Introduction to Molecular Modeling
A Tutorial for RasMol (Revised November 1, 1997)
Gale Rhodes
Department of Chemistry
University of Southern Maine
Portland, Maine, USA 04104-9300
My students tested this tutorial, and as a result, I made many improvements.
In particular, I believe that the assignments are now clearer. Thanks to the 1997
biochemistry class, and to Spencer Anthony-Cahill of Western Washington University and
Cygnus X-1, for helping to improve this tutorial.
Please send comments, corrections, and suggestions to Gale Rhodes.
TEACHERS: Please request permission to use this tutorial in your courses. Here's why.
Contents
Overview of The Tutorial
Tutorial for RasMol
1. Getting Started
2. Windows and Help
3. Rendering
4. Manipulations
5. Picking, Selecting, and
Restricting
6. Viewing in Stereo
7. Exploring
8. Oligomeric Proteins
9. Saving Your Work:
Scripts
10. Summary
11. Assignment and File List
12. Useful Addresses on
the World Wide Web
USM Students: See Special Instructions.
NOTE: Accessory pages like Special Instructions appear in a new browser window; simply close the window to return to this tutorial.
Overview of The Tutorial
This tutorial gives
you an opportunity to learn how to explore the structure and action of biomolecules using
computer graphics. You will learn how to obtain macromolecular structure files from the
Protein Data Bank, and to display and manipulate molecular structures using the program
RasMol. With the skills you learn here, you can independently examine any macromolecular
structure available. Many structural biology sites on the World Wide Web provide
structures, illustrations, and even animations that you can view with RasMol. You can
obtain RasMol free for use on your own computer. See Getting RasMol
if you do not already have RasMol installed on your computer (but first read below about Conventions used in the instructions of this tutorial).
RasMol for the Macintosh is called RasMac, but I refer to the program as
RasMol throughout the tutorial. I wrote the tutorial using RasMac 2.6b2 for Macintosh.
Most of the instructions should be the same for Windows versions of RasMol. Where I am
aware of differences between Mac and PC versions of the program, I added notes in
parentheses for PC users.
The name RasMol comes from raster display of molecules. Raster
is a type of computer display especially useful for showing solid surfaces. It may not be
a coincidence that the letters Ras are also the initials of RasMol's creator, Roger
A. Sayle of Glaxo Corporation and the University of Edinburgh, Scotland. Sayle began
developing this program as part of this graduate work in computer science, and continues
to expand it with support from Glaxo. Thanks to Roger A. Sayle and Glaxo for giving this
powerful tool to the world structural biology community.
Conventions
In this tutorial, instructions for giving commands or using menus
will appear in a consistent format. Here are some sample instructions and their meanings:
< return > (keyboard entry)
means press the key labeled return on the computer keyboard. All key-press instructions
are surrounded by < >'s.
Display: Backbone (menu command)
means pull down the Display menu and select Backbone. All menu instructions
are in bold type with a colon between menu name and command name.
RasMol >
select alpha < return > (command on RasMol Command Line)
means that you should type the command "select alpha" and then press the returnkey.
Any words that you type while RasMol is running appear in the Command Line window beside
the RasMol prompt: "RasMol >." When you type the command and press return,
RasMol executes the command.
button: Open (on dialog windows)
means that you should click once on the button labeled Open.
link: Other Sites of Interest (on web pages)
means that you should find and click the link, usually a word or phrase in colored
text, that says, "Other Sites of Interest".
Filenames are in italics -- for example, 3b5c.pdb.
Tutorial for RasMol
1. Getting Started
NOTE: Plan to work at least
through section 4 of this tutorial without stopping. Thereafter, the end of any section is
a convenient stopping point. See the end of section 4 for instructions on how to resume
the tutorial after stopping.
RasMol works better with Netscape if it is located on the
desktop. Make a copy of version 2.6b2 of RasMol (for PC) or RasMac (for Macintosh) and the
file rasmol.hlp on your desktop. Configure your web browser to use RasMol or RasMac
(hereafter referred to as RasMol) as a helper application for files of MIME type chemical-
x/pdb, with suffix .pdb. For help with this task, see Configuring Netscape.
Use your web browser to obtain the atomic coordinate file (or PDB file) with
code 3b5c from the Protein Data Bank. For help with this task, see Getting PDB Files. Place the file on your desktop and name
it 3b5c.pdb.
The web page PDB File Contents
provides a complete description of the contents of a PDB file, thus allowing you to learn
what information RasMol uses to create molecular graphics displays. But first, get started
with viewing. After you have learned some basics, the contents of the PDB file will mean
more to you.
