version 01/23/00
Rasmol tutorial
Subject: TATA Box Binding Protein - DNA Complex

This page is a tutorial for the program Rasmol. One of the goals is to allow you to become familiar with the program so that it will be useful for study of a variety of macromolecules. Specific questions that students must answer and hand in are highlighted in red font.

RasMol is a widely used molecular graphics program for visualizing three-dimensional structures of proteins, nucleic acids and small molecules. It is easy to use, runs on many platforms, requires meager computational resources, is extremely powerful, and is free! The program can be used for analysis, display, teaching and generation of publication quality images. RasMol runs on PC, MACs, SGIs etc. The program reads molecular coordinates such as the x,y,z coordinates in a PDB file, Tripos Associates' Alchemy and Sybyl Mol2 formats, Molecular Design Limited's (MDL) Mol file format, Minnesota Supercomputer Center's (MSC) XYZ (XMol) format and CHARMm. Rasmol interactively displays molecules in many representations and colors. Molecules can be shown as wireframe bonds, cylinder 'Dreiding' stick bonds, alpha-carbon trace, space-filling (CPK) spheres, macromolecular ribbons (either smooth shaded solid ribbons or parallel strands), hydrogen bonding and dot surface representations. Various parts of a molecule can be independently represented and colored. A molecule can be rotated, translated, zoomed and z-clipped (slabbed) using either the mouse, the scroll bars, or the command line. Images may be written out in a variety of formats including either raster or vector PostScript, GIF, PPM, BMP, PICT, Sun rasterfile or as a MolScript input script or a Kinemage. Rasmol has no capability for docking and model building.

Here is a list of mouse commands that you can use for manipulating structures. Just click and drag across the main window to rotate, translate, and zoom. X and Y are in the plane of your screen. Z is perpendicular to the screen.

Action	Windows	Macintosh
Rotate X,Y	Left	Unmodified
Translate X,Y	Right	Command
Rotate Z	Shift-Right	Shift-Command
Zoom	Shift-Left	Shift
Slab Plane	Ctrl-Left	Ctrl

Before beginning the tutorial, use your web browswer to download and install the UC, Berkeley enhanced version of RasMol. Launch the program. You should see a main window (empty and black), a command window and a molecules window. If one of these windows is not open and visible, pull down the "Windows" menu and open the missing window. Move the windows so none are obscured.

If you have completed the PDB tutorial, omit the rest of this paragraph. Otherwise open the file containing the coordinates of the TATA-BOX Binding Protein (TBP-DNA complex) with your web browser. Save the coordinates, as text only, to your hard drive. Name the file 1CDW.pdb. It would be convenient but not necessary to put 1CDW.pdb in the same directory as the Rasmol program.

Launch Rasmol.  In the Rasmol program, pull down the File menu and open the file "1CDW.pdb". If you cannot see 1CDW.pdb in the Rasmol "Open File" window, and are definitely looking in the proper directory, then maybe you accidentally saved the file in the wrong format, as an HTML or a word processing document. Those files are invisible to Rasmol. If so, fix the file by opening it in your word processing program and saving it as "text only", or by repeating the procedure of the preceeding paragraph. If that does not solve the problem try to open 1CDW.pdb by dragging and dropping the file onto the Rasmol icon.

Once you have opened 1CDW.pdb in RASMOL, rotate, zoom and translate the DNA-protein complex around until you become convinced that you are not going to learn much by doing those things. The complex is just too complicated. There are too many atoms. Chances are you cannot even tell if the protein has a-helices or b-sheets.

Controlling the Rendering

One of the basic tasks here is to simplify the representation in specific ways to reveal various characteristics, interactions, etc. First strip away most detail to visualize the global fold and secondary structure of the protein.

Pull down the display menu and change the rendering to "cartoons" (if cartoons does not work, use "ribbons"). You should be able to see that you have beta-sheet, alpha-helix, and a short fragment of DNA.

Pull down the colors menu and change the coloring to "structure". The colors help show that TBP is composed of a-helices and b-sheets.

