RasMol Training Tutorial

This Jmol tutorial will serve as an overview/review of RasMol commands that are important in designing models to be built using rapid prototyping technology. It is not meant as a substitute for the RasMol Training Guide, but as an abbreviated ready reference with images that correspond with the RasMol commands you enter. You should arrange your workspace so that you can simultaneously view the RasMol molecular visualization window, RasMol command line, and toggle between RasMol and these instructions. These instructions are written for use on a PC.

This tutorial explores the insulin dimer using the PDB file 1TRZ.pdb. You should download the file from the PDB website at www.pdb.org. Enter the filename 1TRZ in the search window. You'll want to explore the structure summary page to determine the resolution, number of chains, and names of any hetero groups that are associated with the protein. If you were going to design and build a model of this protein, you would want to get a copy of the original research paper to get details of features you might wish to model. On the left navigation bar, open the heading 'Download file' and right click on 'pdb text'. Select 'save target as' and save to a folder. Be sure to add a copy of RasMol to this folder.

Open RasMol and arrange the command line (CL) and molecular visualization (MV) windows on your desktop. In the MV, click on file, open then select the file ITRZ from the list. (You may have to browse to find the correct folder the first time you open the file.) Your screen should look like the image at the right.

Although a black background sometimes makes the image more visible, it is often easier to see the image with a white background. When saving a gif image for a poster, you should always use a white background to save on ink. The command for changing the background to white is:

background white

You may also change the background to any color you wish using either the names of colors recognized by RasMol (check the quick reference guide for a list) or RBG colors, such as (255, 14, 3).

Model displaying a white background. Insulin is shown here in a wireframe format. The CPK coloring scheme is applied to the model (carbon is gray, oxygen is red, nitrogen is blue, and sulfur is yellow). The background is white.

Next, let's change to looking at a backbone model. For a sturdy model, you should assign the thickness of the backbone to 300. Use these commands:

backbone 300

wireframe off

spacefill off

Backbone model of insulin. Your design should now look like this. A backbone model connects the dots between the alpha carbons of each of the amino acids in the chain. Each 'elbow' in the model is the location of the alpha carbon atom of one of the amino acids. If you click on an elbow, an identification line will appear in the Rasmol CL. It will look similar to this:

Atom: CA 794 Group: LYS 29 Chain: D

CA is the symbol used for the alpha carbon. This is the alpha carbon of lys 29 on the D chain. If you click on two adjacent 'elbows' in the backbone, you will see that the groups are numbered consecutively. Click on various chains to determine their names (A-D).

Next, let's identify the alpha helices and beta sheets in this model. Let's color the helices red and the sheets yellow.

select helices

color red

select sheets

color yellow

Helices and Sheets. Your design should now look like this. If you look closely, you should be able to see that this PDB file contains two identical insulin molecules, and that the two molecules align to form a beta sheet.

Each insulin molecule consists of two chains, and the chains are held together by disulfide bonds. Let's add the cysteine sidechains to see if we can identify the location of these bonds. Standard values for building models: wireframe 225 and spacefill 275. We'll also color the sidechains in cpk coloring so we can identify the atoms.

select cys

wireframe 225

spacefill 275

color cpk

Cysteine sidechains displayed (bumpy backbone). Your design should now look like this. But if you look closely, you'll see that the backbone is 'bumpy' for each of the sidechains displayed. This is because we selected the ENTIRE amino acid, so it displayed the nitrogen and carbonyl carbon of each amino acid we selected. This will make an unpleasing model. Here are the commands to correct a bumpy backbone.