2. Windows and Help
Start RasMol by
dragging the file icon of 3b5c.pdb onto the RasMol icon. After the file is loaded, two windows appear. RasMol's
Main Window displays a wireframe model of cytochrome b5. Behind the Main Window is the
Command Line window, which you use to issue commands to RasMol. (If you are working in
Windows, the Command Line window starts out minimized to an icon at the bottom of the
screen. If you don't see it, hold down the Alt key while you press the Tab key repeatedly.
When the banner says "RasMol Command Line", let up both keys and the Command
Line window will open.) When RasMol needs to tell you something, its messages also appear
in the Command Line window. When RasMol is waiting for a command, the prompt,
"RasMol > " appears on the command line, which is the last line in the
window.
Arrange the windows conveniently, as follows:
Windows: Command Line
The Command Line window comes to the front. You can also accomplish this by simply
clicking on any exposed part of the window. Grab the window, by placing the mouse pointer
on its top border and holding down the mouse key, and drag it to move it. Place it so that
its lower left corner is very close to the lower left corner of the screen.
< return >
< return >
< return >
Each time you press return, the cursor moves down the window and prints the prompt,
"Rasmol >" on the command line. Keep pressing return until the prompt
appears at the very bottom of the window. Now use your first command to get a brief
introduction to the program:
RasMol > help < return >
Read the introduction.
RasMol > help commands < return >
This information includes a list of the most frequently used RasMol
commands. Any time you would like to know more about a command, just type help
followed by the command. This help is somewhat sketchy compared to the RasMol Manual,
but is quick and handy.
Windows: Main Window
The Main Window returns to the front. You can also accomplish this by
simply clicking on any exposed part of the Main Window. Click on the small square at the
top right of the window. The window expands to fill the screen. Then drag the small square
at the bottom right of the window straight upward to shrink the window just enough to
expose the last two lines of the Command Line window. With this arrangement, the
Main, or graphics, window is as large as possible, but you can also see the command line
and RasMol's messages.
In the Main Window, you are now seeing a wireframe model of the protein
cytochrome b5, a protein involved in redox reactions in the liver. This small protein
contains 86 amino acid residues and a heme prosthetic group with a central iron ion that
is the site of oxidation or reduction. The structure you are seeing is the oxidized (Fe3+ or iron [III]) form.
When RasMol starts, it always shows a wireframe model of all the atoms in the
protein and in any other molecules (such as cofactors or inhibitors) that are included in
the PDB file.
Now you will simplify the display and learn how to manipulate the model.
3. Rendering
Display: Backbone
Now you see only the alpha carbons of the protein, connected by straight
lines. Lines connecting alpha carbons are sometimes called "virtual bonds." You
can begin to see secondary structural elements such as alpha helices and strands of beta
pleated sheet. Try some of the other Display commands. Each command in the Display menu
changes the way RasMol represents, or renders, the model. End by displaying the
backbone model. Now you will make the secondary structural features even more obvious.
Colours: Structure
This command colors alpha helices hot pink, beta strands yellow, beta turns blue, and
other parts of the model light gray (not my choice of colors!).
RasMol > select hetero and not hoh < return >
This command selects only the hetero (non-protein) groups in the file, excluding the water
molecules that are frequently included in crystallographic models. Nothing happens until
you issue another command. Commands only affect currently selected atoms. Also
notice that you do not need to bring the Command Line window to the front in order to type
commands.
Display: Ball & Stick
RasMol displays a ball-and-stick model of the selected atoms. Notice that this command
does not affect the protein chain, because it is not selected.
Colours: CPK
RasMol colors the selected atoms according to widely used chemical conventions: carbon
is light gray, nitrogen is light blue, oxygen is red. Notice again that the menu command
affects only the currently selected atoms.
Click on any alpha carbon of the protein and watch the command line. This is
called picking an atom. When you click on an atom in the Main Window, RasMol
identifies it. You should see something like this:
Atom: CA 652 Group: PRO 81
This tells you that you clicked on the alpha carbon (CA) of residue 81 in cytochrome b5,
which is a proline. RasMol calls a residue a group, and a nonprotein portion of the
molecule, including a water molecule, a hetero. If you clicked on some other atom,
use picking to find this one.
If you can see a yellow atom in the space filling model, click on it. You
should see
Atom: FE
754 Hetero: HEM 201
The yellow atom FE is iron (Fe3+, actually) in the center of the heme group of cytochrome b5. By using the
command select hetero and not hoh, you can quickly find nonprotein groups in the
PDB file. By picking any atom in the group, you can learn the PDB name of the group, and
thus know how to specify it in commands.
