Assignment 1, part 1A 5

EXERCISE 1: SOME BASICS OF SECONDARY AND TERTIARY STRUCTURE

In this lesson you will learn the following Jmol skills:

• How to download a protein structure file from the Protein Data Bank (PDB) web site
• How to read the information on the PDB page for a protein structure
• How to display an interactive image of the protein in Jmol
• How to manipulate images of a protein with your mouse
• How to use the Jmol menus to change the way the protein is displayed
• How to use commands in the Script Console to change the way the protein is
displayed
• How to change the display style of selected parts of a protein
• How to display hydrogen bonds

You will use these skills to:

• Examine a short α-helical peptide and a small protein with both an α-helix and a β-
pleated sheet
• Gain a better understanding of different types of protein secondary structure
• Gain a better understanding of the principles of the tertiary structure of globular
proteins
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DOWNLOADING STRUCTURES AND LAUNCHING JMOL: Download Jmol to your computer as

described in "Downloading Jmol." Then launch your web browser (e.g. Internet
Explorer, Firefox, Netscape, Safari, etc.).

Go to the web page of the Protein Data Bank at https://www.rcsb.org/. In the search box
at the top of the page, type 1AL1. Click on Go or hit Return. You will get a page with
information about the molecule, including the citation to the journal article in which the
structure was published. This is the Structure Summary tab. It has the title of the
journal article in which the structure was first published, information about the authors
and the date the data was deposited, the method of structure determination (X-ray
diffraction in this case) and the resolution (2.7 Å). The R-value is a measure of how well
the 3-D model fits the data. The wwPDB Validation has information of interest to
crystallographers about the quality of the structure – including how well it fits the X-
ray crystallography data, how well the bond lengths and angles conform to standard
values and how well it avoids steric clashes.

There is a description of the molecule in the Macromolecules box; in this case the
molecule is an : ELLKKLLEELKG. The Small Molecules box tells us about non-amino
acid compounds or functional groups present in the structure. The Ligands box tells us
that in addition to the peptide there is a sulfate ion; the Modified Residues section tells
us that there is an acetyl group.

At the top left is an image of the molecule, consiting of a blue helix with a green
extension (the acetyl group) at one end and a sulfate ion bound at one end.

At the upper right, click on the Download Files and then on PDB File (gz) in the drop-
down menu. Save the file 1AL1.pdb.gz to your computer, and remember where you
saved it. (Note: The extension ".gz" indicates that this is a compressed ("g-zipped")
version of the file. It is not necessary to expand the file to the non-zipped version
1AL1.pdb before opening it in Jmol. The compressed files will take up less storage
space on your computer.)

Before you go further, you must download Jmol by following the instructions in
"Downloading Jmol."

Launch Jmol by double-clicking on the file jmol.jar. From the File menu choose Open,
locate the file 1AL1.pdb.gz and select it. Alternatively, you can locate the 1AL1.pdb.gz
icon, double-click on it, and direct your computer to use Jmol to open it.
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In order to proceed further you must have the program Jmol running with a cartoon
image of the molecule in a Jmol window.

Figure 1

If the image of the molecule appears in a browser window instead of a Jmol window,
you will not be able to perform the exercises that follow. Go back and follow the
instructions again.

The window shows a cartoon image of an alpha helical peptide with a sulfate ion at one
end. The sulfate ion forms a salt bridge with the positively charged α-amino group.

Before leaving the PDB page, click on each of the tabs across the top of the page to see
what's there. Notice all of the choices available in the Links tab and try one or two.
Also note that under the image of the molecule in the Summary tab there is a View in
3D link that will allow you to view the structure in your web browser using Jmol
(running as a web-based applet JSMol), as well as links to view the structure in
programs called Simple Viewer, Protein Workshop, Kiosk Viewer and Ligand Explorer.
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ALPHA HELICAL PEPTIDE 1AL1:

GETTING FAMILIAR WITH THE JMOL WINDOW:

Practice manipulating the molecule with your mouse:

-- You can rotate the molecule in the X and Y directions by left-clicking on the
molecule and dragging it. To rotate in the Z direction (in the plane of the screen), hold
down the shift key and move the mouse to the left or right.
-- You can move (translate) the molecule by holding down the shift key, left-
double-clicking on the molecule, holding the mouse button down on the second click,
and dragging the molecule.
-- To zoom in or out, hold down the shift key, left-click on the molecule, and
move the mouse up to zoom out or down to zoom in. If your mouse has a scroll wheel,
you can use it to zoom in and out on the molecule.

-- See http://wiki.jmol.org/index.php/Mouse_Manual for further instructions.

Initially the image of the molecule will be a cartoon showing a magenta-colored helix,
along with ball-and-stick representations of the two non-amino acid groups, the acetyl
and sulfate groups mentioned above.