select cys

wireframe off

spacefill off

select cys and (sidechain or alpha)

wireframe 225

spacefill 275

Cysteine sidechains displayed (clean backbone). Your design should now look like this. In the future, when you want to display a sidechain, you can do it right the first time (no bumpy backbones) by simply using the last three commands above. Note that Rasmol uses Boolean operators. The term 'and' means that BOTH conditions must simultaneously be met. The term 'or' means that either of the conditions must be met. Parentheses are used to indicate which function operates first. So the command 'select cys and (sidechain or alpha) tells Rasmol to select only the alpha carbon and the sidechain of all the cysteines. Note that ALL of the cysteine sidechains in this molecule are involved in disulfide bonds. This is not always true for every protein. If you look closely, the disulfide bonds are not displayed. To show the disulfide bonds, use this command:

ssbonds on

ssbonds 225

Disulfide bonds. Now your model will look like this. We still need to add more stability to the model. We'll start by adding hydrogen bonds. Although you won't typically add hydrogen bonds found in helices to a final design, let's take a look at what they look like:

select helices

hbonds on

hbonds 225

set hbonds backbone

color hbonds white

select helices

hbonds off

Hydrogen bonds within a beta sheet DO add structural stability, so we will add these with the commands:

select sheets

hbonds on

set hbonds backbone

set hbonds 225

color hbonds white

Note that the command 'hbonds 225' adds thickness to the hydrogen bonds; this command only works in RP-RasMol. If you are using RasMol, RasWin or RasMac, the hydrogen bonds will appear as dotted lines. Note that you need to use the command 'set hbonds backbone'; otherwise the hbonds will just float. Since hydrogen bonds and monitor lines are for support, we don't want to draw attention to them. Therefore we choose light colors in our model design for these structural elements. Unfortunately, the only beta sheets in this model are form where the two subunits pair, and the hydrogen bonds are not indicated in the PDB file, so no hydrogen bonds appear in the MV window. Although the following commands are not typically used, we will use them to display the hydrogen bonds that are not in the helices, and we will color them black so they are easily visualized. Enter these commands:

select backbone

color cpk

select not helices

hbonds on

set hbonds backbone

color hbonds black

hbonds 225

Your model should look like this:

Insulin 1TRZ with triangle bonds

The only hydrogen bonds that will display (in this particular file) are triangle bonds. These are hydrogen bonds that connect two amino acids that are separated by a single amino acid (N and N + 2). Since these triangle bonds don't provide stability or aesthetics to the model, these should be removed. To remove these bonds, click on the alpha carbon of each of the amino acids. You will need to know the chain and group numbers for these amino acids. As an example, I clicked and discovered:

Atom: CA 377 Group: THR 27 Chain: B

Atom: CA 354 Group: PHE 25 Chain: B

To remove the triangle bond between these two amino acids, I would enter a command in the form: select *chain and (N or N+2), which, in this case, would be:

select *B and (25 or 27)

hbonds off

You should watch the image in the RasMol MV to see if the hydrogen bond you selected disappears when you hit enter on the last command. If not, look back over your commands to make sure you entered them correctly. Remove ALL triangle bonds before proceeding. You should have NO black lines on your image when you are done!

At this point, you've probably discovered that you can't click an 'undo' button in RasMol. In fact, you may have had to start over several times. This is probably a good thing, because repetition will help you solidify these new commands in your memory. But now is a good time to learn to save your work. You will be saving a script file, and the general command is:

save script filename.spt

You can use any name instead of 'filename' - in fact, it is a good idea if you choose a short, descriptive name while you are designing your model. We often number our model designs - just in case we want to go back to an earlier version of a model. You might save this file as:

save script insulin1.spt

Just to see what happens if you enter the wrong command when saving a script file, save it again, twice, using the following incorrect commands:

save insulin2.spt [this one is missing the word 'script']

save script insulin3 [this one is missing the '.spt' extension]

Now, enter the command:

zap

This will clear the RasMol MV window. To reopen a script file, enter the command:

script filename.spt

In the example above, you would enter:

script insulin1.spt

The design should reappear in your RasMol MV window.

