Download LS1a Fall 2014 Lab 4: PyMOL (Nucleic Acid and Protein Structures)

LS1a Fall 2014
Lab 4: PyMOL (Nucleic Acid and Protein Structures)
Goals: The objectives of this lab are to provide you with a better understanding of:
1. the three-dimensional structure of a deoxribonucleotide
2. how two antiparallel strands of DNA optimize the position and orientation of the
nitrogenous bases to form the hydrogen bonds necessary for base pairing
3. how proteins can detect specific DNA sequences by interacting with the unique pattern of
hydrogen bond donors and acceptors at the edge of the bases at the major groove
Required Safety Regulations and Lab Etiquette
 Wear a lab coat and safety glasses at all times
 Wearing appropriate clothes that protect the leg, foot, and ankle (e.g., jeans, socks, and
sneakers)
 No food is allowed in the lab space
Introduction
As you learned in lecture, nucleic acids are polymers of nucleotides that are strung together to
give rise to both DNA and RNA. DNA and RNA differ greatly in the functions they serve inside the
cell because of their structural differences. These functional differences are a direct result of
their unique chemical compositions. In this lab, you will have a chance to visualize the threedimensional structures of the DNA nucleotide monomers, the DNA double helix, the hydrogen
bonds that form between the nucleotide bases, as well as a special type of RNA (tRNA) that lies
at the heart of protein translation. You will also have a chance to observe the structure of a
protein-DNA complex.
Review of Basic PyMOL Functions
Please recall the basic commands that are used to manipulate molecules on the screen using the
computer mouse:




Rotate (= left mouse button): To rotate the molecule, click and hold (“click and drag”) the
left mouse button and move the mouse left, right, up or down.
Move (= middle button): To “move” an object on the screen, you must click on the object
using the wheel as a button. Make sure to hold the button down as you move the mouse
and try to not turn the wheel. Turning the wheel simply controls the “Z-axis clipping plane”
for the viewer. If you rotate the wheel by accident and clip the image, turn it back in the
other direction until the entire molecule reappears.
Zoom (= right mouse button): Click and hold (“click and drag”) the right mouse button and
move the mouse up or down in order to zoom in or out on the molecule. Go easy at first! It
doesn’t take much to “lose” the molecule if you go too far.
Type in “reset” if things get to crazy, or just close and open the file again.
Also, recall that the rectangular buttons on the right side of the viewer window are used to
select “objects” from the list (see example below). Select the object of interest by clicking on
the rectangular button to turn it on, or remove it from the screen by clicking on it again to turn
it off. We will not be using any of the small square buttons ([A], [S], [H], [L], and [C]) in this lab.
Rectangular buttons select objects from the
viewing list
Procedures for today’s lab
This lab is composed of four sections; each section will utilize different files. The lab questions
(starting in Section 1) are highlighted with a “►” symbol in the left margin of the page and
should be answered as you work your way through the different exercises. Answer your
questions on the “answer sheet” provided. Your answer sheet will be due a week from today’s
lab.
To access today’s files:
 Open the “Documents” folder
 Choose the “Life Science Labs” subfolder and open it
 Click on the “LS1a_PyMOL_Lab4.zip” filed to open it.
 The .zip file will open a new folder called “PyMOL Lab4.”
 Click on this new folder.
OR
 Download and open the appropriate files from the course website.
Section 1: Nucleic Acids Basics
 Open up the folder titled “1_Nucleic_Acids_Basics,” then double-click on the PyMOL file
labeled “NucleicAcids.pml.” This will automatically open up the PyMOL program and load
up the coordinates for the DNA structure. OR
 Download and open “1-NucleicAcidBasics.pse” from the lab page of the course website.
When you download the .pse files from the website, you will be able to scroll through the
“scenes” listed in the protocol.
The first image you will see is that of 2’ deoxyribose, a derivative of the sugar ribose.
Try orienting the molecule so that the perspective aligns with those shown in the drawings
below. These two-dimensional drawings try to represent a three-dimensional structure by using
a solid wedge (
) to show that a bond is pointing towards you (“out of the page”) and a dash
(
) to show that a bond is point away from you (“into the page”).
We use a superscript prime (’) next to a number to designate the positions of the carbon atoms
of the sugar (either ribose or deoxyribose) to distinguish the sugar carbons from those of the
nitrogenous bases (which do not have primes, as discussed below). Both ribose and deoxyribose
adopt the shape of a five-membered ring. Oxygen is colored red and carbon is colored green. Try
to familiarize yourself with the relative locations of the 1’, 3’ and 5’ positions in the deoxyribose
molecule. It is customary to use the “prime” designations when referring to groups attached to
the sugar carbons. For example, a 3’ hydroxyl group refers to an OH group that is connected to
the 3’ carbon atom of the sugar.