Note: Picking works best with Wireframe and Sticks displays, does not
work at all with Ribbons, Strands, or Cartoons, and is somewhat
unpredictable with Spacefill and Ball&Stick. This is not a great
inconvenience, because Wireframe is the most useful display for exploring
unfamiliar models in detail.
Listed below is the sequence of commands you have used so far. You can use
this sequence to give you a quick overview of any macromolecule for which you have a PDB
file, and to reveal any hetero groups (cofactors, inhibitors, water, and so forth) that
are present in the file. The sequence is
Display: Backbone
Colours: Structure
Rasmol > select hetero and not hoh < return >
Display: Ball & Stick
Colours: CPK
Now you will learn how to manipulate the model. You can rotate it, move (translate) it to
a different part of the screen, and zoom in or out.
4. Manipulations
To rotate, place the pointer
anywhere on the Main Screen, hold down the mouse button and move the mouse. This is called
dragging the pointer (as opposed to moving the pointer without holding down the
mouse button). Dragging up and down rotates the model around a horizontal (x) axis
through its center. Dragging left and right rotates around the vertical (y) axis.
Hold down shift and command keys while dragging left and right to rotate the
model around the z-axis, which is perpendicular to the screen.
To move the model around on the screen (called translation), hold down
option and drag. The model moves across the screen, following your mouse motion. To
translate or to rotate about the z-axis, you don't even have to hold down the mouse
button
To zoom, hold down shift and drag the pointer down the screen to bring
the model closer, or up to move it farther away.
These mouse motions allow you to move the model around and orient it so that
you can see its features clearly. Such motions are a fundamental part of any molecular
modeling program. Take time to play with the the model by rotation, translation, and
zooming. You can always return to the original orientation like this:
RasMol > reset < return >
You will often need to rotate, translate, or zoom the model in order to carry out the
instructions that follow. Movement helps you to see the molecule better, especially to
distinguish near from far.
You may conveniently take a break from this tutorial at the
end of this or any later section. To resume your work at the beginning of any section,
start the desktop copy of RasMol and open 3b5c.pdb. Arrange the windows as in
section 1.
5. Picking, Selecting, and Restricting
Use picking to find residues 64
and 72. You may need to manipulate the model to bring these residues into view. Notice
that residues 64 through 72 constitute an alpha helix. Now you will examine the helix more
closely.
Rasmol > restrict 64-72 < return > The restrict
command selects all atoms specified, and removes all others. It does not alter the
display of the selected atoms. Subsequent commands will affect only the selected atoms.
Display: Sticks
Colours: CPK
RasMol draws residues 64 through 72 as a stick model. Try rotating the model so that
all main chain carbonyls point upward. You will find that the rotation behavior is
strange, because RasMol is still rotating about the center of the whole molecule. Fix this
as follows:
RasMol > set picking center < return >
Now find the lysine residue at one end of the helix. Click on its alpha carbon. The
command line should read
Rotating about lys72.ca (578)
If some other atom is listed, search again for the alpha carbon of lysine 72 and click on
it to get this message. RasMol will now rotate the model around this atom.
RasMol > set picking ident < return >
This resets picking to identify picked atoms without changing the centering of rotation.
After issuing a command, always check the command line to see if RasMol gives back a
message. If there is no message, it means that RasMol recognized the command. RasMol
reports syntax errors in commands if it cannot recognize them.
Now you will reduce the display further to just one residue.
RasMol > restrict 72 < return >
Now only the lysine residue is shown. Bring the residue to the center of the display.
Click on the atoms of this one residue, starting with the alpha nitrogen (blue, near the
red oxygen), then the alpha carbon attached to it, the carbonyl carbon, and the carbonyl
oxygen (red). Note their atom names on the command line. In the same order, they are N,
CA, C, and O. In the PDB file, these are the names of the main chain atoms of each
residue. Now find the atoms of the side chain and click on them, noting their names on the
command line. Starting next to the alpha carbon, they are the beta carbon (CB), gamma
carbon (CG), and so forth, out to the zeta atom, which is nitrogen (NZ). PDB files contain
lists of all atoms in a protein, named in the same way as this residue, along with the
data needed to display them as a graphics model.
In addition to display data, a PDB file contains other useful information.
This is a good time for you to take a break and look at PDB
File Contents.
Now you will bring the entire alpha helix back into the display, and center
the rotation about the alpha carbon of the central residue, arginine 68.
