAPs and SYMmetry"). You can then quickly move through the molecule. Whenever you do want to update the maps and symmetry objects, simply click on the "@map_sym" command. If you want to restore the automatic updating, click on "@auto_map_sym".
   • "@big_sym_sph" and "@small_sym_sph" - by default, O displays parts of symmetry-related molecules that lie within 10 Å from the point on which you are centred. You can increase this to 20 Å by clicking on "@big_sym_sph". To go back to a value of 10 Å click on "@small_sym_sph". (Note: if you want to use a different value, e.g. 15 Å, use the command: "symbol symrad 15".)
   

5. Miscellaneous. A number of minor changes have been made as well, e.g. the map windows are positioned more logically, maps are drawn with a default radius of 25 Å, the reading of PDB files has been made compatible with O version 11, etc.

If you should have suggestions on how we could further improve the "EDS experience" in O, please do not hesitate to contact us !

Latest update at 30 November, 2006.