Clear the screen, then try opening your two files that were saved incorrectly. What happened in each case? You'll note that when you forget the extension, the file will still save and open correctly, but you won't communicate to other model designers that this is a script file. This is easy to remedy; you simply save the file again, adding the .spt extension. On the other hand, if you forget the word 'script' when saving a file, and you try to open it again later, you will see all kinds of numbers and letters flying by in the RasMol MV window. The file is unreadable by RasMol, and you simply have to start your model design over again. This is why a lot of model designers like to jot down notes on the commands they use when designing a model. If they need to redesign, they can do so quickly by referring to their notes. Taking notes also helps when you are writing a model description sheet, because you can quickly verify the coloring scheme and your rationale for featuring various characteristics of your macromolecule.

Clear the screen, then open your correctly saved script file. Before go go further, let's color the chains so we can clearly distinguish the A and B chains of each of the two monomers. Enter the following commands:

select backbone and (*a or *c)

color red

select backbone and (*b or *d)

color blue

Model with colored backbone.

If you look carefully at this model design, you'll quickly realize that, although you have a dimer in the RasMol MV window, there is nothing holding the two insulin molecules together. If this model were built, you would have two individual insulin molecules. Furthermore, if you imagine squeezing the model, you will see that the helices and loops would wiggle (or break), because they are not well anchored. To resolve both of these difficulties, you need to add monitor lines. These are stabilizing lines that join dimers, or join loose strands to the rest of the protein. There are two ways to add monitor lines, and each way is preferred by some model designers. I'll describe both methods, as well as advantages and disadvantages of each.

The first method is to click on the two atoms you wish to join with a monitor line. Their identy will be listed on the RasMol CL screen. Then you enter the command

monitor atom number, atom number

For example:

monitor 339, 780

You should see a dotted line between the two atoms you selected, and a number. The number is the distance, in Ångstroms, between the two atoms you selected. If at all possible, monitor lines should be no longer than 9 Ångstroms, but the shorter, the better. The advantage of using this method for adding monitor lines is that if you make a mistake and get a monitor line you don't like, you can 'erase' it by reentering the same command again. This will toggle the monitor off. Some model designers will save a copy of their design, then go through the model, adding monitor lines from one end to the other. They keep track of where they have added monitor lines by recording the atom numbers and coloring the backbone as they go. (You can even add thickness to the monitor lines - set monitor 225 and color them some bright or dark color to keep track of what you have done. Once you have finished, you can go back to your perfectly designed model and simply add the monitor lines based on the notes you have taken.)

The second method is to set the picking (term used when you click on an atom) to automatically add the monitor lines. The command is:

set picking monitor

Now, when you click on two atoms consecutively, RasMol will automatically add monitor lines. This method is a little more direct than the first method, but it has its disadavantages. If you don't click directly on the atom you want, there might be another atom lying behind it (whether visible or not) that RasMol will select. You can sometimes get very long monitor lines that extend from one side of the model to the other. These monitor lines have to be removed by clicking on EXACTLY the same two atoms again. It can be frustrating in a large protein, when you generate three or four MORE monitor lines when trying to remove one. The key if you use this method for adding monitor lines is to save early and often! If you want to go back to the first method of adding monitor lines, you can turn off the picking by entering the command:

set picking ident

Now when you click on an atom, its identity will appear in the RasMol CL window.

Strategy for adding monitor lines: You'll want to use monitor lines to stitch together subunits, attach substrates or cofactors, and stabilize helices and loops. But you don't want to add so many monitor lines that they detract from your model. A good rule of thumb is to attach substrates and cofactors with one or two monitor lines and connect helices and loops near each end. If you have an especially long helix (8 or more turns), you may want to add a third monitor line in the middle.

Let's analyze the insulin dimer before adding monitor lines. First, the two monomers need to be joined along the beta sheets. This can be done by adding a monitor line at each end of the blue beta sheets. If you want to show hydrogen bonding (which is not indicated in the pdb file), you could add a monitor between the two strands at every other sidechain. You might also join the blue alpha helices at the top and bottom to stabilize the dimer. This protein is a nice compact protein with lots of disulfide bonds and short helices on the red chains. The only loose ends on the red chains can be joined to the blue chains with a single monitor line for each dimer. Be sure to save your file correctly once you have added your monitor lines!