RasMol > select 64-72 < return >
Display: Sticks
RasMol > center arg68.ca < return >
The last command is another way to change the center of rotation. Notice the syntax of the
atom expression that follows the word center. An atom expression describes a
particular atom or set of atoms. This expression for a single atom consists of the
abbreviation of the residue (arg), followed -- without a space between -- by the residue
number (68), a period, and the atom name (ca for alpha carbon). An atom name is always
preceded by a period. The previous command also contained an atom expression, 64-72.
RasMol is pretty picky about the syntax of these expressions, so take careful note when
you encounter them. You can use these expressions in select and restrict
commands as well.
For more information about expressions, type help expressions, help
primitives, and help examples. Although atom expressions may seem daunting to
you now, you will see many examples before you complete the tutorial.
Arrange the helix so that it takes up most of the screen, with carbonyl
carbons pointing upward. Press the cursor-up key next to the keypad and watch the command
line. Each time you press cursor-up, RasMol reprints your previous command. Keep pressing
until you bring your restrict 64-72 command back to the command line. If you go
past it, use cursor-down to retrieve more recent commands. When the command is on the
command line, add and mainchain to the command, and press return. In other words,
RasMol > restrict 64-72 and mainchain < return >
The side chains disappear, giving you a clear view of the main chain conformation in an
alpha helix. You have restricted the selected atoms to residues 64 through 72, and
to the mainchain atoms of the protein. Mainchain is one of many sets that
you can specify in RasMol commands. The mainchain set includes only atoms N, CA, C,
and O from each residue. For a listing of the names of other sets, type help sets return.
For a listing of residues in each set, see the RasMol Manual.
RasMol > hbonds < return >
RasMol draws dotted lines for all the main-chain hydrogen bonds of currently selected
residues. Now use picking to confirm that most hydrogen bonds in an alpha helix are
from the carbonyl oxygen of residue n to the N-H of residue n + 4. Note that
hydrogen atoms are not present in this file. In the x-ray crystallographic image of
proteins, it is usually not possible to resolve the hydrogen atoms, but we deduce their
locations from principles of structural chemistry.
RasMol only looks for hydrogen bonds between mainchain atoms, such as those
in helix and sheet. What is more, it "finds" the bonds by simple criteria of
geometry and distance, criteria that are sometimes met more than once by the same atom
(for instance, notice the two hydrogen bonds drawn to the nitrogen of lysine 72). In this
case, the real bonding situation is unclear.
6. Viewing in Stereo
(If you are resuming the tutorial
after a break, start your desktop copy of RasMol, arrange the windows as in section 1, and
open 3b5c.pdb. Restrict the view to residues 64-72, mainchain only. Turn on
hydrogen bonds.)
Options: Stereo
RasMol displays the model as a stereo pair. Viewed properly, a stereo pair
gives you a three-dimensional (3D) image of the model. Take time now to begin learning
this skill. It takes some practice, and you may find it slightly uncomfortable at first,
but it will become easy and comfortable, and your effort will be richly rewarded by
increased power to see spatial relationships and structural details that are much harder
to see any other way.
Here's how to view in 3D. Gaze at the screen, keeping your head level (don't
tilt it left or right), and cross your eyes slightly. As you know, crossing your eyes
makes you see double, so you will see four images. (By the way, you can't hurt your eyes
or eye muscles by crossing your eyes, and you can't get them stuck that way.) Try to cross
your eyes slowly, so that the two images in the center come together. When they converge
or fuse, you will see them as a single 3D image. The fused image will appear to lie
between two flat images, which you should ignore. When you are viewing correctly, you see three
images instead of four. The center image is three-dimensional. At first, the 3D image may
be blurred. Keep trying to hold the stereo pair together while you focus. The longer you
can hold it, the more time your eyes have to adjust their focus. Usually, even before you
begin to get the hang of focusing, the two central images lock together, because your mind
begins to interpret them as a single 3D object.
Having trouble? Here's another approach. With your head level and about 2.5
feet from the screen, hold up a finger, with its tip about 6 inches in front of your face,
and centered between the stereo pair on the screen. Focus on your finger tip. Without
focusing on the screen, notice how many images you see there (they will be blurred). If
you see four images, move your finger slowly toward or away from you eyes, keeping focused
on your finger tip, until the middle pair of images converge. With your finger still in
place, partly covering the converged pair, change your focus to the screen. The image
partly hidden by your finger should appear three-dimensional. Your finger should still
appear single, but blurred. With some practice, you can remove your finger and still keep
the screen images converged into a stereo image.
Too often, people try only briefly and halfheartedly to view in stereo, and
never try again. Almost anyone can view in stereo with a little effort and practice. The
only ones who simply cannot are those who have acute amblyopia (one very weak eye). And
those who say they can see just as much without stereo simply cannot imagine what they are
missing. You can continue this tutorial with or without stereo viewing, but if you ever
need to explore macromolecules on your own, you can be a much more effective explorer if
you learn to see in 3D.