We can display the molecule in many different ways. One way to do this is by using
the Jmol pop-up menu. On a PC, right-click anywhere in the window (on a Mac click
while holding down the Control key) to bring up the pop-up menu.

Figure 2
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• Choosing Style à SchemeàCPK Spacefill displays each of the atoms as a

sphere with a radius equal to the van der Waals radius of the atom and colored
using the Corey-Pauling-Koltun scheme (carbon colored gray, oxygen colored
red, nitrogen colored blue, hydrogen colored white, sulfur or phosphorus
colored yellow). Hydrogen atoms are generally not detected in X-ray
crystallography and are not present in PDB structures.

• Style àScheme à Ball and Stick displays the atoms as spheres with a radius
that is 20% of the van der Waals radius; the bonds are displayed as lines 0.15 Å in
diameter. You can control these parameters: Choose StyleàBondsà0.25 Å to
make the bonds thicker or StyleàAtomsà50% van der Waals to make the
atoms larger. StyleàAtomsàOff gives a representation with bonds only. A
second way to control these parameters is through the Display menu at the top
of the Jmol window.

• Also try StyleàSchemeàWireframe and StyleàSchemeàTrace (which shows

a trace of the N and C atoms of the polypeptide backbone) or
Style/Scheme/Cartoon to restore the cartoon view of the helix.

What else can we do with the pop-up menu? Let's look at a few more ways to
manipulate the molecule.

• Choose SpinàOn to cause the molecule to rotate and SpinàOff to stop it.
Now try SurfacesàDot Surface. Next SurfacesàVan der Waals Surface. Both
of these show the outer surface of the protein obtained when all the atoms are
solid spheres with radii equal to the van der Waals radii. Next try
SurfacesàMake Opaque. To return to the original view, choose SurfacesàOff.

USING THE SCRIPT CONSOLE:

Another way to manipulate the molecule is through the Jmol Script Console. You may
already see a second window open on your desktop titled Jmol Script Console. If not,
from the File menu choose Console…. A new window opens called Jmol Script
Console, and a $ prompt is displayed there.
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Figure 3

We can type commands into this window using a simple language that is easy to learn.
Throughout this manual we will do most of our manipulations using the Script
Console. At first it may seem as if there is a lot to learn, but most Jmol users find that
for most manipulations of the molecules, it is easier and faster to use Script commands
than to use either the main Jmol menu or the popup menus. As you go through these
exercises, you will learn the commands gradually and slowly become competent at
using them.

For example, we can use the Script Console to display the molecule in different ways.
We can see the spacefill representation by typing the word spacefill. To return to the
original display, type undo. (Often you can do this by clicking on the Undo button of
the Script Console.) You can also type spacefill off.

Let's start over with the molecule in Style àScheme à Ball and Stick. Now type
ribbon. To see just the ribbon and not the balls and sticks, type ribbon only. For a
narrower ribbon type ribbon 200.

Now type ribbon off and then backbone (or just type backbone only). The backbone
trace shows straight lines connecting the alpha carbon atoms. Type backbone 100.
Rotate the molecule so that you are looking down the helix axis, which is a line running
lengthwise down the center of the helix. You should see that there are between three
and four amino acid residues per turn of the helix.
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Now use the drop-down menu to return the molecule to Style àScheme à Ball and
Stick. If you are still looking down the helix axis, you can now see the side chains
projecting outward from the helical backbone.

We can also use the Script Console to control the colors of the atoms and bonds.
Type select hydrophobic (hit return after each line)
color green
select positive
color yellow
select negative
color magenta
You will now be able to see the relative locations of the different types of amino acid
residues in the molecule. (Note that you can also change atom colors by using the
Select and Color commands in the pop-up window -- but using the script console is a
little easier.)

Let's focus just on the chemical nature of the sidechains. Type select backbone and
then color cpk. (You can also type the two commands on a single line: select
backbone;color cpk. Now only the sidechains are coded by chemical type.
It might be interesting to see what the molecule looks like in spacefill display. Type
spacefill. Oops! What happened? Since only the backbone was selected, only the
backbone atoms were changed into sticks. . Choose Select →All from the Jmol
Display menu. (You can also type “select all” on the command line.) Now type
spacefill again. That’s much better. Save this picture and include in your report.

(Note: Instead of typing spacefill in the script console, you could have chosen
StyleàSchemeàCPK Spacefill in the pop-up menu, but then you would have lost the
color coding and displayed all the atoms in CPK colors. Choosing any of the style
schemes automatically also chooses an associated color scheme. For this reason it is
probably a good idea to use Style in the pop-up menu only for the very initial stages of
displaying a molecule and to use script commands for subsequent changes of display
style.)

Type show info. The script console displays the name of the molecule, along with
information about its sequence, chain nomenclature, size, and secondary structure.