Question #1a: How many different a-helices are there in TBP?

Question #1b: Are the helices right-handed or left-handed?

Question #1c: How many different b-strands are there in TBP?

For this tutorial, you type the bold text that is contained within the quotation marks. Do not type the quotation marks. To enter text-based instructions, use the command line. Activate the Rasmol Command Window by clicking on it. The up arrow on your keyboard allows you to scroll up and repeat or edit your previously-typed commands.

Click on the Rasmol Command Window and type.

"restrict protein" this command cuts away all atoms except protein atoms

"select DNA" selects DNA

Pull down the display menu and set the display to ball and stick. Note that this changes the rendering only of the selected atoms (in this case, the DNA).

"color blue" changes the color of the selected atoms to blue. Try green, red, cyan, etc.

"select hetero" selects non-DNA, non-protein atoms

Pull down the display menu and set the display to spacefill. Now you can see the oxygen atoms from water molecules. In an x-ray diffraction experiment, hydrogen atoms are generally not observable, so they are left out of the coordinate file.

"color red" changes the water oxgen color to red

Let's take a look at the DNA in the absence of everything else.

"restrict DNA"

Pull down the display menu and set the display to spacefill.

"select c or g"

"color red"

"select a or t"

"color green"

Question 2a: How many AT base pairs are there in this DNA fragment?

Question 2b: How many CG base pairs are there in this DNA fragment?

Question 2c: Does the DNA seem to be linear or bent?

Question 2d: What are the terminal base pairs?

Let's take a closer look at a part of the DNA.

Pull down the display menu and set the display to sticks.

"restrict t2, g3, c114, a115" turns off everything but a dinucleotide step (i.e., two base pairs plus the backbone)

"select all"

"color red"

"select c114"

"color cyan"

"center g3" sets the rotation center to residue g3

Zoom in to get a closer view of this dinucleotide fragment. Rotate it around and admire it. If you click on an atom, some information about that atom pops up in the command window. Again notice that the hydrogen atoms are missing.

Question #3a: What base is paired with cytosine 114. What base is stacked on cytosine 114.

"color cpk" sets the color of the DNA to Corey-Pauling-Koltun; N is blue, O is red, C is gray, etc)

Pull down the display menu and set the display to spacefill.

Question #3b: How many of each type of atom (nitrogen, oxygen, carbon, iron, phosphorus, etc., omit hydrogen) are there in the dinucleotide?

Click the [Distance] button in the Molecules Window to toggle that function on. Click on any two atoms of the dinucleotide. Note that the distance between them pops up in the Command Window. If you toggle on [Keep labels on screen] in the Molecules Window, the names of the atoms and the distances between them remain on the screen.

[Alternate method: in the command line type "set picking distance"]

Question 4a: Measure the lengths of all the the hydrogen bonds in these two base pairs. Construct a table with columns: residue name 1, atom name 1, residue name 2, atom name 2, distance.

Click on the [Distance] button a second time to toggle that function off.

A bond angle is determined by the relative positions of three atoms. Click the [Angle] button in the Molecules Window to toggle that function on.

[Alternate method: in the command line type "set picking angle"]

Click on any three atoms of the dinucleotide. Note that the angle between them pops up in the Command Window.

Question #5a: For carbon 4 of the guanosine, measure the bond distances and bond angles relating all the atoms that are bound to it. What is the hybridization of carbon 4?

Question #5b: For the carbon 4' (4 prime; primes are indicated by * in Rasmol; the primed atoms are located in the sugar) of the guanosine, measure the bond distances and bond angles relating all the atoms that are bound to it. What is the hybridization of that carbon?

Question #5C: Measure all the angles involved in two of the hydrogen bonds. You should arrive at two angles for the external hydrogen bonds and four angles for the internal hydrogen bonds.

Click on the [Angle] button a second time to toggle that function off.