(This viewing method is different from that needed for viewing printed stereo
pairs in textbooks and journals. To view most printed images, you need to view the left
image with the left eye, and the right image with the right eye (called divergent
viewing). In addition, with printed views, the distance between the images must be less
than the distance between your eyes, so the images must be small. Viewers are available to
magnify the image and to guide your eyes. To view larger stereo pairs, such as those on a
computer or projection screen, you must use the method described above, and cross your
eyes slightly to look at the right hand image with your left eye, and at the
left-hand image with your right eye (called convergentviewing). For most
people, convergent viewing is easier to learn, but most structural biologists learn to
view both ways without viewers. For more help, see Stereo Viewing.)
One unfortunate quirk of the current version of RasMol is
that picking is sometimes unpredictable in stereo. You should always pick on the left
image, but if you do not get proper response to picking, turn off stereo for picking by
selecting Options: Stereo. (The check mark appears by the word Stereo when
the stereo display function is turned on, and disappears when it is turned off -- this
type of on/off command is sometimes called atoggle.)
7. Exploring
(If you are resuming the tutorial
after a break, start your desktop copy of RasMol, arrange the windows as in section 1, and
open 3b5c.pdb.
Restrict the view to residues 64-72, mainchain only. Turn on hydrogen bonds.)
Display: Spacefill
Rotate this model to view it end-on. Notice that stick models make protein structures
appear very open and empty, but even an isolated helix is quite densely packed.
RasMol > restrict 64-72 < return >
RasMol > hbonds off < return >
Display: Spacefill
Now you see a space filling model of the entire helix. The side chains reappear
because this restrict command includes them.
RasMol > select < return >
Now all atoms are selected.
Display: Backbone
Colours: Structure
RasMol > reset < return >
This returns the full backbone to the screen, centers rotation on the center of the whole
model, and returns it to the original orientation. The last four commands are useful when
you get lost and need to redisplay everything and get your bearings again.
RasMol > restrict sheet < return >
This command removes all residues except those in pleated sheet (sheet is another set).
Center rotation on one of the alpha carbons in the middle of the sheet (look back at
previous commands if you don't remember how, and remember to set picking back to ident
after you change the center of rotation). Display the model as sticks and color it CPK.
Display hydrogen bonds. To help you see the sheet structure more clearly, remove the
sidechains as follows:
RasMol > restrict sheet and mainchain < return >
Decide whether the three central strands in the sheet are parallel or antiparallel. What
about the edge strands? Are they parallel or antiparallel to their neighbors? Here's how
to check your answers:
Display: Cartoons
Cartoon displays show sheet strands as arrows pointing toward the C-terminal end of
the chain. A pair of chains with arrows at the same end are parallel. If arrows on
neighboring strands are at opposite ends, the strands are antiparallel.
RasMol > select < return >
RasMol > hbonds off < return >
Display: Cartoons
Colours: Structure
Now you see the whole protein as a cartoon. This is a vivid display that is easy to
interpret, but it has one disadvantage for further exploration: picking doesn't identify
atoms. Remember that you must us a display function that shows the exact location of atoms
in order to identify atoms by picking.
RasMol > restrict turn < return >
Display: Backbone
Now you see the beta turns in the model.
RasMol > restrict 17-21 < return >
This shows just one turn. Display it as sticks in CPK colors, and turn on hydrogen bonds.
Notice that residue 19, the middle residue, is lysine.
RasMol > center lys19.ca < return >
Confirm that the hydrogen bond in a beta turn connects the carbonyl oxygen of residue n
with the N-H of residue n+3. From information in a standard biochemistry textbook,
decide whether this is a type I or type II turn. One way to look at it: if the three
carbonyls that make the turn (residues 17, 18, and 19) all point in the same general
direction (up or down when you look at the turn edgewise), then it the turn is type I. If
the middle carbonyl points in the opposite direction from the other two, it's type II.
RasMol > reset < return >
RasMol > select < return >
RasMol > hbonds off < return >
Display: Cartoons
Colours: Structure
RasMol > select hetero and not hoh
Display: Spacefill
Colours: CPK
Now you see the heme prosthetic group in its cartoon-protein binding site. You will
now use RasMol to explore the binding of the heme to the protein.
Rotate the molecule so that you see the heme edge-on, protruding from the
right- hand side of the protein. Notice that the binding pocket is composed of four alpha
helices above and below the heme, and a four-strand pleated sheet on its inside edge. The
molecule looks somewhat like a pair of jaws holding a heme. The teeth are four alpha
helices, and the throat is pleated sheet. However, this cartoon view does not display any
of the chemical details of heme binding.