Turn off “Deoxyribose” and turn on “Nitrogen_Base” (OR select Scene 02)
This will give you a basic view of adenine, one of the four nitrogen bases. Note that it is
essentially planar in shape, as depicted in the drawing below (in which the numbers of the
atoms are colored brown). You’ll also note that nitrogen is colored blue, another convention
we’ll use throughout the lab. We’ll discuss the different nitrogen bases of DNA in detail shortly.

Turn off “Nitrogen_Base” and turn on “d_Nucleoside” (OR select Scene 03)
This molecule illustrates for you how nucleosides are assembled by linking a nitrogenous base
to the 1’ carbon of a sugar. The bond linking the sugar to the nitrogenous base is called a
glycosidic bond. DNA makes use of four possible deoxyribonucleosides: deoxyadenosine;
deoxyguanosine; deoxythymidine; and deoxycytidine. (Note that the label “d_Nucleoside” refers
to these being “deoxyribonucleosides.”) Which one are we looking at right now?
Note that while the 1’ hydroxyl of deoxyribose has been replaced by the nitrogenous base, the
5’ carbon of a nucleoside is still attached to a hydroxyl group (and not to a phosphate group).
► Q1)

Turn off “d_Nucleoside” and turn on “d_Nucleotide” (OR select Scene 04)
If the 5’ hydroxyl of the sugar is replaced by a phosphate group, the molecule is now called a
nucleotide, which is the basic building block (i.e., “monomer”) of a nucleic acid. This particular
one is “deoxyadenosine monophosphate,” since adenine is used as the nitrogenous base. Also
please note that phosphorous is colored orange.

Turn off “d_Nucleotide” and turn on “Dinucleotide” (OR select Scene 05)
This molecule depicts how two nucleotides are linked together through a phosphodiester bond.
► Q2)
Now that you have a basic understanding of how nucleotides are linked together, one can
imagine linking several of them together to form a long, polymeric strand of nucleic acid.
Nucleotides always link together in the same way that we saw above. This means that when an
extended strand is formed, one end of the strand will have a nucleotide containing a “free”
(unlinked) 3’ hydroxyl group, while the other end will contain an unlinked 5’ phosphate group.
These ends are referred to as the 3’ end and the 5’ end of the strand, respectively.

Turn off “Dinucleotide” and turn on “Strand_A” (OR select Scene 06)
Notice that as the strand length increases, the strand begins to take on a helical shape. This is
because “strand A” is base-paired to a “strand B” which is currently hidden. Also note that the
nitrogenous bases all point towards the “inside” of the helix. This forms the interface for the
second strand of DNA (“strand B”).
► Q3)

Turn on “Strand_B” (leave “Strand_A” on) (OR select Scene 07)
As discussed in lecture, DNA is double stranded; the helical nature of the strands accommodates
two strands running together.
► Q4)

Turn on “Cartoon” (leave “Strand_A” and “Strand_B” on) (OR select Scene 08)
To simplify the backbone structure, scientists often use a representation called a “cartoon.”
This makes the general structure of the DNA molecule more easily discernable. Notice that the
lines appear along the sugar-phosphate backbone.
As you may recall from our first PyMOL lab, cartoons are particularly useful for viewing larger
structures when “sticks” can get too cumbersome.

Turn off “Strand_A” and “Strand_B” (OR select Scene 09)
Notice how the bases are indicated as skinny sticks that jut into the interior the double helix in
cartoon representation.