A torsion angle is determined by the relative positions of four atoms. Phi and psi are torison angles within peptides and proteins. The conformation of each amino acid residue is characterized by phi and psi. Phi and psi, and their significance, are explained in Chapter 7 of Voet and Voet, 2nd Edition.

Click the [Dihedral] button in the Molecules Window to toggle that function on.

[Alternate method: in the command line type "set picking torsion"]

Click on any four atoms atoms, which are bonded together linearly. Note that the dihedral angle relating them pops up in the Command Window. The dihedral angle relating four atoms in a plane is zero.

Question #6a: Locate O4', C1', N1, and C2 of T2 (which is a pyrimidine; note that in some structures the O4' is called the O1'). With the dihedral function toggled on, click on these four atoms in the order given.  Measure the torsion angle.  This torsion angle is called the glycosidic torsion.

Question #6b: Measure each glycosidic torsion angle of the dinucleotide. There are four of them. For purines (A and G) the relevant atoms are O4', C1', N9, and C4.

Click on the [Dihedral] button a second time to toggle that function off.

Measuring Phi and Psi, Constructing a Phi/Psi Map

Here we will make a phi/psi map of an a-helix and a b-sheet. Check here [phi and psi] for the meaning of the torsion angles phi and psi. To help perform this task look at an example of a Phi/Psi map (as shown in Figure 7-7 in Voet and Voet, 2nd edition).

Part 1: a-helix

First simplify the representation and make each type of atom in the backbone a different color.

"select all"

Set display stick.

"restrict backbone and 314-330" turns off sidechains and everything but one helix.

"center 320"

"color red"

"select *.n"

"color blue"

"select *.ca"

"color green"

"select *.c"

"color yellow"

Click the Dihedral button in the Molecules Window to toggle that function on.

To measure phi, for one of the residues click on four atoms in order

C' (yellow) then Ca (green) then N, (blue) then C' (yellow).

To measure psi, click on four atoms in order

N, (blue) then C' (yellow) thenCa (green) the N, (blue).

Click the Dihedral button in the Molecules Window to toggle that function off.

Question #7a: Measure all the phi and psi angles for this fragment. You may want to change the center for each pair, to walk down the peptide. Check the sign by viewing directly down the appropriate bond (some versions of Rasmol contain a bug that give an error in the sign). Make a table of residue name, phi and psi.

Part 1: b-sheet

"select all"

Set display stick.

"restrict backbone and (160-169 or 253-259)" turns off sidechains and everything but two b-strands.

"center 164"

"color red"

"select *.n"

"color blue"

"select *.ca"

"color green"

"select *.c"

"color yellow"

Question #7b: Measure all the phi and psi angles for these two fragments. Again you may want to change the center, and check the sign. Add these values to your table from question 7a.

Question #7c: Graph phi versus psi. Set the limits of both axes from -180 to +180 degrees. How does this compare to the phi/psi plot in Voet and Voet?

Studying Interactions between DNA and Protein.

Now let's study how this protein binds to the DNA.

"select all" selects all atoms

Set display to Cartoons.

"color cyan"

"select DNA"

"color white"

Set display to Spacefill.

Zoom back and rotate the complex, and you should see that some of the b-sheets interact with the DNA (are near it) and while the a-helices are remote from the DNA. Let's use some of RasMol's powerful features to find in detail out how the protein interacts with the DNA.

"select protein"

Set display to stick.

"select protein and backbone"

Set display to cartoons. this series of command allows you to visualize secondary structure (helices and sheets) and the protein side chains, too.

"select within (10.0, DNA) and protein"selects all protein atoms that are within 10.0 Angstroms from the DNA.

"color yellow"

"select within (6.0, DNA)"selects all atoms that are within 6.0 Angstroms from the DNA. You can use the up arrow on the keyboard to revisit previous commands.

"color blue"

"select within (4.0, DNA)"

"color red"

This series of commands has colored your protein by layers from the DNA, like an onion. The red atoms are essentially in van der Waals contact with the DNA. The blue atoms are a little further away, and the yellow further still.

Click on some of the red atoms to find which residues they are. It may be easier to see if you:

"select DNA"

Set the display to stick.