RasMol > restrict within (7.0, hem) < return >
Display: Wireframe
Colours: CPK
RasMol > center hem.fe < return >
RasMol > select hem < return >
Display: Ball & Stick
Now you see the heme as a ball and stick model surrounded only by atoms that lie
within 7.0 angstroms of heme atoms. The restrict within and select within commands
are powerful tools for directing your attention to specific interactions within
macromolecules.
Look for possible electrostatic interactions and hydrogen
bonds between the heme (ball & stick) and the protein (wireframe). First, look at the
yellow iron (III) ion in the center of the heme. The ion is part of an octahedral
transition metal complex. Four of its six ligands are the light blue nitrogens of the heme
porphyrin ring. What are the other two ligands? Use picking to identify them. When you
have learned the residue numbers of the side chains that contain the ligands, display them
in stick form as follows:
RasMol > select (#1 or #2) and sidechain < return >
(substitute the residue numbers of the ligands for "#1" and "#2").
Display: Sticks
Now you should see two histidine side chains providing nitrogens as the fifth and
sixth ligands of iron(III). The stick display does not include the alpha carbon in the
main chain, because the alpha carbon is not included in the selection sidechain,
which selects CB but not CA. You can, however, include CA in the display, and complete the
stick model of the histidine side chains without changing the selection, as follows:
RasMol > set bondmode or < return >
Display: Sticks
Notice that one bond, the CA-CB bond, is added to the display for each histidine.
RasMol has two bond modes, called and and or. In bond mode and,
RasMol draws bond CA-CB if both CA and CB are selected. In bond mode or,
RasMol draws bond CA-CB if either CA or CB is selected. When you start RasMol, the
bond mode is and until you change it. Consider your last two sticks
commands, with CB selected and CA unselected. On the first try, in the and bond
mode, RasMol did not draw the the CA-CB bond as a stick because CB was selected and CA was
not. Then on the second try, in the or mode, RasMol drew the CA-CB bond as a stick
because CB was selected. Changing the bond mode does not change the current display, but
it does change the behavior of subsequent display commands.
RasMol > set picking monitors < return >
Working without stereo, click first on the iron (III) ion, and then on one of its
histidine nitrogen ligands. A dotted line appears between the atoms, along with a label
showing the distance between the atoms in angstroms. The label is colored that same as the
first atom picked. If you accidentally pick the wrong atoms, you can remove the dotted
line and label by picking the same two atoms again. Now measure the distance between iron
(III) and the other histidine ligand. This distance, about 2.0 anstroms, is the length of
the bond between iron (III) and nitrogen in the transition-metal complex. Such bonds were
once called coordinate-covalent bonds, because one of the two bonded atoms, in this case
the ligand nitrogen, donates both of the electrons to form the bond.
The heme has two chains that extend from the edge that sticks out of the
binding pocket. Both chains end with carboxyl groups. Can you find any heme-protein
interactions that involve the heme carboxyls? There are two hydrogen bonds involving
serine 64. Find and measure them. To make all your measurements easier to see, use the
cursor-up key to retrieve and execute your command restrict within (7.0, hem). Then
display all the atoms of the heme and its binding pocket in wireframe. Another quirk of
RasMol is that the heme iron is not displayed in wireframe. Select only the iron (hem.fe)
it and display it as a ball using Ball & Stick.
Now you will study the remaining interactions that hold the heme in place,
which are primarily hydrophobic.
RasMol > set picking ident < return >
RasMol > monitors off < return >
This sequence resets picking to the default and removes all measurement lines and labels.
RasMol > select hydrophobic and sidechain < return >
RasMol > color yellow < return >
This sequence selects only the sidechains of hydrophobic residues, and colors them yellow.
The hydrophobic set is another set of atoms you can use in select commands.
The color command does not add atoms to, or remove atoms from, the display. To see
the names of other colors you can use in commands, look at help for color
and colors. (In the colors help information, RGB stands for red-
green-blue.)
RasMol > select hem.c?? < return >
RasMol > color cyan < return >
This sequence selects all carbons in the hem and colors them cyan. The question
marks stand for unspecified characters. The selection hem.c?? means,
"atoms of heme designated c followed by up to two additional unspecified
characters." Now you will display the heme and its binding pocket as a space filling
model.
RasMol > restrict within (7.0, hem) and not hoh < return >
Arrange the model so that you are looking into the opening of the hem pocket, with the two
heme carboxyls pointing at you. It is much easier to do it stereo. If you can't tell front
from back, try displaying in ball and stick, which gives more depth cues.