Turn off “Cartoon” and turn on “Helix” (OR select Scene 10)
To clarify the structure of the double helix, here is a “spheres” representation of the DNA
strands, where we’ve colored one strand blue and the other red. This representation can also
make the general shape of the helix easier to see while also depicting the van der Waals radii of
each atom in the molecule.
Since there are two strands in a DNA helix, you’ll also notice that there are two “grooves” (the
spaces between the two strands) that run the length of the DNA helix. One of these grooves is
larger than the other, and we call it the “major” groove. The smaller groove is called the “minor”
groove.
As a general rule of thumb, most proteins typically bind through the major groove, though they
can also bind to the minor groove. Why do you think this might be?
► Q5)

Turn off “Helix” and turn on “Backbone” (OR select Scene 11)
To clarify the structure of the DNA backbone, we’ve eliminated the bases and have colored all
the oxygens of the backbone red to emphasize their solvent accessibility.

Turn on “Bases” (leave “Backbone” on) (OR select Scene 12)
Here, we see how the nitrogen bases fill out the interior of the DNA double helix. We’ve colored
each of the four bases a different color (one color for each of the four bases).
Notice the stacking between the bases in the center of the helix and how close the planar bases
are stacked on top of one another. This is an important feature of DNA which contributes
favorably to the formation of the double helix.
Section 2: DNA Hydrogen Bonding Basics


Close the file you were working on and open up the folder titled “2_DNA_H_Bonding,” then
double-click on the PyMOL file labeled “H_Bonding.pml”. This will automatically open up
the PyMOL program and load up the atomic coordinates for the next DNA structure. OR
Download and open the file “2-DNA_Hbonding.pse” from the course website
The program will start with an image of a DNA double helix (including “Strand_A” and
“Strand_B”), just as you saw in the previous file. However, in this structure we’ve included the
hydrogens (white) into the structure for clarity. These hydrogens will allow us to see hydrogen
bonding patterns a bit more easily.

Take a moment to view DNA with the hydrogens represented.

Turn off “Strand_A” and “Strand_B”, and turn on “d_Adenosine” and “d_Thymidine” (OR
select Scene 02)
In this orientation in which both bases are in the same plane as the monitor, how is the relative
orientation of the two strands manifest? Look at where the 5’ carbon is point on for one
nucleotide (towards you or away from you) and where the 5’ carbon is point on the other
nucleotide. You can do the same for the 3’ hydroxyl for both nucleotides.
Note how these two nucleotides, deoxyadenosine and deoxythymidine, always pair opposite
each other (you can check this using “Strand_A” and “Strand_B” to view the entire DNA strand).
This is abbreviated as an AT base pair. Rotate the AT base pair on your screen some and note
how the edges of the base pair contain hydrogen bond donors and acceptors. Many proteins
interact with these exposed edges when binding to specific DNA sequences.

Turn on “AT_H_Bonds” (OR select Scene 03)
This should add the hydrogen bonds that occur between these two bases, which are
represented as dotted yellow lines.
Determine the distance between the deoxyribose attached to adenine and the deoxyribose
attached to thymine.
 First, click on “Wizard” from the top menu, which will generate a drop-down menu.

Next, click on “Measurement.” You will now be able to measure the distance between
the next two atoms which you click on.

Click on the 1’ carbon on the sugar bound to adenine, and then click on the 1’ carbon on
the sugar bound to thymine. You will see a yellow dashed line connecting the two atoms
you chose and a white number in the middle of the yellow dashed line indicating the
distance between the two atoms in angstroms.


Record the value and click “Done” when you are finished.
The measurement you took will appear on the right menu as “measure01.” Feel free to
deselect it to make it disappear.
Now let’s look at the base-pairing for the other set of nucleotides:

Turn off “d_Adenosine”, “d_Thymidine” and “AT_H_Bonds”, then turn on “d_Guanosine”,
“d_Cytidine” and “GC_H_Bonds” (OR select Scene 04)
Determine the distance between the 1’ carbons of a G-C base pair, using the instructions above.
► Q6)
A and T are referred to as complementary bases. G and C are thus complements as well. A
complementary strand is one that has perfect base complementation for each base of the
original strand. Remember that DNA binds antiparallel naturally, so if both strands are written
from a 5’ to 3’ direction, you must remember to account for the reverse in direction.
Section 3: DNA-Protein Interactions