Click on an atom. A message appears in the Jmol script console which gives the type of
amino acid residue followed by the residue number (e.g. [LEU]6), the polypeptide chain
(:A – in this case there is only one chain, the A chain), the type of atom (N = NH
nitrogen, O = CO oxygen, C = CO carbon, CA = alpha carbon, CB = beta carbon, etc.),
the atom number (each atom in the molecule is assigned a unique number), and the x, y,
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and z coordinates of the atom that locate the atom in three-dimensional space in the
model.

Now type spacefill off, then wireframe 100. Click on the various atoms and answer the
following:

Question 1: Which amino acid is in position 8?

Now type show sequence. See whether your answer was correct.

There's one complication that needs to be pointed out. You should see three yellow
amino acid residues, all of them lysine residues. However, only one of them, lysine 4,
has a complete side chain. According to the crystallographers who determined this
structure, for some reason the side chains of lysines 5 and 11 could not be clearly
located in the electron density, so only partial side chains were included in the
structure.

Question 2: What do you observe about the distribution of the various types of side
chains? (Examine the molecule in the wireframe 100 display. Then go to the spacefill
display by typing spacefill. To return, either type spacefill off or click on the Undo
button. (If your Script Console doesn't have an Undo button, try expanding the console
by dragging downward on the lower right corner.) In both display modes, examine the
molecule in both side and end views.) How would this helix be oriented if it were on
the surface of a globular protein?

IMMUNOGLOBULIN BINDING PROTEIN (1PGB):

OVERALL STRUCTURAL ORGANIZATION:

Go back to the PDB web site and obtain the information page for the file 1PGB. This is a
56-amino acid residue portion of a larger protein, namely the B1 immunoglobulin-
binding domain of streptococcal protein G. We will use this small structure for our first
exploration of tertiary structure

Note that there is additional information in the Macromolecules box in the form of a
series of colored diagrams.
-- The top line tells you that this structure comes from entry P06554 in the
UniProt amino acid sequence database.
-- The Mol. Processing line tells us that the original protein was processed by
removal of a signal peptide at the N-terminus and another peptide at the C-terminus.
-- The Motif diagram tells us the location of certain structural motifs in the larger
precursor protein. (Move your mouse cursor over each portion to get more information
about that motif.)
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-- On the UP Sites (Uniprot Sites) line, moving your mouse cursor over the blue
circle reveals that a threonine residue is attached to a murein peptidoglycan by a
pentaglycine linker peptide.
-- The SCOP domains line tells us the location in the large precursror of several
regions whose structure corresponds to conserved domains found in a variety of
proteins.
-- The Secstruc line tells us the location of alpha helices and beta stands within
the protein. (Moving your mouse cursor over each tan or red symbol reveals the
residue numbers of the helix or strand.)
-- The PDB Validation line tells us the location of geometric outliers, residues for
which the bond angles were not within normal limits. With modern structure
minimization methods available, more recent structures have very few outliers (see
www.rcsb.org/pdb/help/featureView.html for more information about this and other
aspects of the Protein Feature.)
-- Finally the last line tells us that the structure in the A chain (the single
polypeptide chain) of structure 1PGB corresponds to amino acids 228-282 of the
precursor protein.
-- Clicking on the button Full Protein Feature View for P06654 brings up even
more information about this protein.

Download the 1PGB structure file and open it in Jmol. You will see a Cartoon view of a
protein colored according to structure. In this color scheme beta sheets are yellow,
alpha helices are magenta, and regions with none of these secondary structures --
sometimes called "coil"-- are white.

There are also small red spheres that represent the oxygen atoms of water molecules
that are part of the crystal structure. To make them disappear, type delete water. The
water molecules are removed from the working PDB file and cannot be restored
without reopening the original file in a new Jmol session.

Alternatively you can type restrict not water. (A "restrict" command causes everything
not selected to disappear from the display, but the selected atoms can be restored later.)

The instructions for the exercises in this manual will assume that you have removed the
water molecules with a delete command.

Type the command show structure and see what information is displayed.

Rotate the molecule to get a sense of the relative orientations of the helix and beta sheet.
Orient the molecule so that the helix axis is pointing straight at you. Notice that the
beta sheet is curved so that it has a convex surface and a concave. Copy this image and
include in the assignment report.
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Figure 4

Question 3: Describe the structural organization of this protein. Use the word “convex”
or “concave” in your description.

Put the molecule into backbone display, in which the polypeptide is represented as a
series of lines connecting the alpha-carbon atoms. To do this type backbone 100 and
then cartoon off (or backbone only; backbone 100). Click on the two ends of the
protein to identify the N- and C-termini. Another way to do this is by the command
color group. In this scheme the colors change from cooler to warmer (blue to red) as we
go from the N-terminus to the C-terminus. Restore the molecule to color structure
before proceeding.