Display: Spacefill
Now you see the heme peeking out of its pocket. One carboxyl points out into space,
and the other is in contact with two atoms of serine 64. The cyan carbons of the heme are
hydrophobic, as are the yellow carbons of the hydrophobic sidechains. In this view, you
see hydrophobic interactions, therefore, as contact between cyan and yellow. But much of
the contact area is buried by the space-filling models. Let's look inside.
RasMol > slab 100 < return >
Now hold down the control key at the bottom left of the keyboard (not the command
key). While holding the key down, move the mouse pointer up the screen. As you move
the mouse, an invisible plane slides back through the model, cutting away everything that
lies in front of it. Thus you can slice into the model to any depth, removing all
foreground as you go. Slide the pointer up and down the screen to change the position of
this cutting plane. (You may find that the action is choppy, because the computer is doing
many calculations to produce each successive view.) As you cut into the model, notice the
contacts between heme carbons (cyan) and atoms of hydrophobic side chains (yellow). By
releasing the control key, you can rotate the model to cut into it from other
directions.
RasMol > slab off < return >
Again orient the model so you are looking at the heme peeking out of its pocket.
RasMol > select hem < return >
Display: Wireframe
RasMol > select hem.fe < return >
Display: Ball&Stick
Now you can clearly see the interior of the pocket, and observe its strongly
hydrophobic nature. You can also see the two histidine side chains that protrude into the
pocket to interact with the iron (III).
RasMol > select hem < return >
RasMol > dots 200 < return >
This display colors the surface of the heme with about 200 dots per atom. Dot displays
give a feeling of solidity, but you can see through them to neighboring atoms. Zoom in and
try to see contacts between heme carbons and other atoms in the pocket. These contacts are
much easier to see in stereo. For a dramatic view of the pocket, try turning the model
around and slabbing in through the back. You will see what the world looks like from the
perspective of the iron (III) ion.
File: Close
Cytochrome b5 is a small protein consisting of a heme and only one polypeptide chain. In
your biochemistry class, you will also study oligomeric proteins, which consist of
more than one polypeptide subunit, as well as protein-protein and protein- nucleic acid
complexes. RasMol has some features that are very useful with models that contain more
than one chain. Now you will briefly examine such a model -- part of an antigen-antibody
complex.
8. Oligomeric Proteins
Obtain the PDB file with code2iff(see section 1 for a reminder about how to get a PDB file). After the
file appears on the desktop, rename it 2iff.pdb. Then drag it onto
the RasMac v2.6 icon to open it. For sections 8 and 9 of this tutorial, you must make sure
that you are using the desktop copy of RasMol; otherwise you may have difficulty finding
files that you create using RasMol.
The file 2iff.pdbis larger than 3b5c.pdb
and takes longer to load. When loading is complete, RasMol provides some information about
the file in the Command Window, and shows a complete wireframe model in the Main Window.
Arrange the windows as described in section 1. Move the model around to get some feeling
for its size and complexity in comparison to cytochrome b5.
Display: Backbone
Colours: Chain
RasMol presents each chain in a different color. This allow you to see immediately how
many different chains are present. Click on any atom in the pea-green chain. (the smaller
of the two chains shown in shades of green). On the command line, RasMol displays
something like
Atom: CA 2574 Group: GLY
117 Chain: Y
This tells you that the green chain is designated Y in the PDB file. With
oligomeric proteins, you need to know chain designation in order to compose specific select
and restrict commands. Other than the need for chain designations, exploring a
multi-chain model is just like exploring a single chain. Identify the other chains by
clicking on them. Chain Y is the enzyme lysozyme from hen egg white. Chains H and L are
the antigen-binding parts of the heavy chain and light chain of a mouse
antibody. Together, they are called an Fab
fragment. The antibody was made by immunizing a mouse with hen lysozyme and then purifying
a specific antibody to lysozyme So in this model, lysozyme is the antigen.
RasMol > select helix or sheet < return >
Display: Cartoons
Colours: Structure
The first command selects all residues that are in either alpha helical or beta
pleated sheet conformations. (The command select helix and sheet selects
nothing. Why?) The Display and Colours commands affect only the selected
residues, so the non-helical and non-sheet portions remain in colors that allow you to
distinguish them as separate chains. You can see that the antigen (Y) contains several
alpha helices and a 3-strand pleated sheet, and that the antibody chains are primarily
beta structure. The two antibody chains each exhibit two domains of the immunoglobulin
fold, a beta structure that is found in all antibodies, as well as some other proteins
whose function is recognition.