Close the file you were working on and click on the Google Chrome or the Firefox icon in the
Dock at the bottom of the screen. Navigate to the Laboratory page in the LS1a course
website and download the file “3_CAP_DNA_RNAP.pse.” Next, open up the “Downloads”
folder, and open the “3- CAP_DNA_RNAP.pse” file. This will automatically open the PyMOL
program and load up the atomic coordinates for the next DNA structure.
We are about to examine a protein called CAP (the “Catabolite Activator Protein,” a protein
that, as Rich has noted, also goes by “CRP”) that can positively regulate the bacterial promoter
for the lac operon. The lac operon contains several genes that encode for proteins necessary for
the bacterium to metabolize lactose in the absence of glucose (its preferred food). The absence
of glucose leads to an increased concentration of cyclic AMP (adenosine 3', 5'-monophosphate;
“cAMP”), which binds to and activates the CAP. Once cAMP binds and activates CAP, CAP can
bind the DNA upstream of the lac promoter and recruit the RNA polymerase. In fact, cAMP
binding is required to enable CAP to bind DNA, and we will see why shortly.
CAP recruits RNA polymerase to the promoter by binding the carboxy-terminal domain (“CTD”)
of one of the polypeptides that makes up RNA polymerase (i.e., the “alpha domain”) to recruit
the RNA polymerase to the promoter. The crystal structure we are looking at is of the CAP
protein bound to DNA upstream of the lac promoter and the carboxy-terminal domain of the
RNA polymerase.
Use the small buttons, labeled 001 through 012, on the bottom left of the screen to toggle
between different scenes. If you cannot see the buttons, type the names of the scenes listed
below (e.g., “scene 001”) in either one of the two PyMOL prompts on your screen. (The “PyMOL
prompt” looks like “PyMOL>” with a blinking cursor afterwards. The two examples of the
prompt are shown below. You can simply type “scene 001,” or “scene 002,” at these prompts
and then hit enter.)
Example images of the PyMOL prompt:
OR
In scene 001, CAP is colored in orange, cAMP is colored in blue, the CTD of RNA polymerase is
red, and the carbons of the DNA are shown in green.
In scene 002, you can observe that CAP actually consists of two identical polypeptide units: one
is colored in orange and one is colored in cyan. Each CAP peptide binds one molecule of cAMP
(the cyan CAP binds the yellow cAMP, and the orange CAP binds the blue cAMP).
Scene 003 displays the electrostatic distribution on the outside surface of the CAP protein.
Negative charge is depicted as red and positive charge is depicted as blue.
► Q7)
Scene 004 returns us to our original cartoon view of CAP, but this time with the protein’s
recognition loop highlighted in magenta. Proteins often use “helix-turn-helix” motifs to bind
DNA, and the recognition loop of CAP is one example. (A “helix-turn-helix” describes a part of a
protein where it has an alpha-helix linked to another alpha-helix via a loop. One of these alphahelices typically interacts with the DNA as in this example of CAP. Another example was shown
in ICE 3.)
Zoom in on the interaction between the recognition helix and DNA by selecting scene 005. The
electrostatic interactions are highlighted in yellow.
► Q8)
Select scene 006. This shows the interaction between the recognition helix, in particular two
arginines and a glutamate, and the DNA. The electrostatic interactions are highlighted in yellow.
► Q9)
To observe how cAMP binds CAP, toggle to scenes 007 and 008. In scene 008, the electrostatic
interactions are highlighted in yellow.
► Q10)
► Q11)
To observe how CAP binds the alpha-CTD of RNA polymerase, toggle to scenes 009, 010, and
011. In scene 011, the electrostatic interactions are highlighted in yellow.
► Q12)
Scene 012 shows how the alpha-CTD of RNA polymerase interacts with DNA. One interaction in
particular is highlight in which the alpha-CTD makes interacts with the minor groove of the DNA.
It does so via a water-mediated hydrogen bond from the arginine of the protein into the minor
groove of the DNA.
Scene 013 highlights the residues of the alpha-CTD (in white) that interact with the sigma
subunit of the RNA polymerase, indicating where the rest of the polymerase would be if it were
present in this crystal structure.
Section 4: RNA Structure