Let's find out which atoms of the protein make short contacts with phosphate oxygen atoms.

"select within (2.9, (*.o1p or *.o2p)) and protein" Six atoms are selected

"color violet"

Set the display to spacefill.

Question #8a: Compile the data for each of the 6 close contacts between protein and DNA phosphate groups. Make a table with 4 columns; protein residue, protein atom, DNA residue, DNA atom, distance.

Studying Hydrophilicity, Hydrophobicity and Amphiphilicity

RasMol can show locations of polar and non-polar residues, and tell you if a-helices are amphiphilic.

"select protein"

set display to stick

"select backbone"

set display cartoon

"select hydrophobic" selects all hydrophobic residues

"color red"

"select polar" selects all polar resides

"color cyan"

"select arg, lys, asp, glu" selects charged residues (not histidine, which is sometimes charged).

"color blue"

Question #9a: Which region of the protein contains the most polar residues?

Question #9b: Which region of the protein contains predominantly non-polar residues?

Question #9c: Find the starting and ending residues of one of the a-helces of TBP. Determine the sequence. Construct a helical wheel of your a-helix. Check here for information on helical wheels.

Question #9d: Is this a-helix amphiphilic? Why?

Finding Hydrogen Bonds

Question #10a: Turn on the hydrogen bonds within your a-helix (from above). Measure the length of each hydrogen bond. Make a table with atom 1, atom 2, length. What is the average hydrogen bond length? What is the dispersion (given by the standard deviation).

Making Illustrations

In this section you will make an illustration showing two hydrogen bonding interactions between the DNA and the protein.

"select all"

Set display to spacefill.

"color cpk"

"restrict (290 and protein) or (6,7,8 and DNA)"

"background white"

Zoom and rotate these fragments of the complex until the hydrogen bonding interactions between the DNA and protein can be clearly observed. You can recognize the hydrogen bonds by the penetration of van der Waals surfaces.

Pull down the Export Menu and save the view as a GIF file (or any other format that you might prefer). Import the file into a graphics program such as Photoshop, and label the residues. Write your name on the bottom right of the figure. Print the figure, preferably on a color printer.

RasMol has built in on-line help.

"help commands" gives a list of commands.

"help colour" [or "help color"] gives a list of available colors.

"help expressions" and "help sets" gives partial lists of expressions for selecting or restricting atoms. Some useful expressions are given here.

Rasmol Expressions (note there are two types of wildcard; * & ?)

select expression	result
select all	selects everything
select hydrophobic	selects all hydrophobic resides
select polar	selects all polar resides
select alpha	selects all a-carbons
select helix	selects all resides in a-helices
select sheet	selects all resides in b-sheets
select ligand	selects ligands
select water	selects water molecules
select DNA	selects DNA atoms
select at	selects adenine and thymine
select cg	selects cytosine and guanine
select 14	selects reside number 14
select 14,21	selects reside numbers 14 and 21
select 14-21	selects reside numbers 14 through 21
select 14-21,35	selects reside numbers 14 through 21 and 35
select atomno=1259	selects atom number 1259
select arg	selects all arginine resides
select arg43	selects arginine 43
select arg43.ca	selects Ca of arginine 43
select arg.ca	selects all Ca atoms of arginines
select *.cb	selects all Cb atoms
select arg.c??	selects all carbon atoms of arginines
select arg43.*	elects all atoms of arginine 43
select *.?e?	selects epsilon atoms of all residues (the 2nd ? is required because some residues have e1 and e2)
select within (8.0, ser)	selects all atoms within 8.0 Å of all serine residues
select within (6.0, 14-21,35)	selects all atoms within 6.0 Å of residues numbers 14 through 21 and 35

There is RasMol help available on the WWW.

Frequently Asked Questions

Q: I noticed that the cartoon command does not seem to work very well in version 2.6 for windows. Am I doing something wrong?
A: Yes there seems to be a bug there. You can use the ribbons rendering, it is almost as good.