RasMol > select :H or :L < return >
Display: Backbone
Colours: Chain
RasMol > select within (6.5, :Y) and not :Y < return >
RasMol > color white < return >
Parts of the antibody backbone that are close enough to the antigen to be involved in the
antigen-antibody interaction are now shown in white. With a few commands, you have focused
on what are called the complementarity determining regions(CDRs) of the
antibody. By restricting your display to these regions and adding all atoms in wireframe,
you could explore in detail the interactions that bind this antibody to its antigen.
9. Saving Your Work: Scripts
During detailed exploration of an
unfamiliar model, you my want to put your work aside, and come back later to start with
your current view. Sometimes you can invest much time and thought in producing a
particularly revealing view of a model. RasMol provides a way to preserve your work. This
is a great feature for illustrating lectures, because you can make vivid classroom views
and bring them to the screen immediately. At any time during operation of RasMol, you can
write a script that RasMol can use to recover the current view. You will now write
a script.
First, rotate, translate, and zoom your model into a position that shows the
CDR's clearly, and that fills the screen nicely.
RasMol > write script CDRs.spt < return >
After a brief pause, RasMol writes the script. This new file will appear in the same
location as the RasMol program icon. If you have been working, as instructed, with RasMol
on your Macintosh desktop, the file will appear there. Its icon is a little scroll. The
file ending .spt (for script) helps to distinguish a script file from
a coordinate file, which usually ends in .pdb or .ent. After one more
exercise, you will try out your script.
Look back at the commands you have used with this file, and notice the syntax
of atom expressions that designate the chain. Observe that just as .ca means atom
CA(an alpha carbon), :Y means chain Y (if you prefer, you can use *Y).
In a select or restrict command, you must specify the chain unless you want
the command to apply to all chains. As an example of the select syntax, select
gly40:L.ca selects one atom: the alpha carbon of glycine 40 in the L chain. To test
yourself on this syntax, try this: restrict the display to aspartic acid 48 of the antigen
chain, display it as ball and stick, center on the alpha carbon, select the gamma carbon
of this residue, and color it green.
Next, exit from RasMol.
File: Quit
This commands stops RasMol, and its windows disappear, revealing the desktop.
Find your script file (CDRs.spt) and double click on
it. RasMol appears, and after a pause (perhaps a long pause!), presents the same view you
were displaying when you wrote the script file.
There are some restrictions to your use of the RasMol scripts. In their
original form, they can only be used from the same location (in this case, the desktop)
into which RasMol saved them. This means that RasMol, the PDB file, and the script must
all be exactly where they were at the time the script was prepared. Fortunately, by
editing the scripts in a word processor, they can be modified and made portable. In the
exercise at the end of this tutorial, you will make and save several scripts, and then
transfer them to a disk to hand in. I will be able to modify them so I can see your
results. If you want to use scripts yourself see ##Making Scripts Portable.
10. Summary
In working through this tutorial,
you have used many RasMol commands and features, but there are many more available. In
fact, this tutorial merely scratches the surface of a powerful and versatile program. The RasMol Manual is
a complete manual for RasMol, designed for display in a World Wide Web browser like
Netscape. The manual is designed for ease of use as a reference. After you download the
manual, save it as a "Source" file named rasman.htm. Drag this file icon
onto your Netscape icon to open it. Wait until it loads completely before clicking any
links; otherwise, you will interrupt loading of the file. Then click on any link in the
manual to jump to more information about that term or command. You can also use Edit:
Find in you web browser to find more information about any command, including those
you have used in this tutorial. Now that you know the basics of RasMol, you will find it
easy to learn more.
For students of biochemistry, you will find PDB files of many of the
molecules studied in a typical biochemistry course at the web site for the course I teach
at the University of Southern Maine. Studying these molecules with RasMol may help you
grasp difficult structural concepts in your own course. To obtain these files, direct your
web browser to my home page.
Happy Modeling!
11. Assignment and File
List
USM students: Click this link to find instructions for your assignment on
this work.
TEACHERS: The assignment includes a list of PDB files appropriate for students who have
completed this tutorial.
13. Useful Addresses on the World Wide Web
Structure Files for Biomolecules:PDB WWW Server -- http://www.pdb.bnl.gov/
Molecular Viewer, Basic: RasMol -- http://klaatu.oit.umass.edu:80/microbio/rasmol/
Molecular Viewer, Advanced: SwissPdbViewer -- http://www.pdb.bnl.gov/expasy/spdbv/mainpage.html
Powerful Computing Tools in Molecular Biology: ExPASy Molecular
Biology Server -- http://expasy.hcuge.ch/www/expasy-top.html