Now start at the N-terminus of the molecule and trace the course of the polypeptide
backbone, rotating the molecule as necessary to see where you are going.

Question 4: Describe the order of the various secondary structure elements as they
occur in the primary sequence, starting as follows: “Residues 1 to 8 are in a beta sheet
conformation. Residues 9 and 10....”

A CLOSER LOOK AT THE BETA SHEET:

Question 5: Number the four regions of beta sheet 1 through 4 to indicate their position
within the amino acid sequence. Now start at one edge of the beta sheet and look at the
order of the strands as you go to the other edge of the sheet. Indicate the order in which
the four beta strands occur. (Hint: Is it 1-2-3-4? 1-3-2-4? Something else?)

Question 6: Beta sheets can be either parallel (with strands running from their N-
termini to their C-termini in the same direction) or antiparallel (with strands running in
opposite directions). Examine each pair of adjacent strands in the beta sheet (there will
be three pairs). For each pair of strands, indicate whether they are parallel or
antiparallel.
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To see the hydrogen bonds (H-bonds), type hbonds calculate. This command directs
the program to identify each pair of NH hydrogens and CO oxygens of the backbone
(for the currently selected atoms) that are close enough to allow H-bond formation. The
H-bonds involving amino acid residues from the sheet will appear as dash lines. But
since the H-bonds connect amide nitrogens to carbonyl oxygens but are themselves not
part of the backbone, they seem to be floating in space. To fix this type set hbonds
backbone. You can make the H-bonds thicker by typing hbonds 20. If you want to
make them a different color, type color hbonds green. They’re a little hard to see
against the black background, so type background white. (If you're in a playful mood,
try background magenta.) Notice how the H-bonds run perpendicular to the strands of
the beta sheet and lie in the plane of the sheet, while the H-bonds in the helix are
parallel to the helix axis.

Question 7: Print an image of the molecule. (See "Printing and Saving an Image" in the
Appendix for instructions on printing.)

Let's display the other atoms as well. Type select all; wireframe only; wireframe 100.
Now type set hbonds sidechain to make the H-bonds connect the N and O atoms of the
backbone. Typing color hbonds cpk will color each H-bond blue at the amide end and
red at the carbonyl end. To see how the H-bonds connect the N and O atoms more
clearly, we'll look first at the beta sheet and then at the helix. Type select sheet; color
cpk, then restrict sheet. Finally type select not sheet; hbonds off. Zoom in on the
sheet. You should now be able to see how each H-bond connects an NH group to a CO
group in an adjacent strand. (The hydrogens are not displayed, so the NH hydrogens
are missing.) Note how within a given strand the red oxygen atoms alternate up and
down, as do the blue nitrogens. Include a copy of this image in your report.

A CLOSER LOOK AT THE ALPHA HELIX:

Now let's restore the helix and examine it. Type select all; wireframe 100; color cpk,
then restrict helix. Now type select helix; hbonds 20; select not helix; hbonds off.

Question 8: Choose two H-bonds. Click on the atoms at each end to identify the amino
acid residue numbers. List the pairs of residues for two H-bonds. In an alpha helix,
how far apart is each pair of amino acid residues?
(Is amino acid residue X hydrogen-bonded to residue X+3? X+4? X+5?)

Type select all; hbonds 20; wireframe off. Only the H-bonds remain. Can you tell
where the helix is and where the sheet is?

Restore the cartoon view (select all; cartoon; color structure) and, just for fun, type
rotate. To stop the rotation type rotate off. (You can also use the commands spin and
spin off.
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**An Important Note About Changing Display Styles**

When you switch from wireframe to spacefill display, you can just type spacefill.
However, when you switch from spacefill to wireframe display, you have to type
wireframe; spacefill off or wireframe only; otherwise the spacefill will block your view
of the wireframe inside.

If you're in wireframe and you type cartoon, you will see the cartoon image
superimposed on the wireframe image. If you just want to see the cartoon image, type
cartoon; wireframe off or cartoon only.

FURTHER EXPLORATIONS

FE 1: Download and display the structure file 1GZI. Which protein is this? Trace the
polypeptide chain and describe the sequence of secondary structure elements within the
protein. Analyze the beta sheet in terms of parallel and antiparallel orientation. Look
at the region from residues 41 to 56. Describe the secondary structure of this region.
Include a cartoon view of this protein in the report.

FE 2: Download and display the structure file 2KT2. Which protein is this? Trace the
polypeptide chain and describe the sequence of secondary structure elements within
the protein. Analyze the beta sheet in terms of parallel and antiparallel orientation.
Include a cartoon view of this protein in the report.

FE 3: On the basis of your observations in exercises FE1 and FE2, describe the role of
beta sheets in bringing different parts of the polypeptide chain together.