Close the file you were working on and navigate back to the PyMOL Lab 4 file folder in the
Dock. Open up the folder titled “4_tRNA,” then double-click on the PyMOL file labeled
“tRNA.pml.” This will automatically open up the PyMOL program and load up the
coordinates for tRNA. (OR download the file “4- tRNA.pse” from the course website.)
Having just viewed DNA (deoxyribonucleic acid) in detail, we now turn our attention to RNA
(ribonucleic acid). The first item we are viewing is the basic unit of RNA, a nucleotide. Take time
to examine the nucleotide, locating the ribose sugar, the 5’ phosphate, and the nitrogen base.
► Q13)
Although the difference may seem trivial, remember that subtle changes in chemical structure
can result in dramatic changes in chemical properties. For instance, DNA is stable in water for
long periods of time whereas RNA degrades much more quickly. This is why DNA is used as the
central form of genetic information storage; it is more stable to degradation and serves as a
better “genetic blueprint”.
[If you have extra time, open “tRNA.pml” and “NucleicAcids.pml” (from the first section) in two
windows next to each other. Check out the ribose ring: How are the 2’ and 3’ carbons oriented
differently in the two molecules? Why might that be?]

Turn off “Nucleotide” then turn on “RNA” (OR select “scene 002”)
You will need to zoom out to see the entire structure. As you’ve seen in DNA, nucleotides in
RNA link together to form long, polymeric strands. However, you will immediately notice that
the RNA structure is somewhat less regular than that of DNA. The particular molecule we are
looking at is a “transfer RNA”, or tRNA for short. This macromolecule plays a very important role
in the translation of genetic material into protein strands.
To clarify the backbone structure, let’s add a cartoon.

Turn on “Cartoon” (leave “RNA” on) (OR select “scene 003”)
Notice that the general structure of the RNA is still helical, but that the helices orient themselves
in different ways to generate diverse secondary structural elements (“motifs”). This
characteristic of RNA (the ability to form more diverse structures than DNA) enables RNA to
perform a greater variety of functions within the cell.

Turn off “RNA” and “Cartoon”, then turn on “Backbone” (OR select “scene 005”)
Just as with DNA, RNA contains a highly negatively-charged backbone. As we’ve discussed with
DNA, this backbone attracts positively-charged species, some of which help to stabilize RNA’s
unusual secondary structure elements.

Turn on “Magnesium” (leave “Backbone” on) (OR select “scene 006”)
In this particular tRNA structure, we see several magnesium ions (shown as magenta spheres in
the program) bound to the sugar-phosphate backbone. Magnesium ions, as you’ll recall, have a
+2 charge. Notice how the Mg2+ ions are positioned next to the oxygen atoms of the phosphate
backbone. The drawing below illustrates one example of how scientists depict this interaction in
two dimensions.
phosphate
O
O
P
O O
Mg2+
O
O
P
O O
phosphate
► Q14)

To get a clearer view, turn off “Backbone” and turn on “RNA” to see the interaction in
“sticks” representation. (OR select “scene 007”)
At this point, let’s look at some unique structural characteristics of this RNA. First, recall that
the base “uracil” (U) is used in RNA instead of “thymine” (T). Just like T was the complement for
A in DNA, U is the complement for A in RNA.

Turn off “Magnesium” and “RNA”, then turn on “Uracil” (OR select “scene 008”)
► Q15)
Note: To help you see the difference, you can click on “Thymine” to see its structure (OR select
“scene 009”). You will need to zoom out a bit and bring thymine to the center to see it. It is
difficult to view both bases up close on the screen at the same time, but you can draw the
structure of one of them in order to remember its structure if you can’t see the difference.
END OF LAB
Sidebar: If for whatever reason your background display goes haywire and appears red:
Here’s what to do:
1. Click on the “Display” menu at the top:
2. Scroll down to “Background”
3. Select “Background” and scroll down to “Black.”
4. All better.