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CH/BI 421/621/527 F16
Molecular Visualization Worksheet for LDH
MOLECULAR VISUALIZATION LAB (replacing Ch 11)
OF LDH USING PYMOL
SETUP (needs to be completed BEFORE LAB TIME)
Before coming to your pre-lab section during the week of Oct 24-Oct 28th, make sure that you
have a working PyMOL program on your laptop computer. If you don’t have a laptop you can
bring to class, email me for special arrangement. Also, please make sure to bring a threebutton mouse.
To download PyMOL:
DOWNLOAD URL: http://pymol.org/ep
USERNAME: dec2015
PASSWORD: benzene6
To check that the program is working:
Open up the program and make sure that you get two windows (one black display and one
white text window ) On the text window type:
fetch 4OKN (the second character is a letter not a number)
If you see a molecule appearing on the display window, your PYMOL is working.
For Mac Users:
To use the pdb-loader service plug-in, they simply need to rename the executable, and they
need to have X-Quartz installed.
http://xquartz.macosforge.org/landing/
Using MacPyMOL
The standard OS X Pymol application, MacPyMOL.app does not run with the Tcl/Tk interface
which is required for plugins to work. However, a quick renaming of the program from
MacPyMOL.app to PyMOLX11Hybrid.app makes the application run as an X11 application, and
plugins are now available.
To rename the executable, right click (or control click) on MacPyMOL and choose "Get Info" in
the Panel. Change the Name & Extension to PyMOLX11Hybrid.app. This name can also be
changed using the mv command in Terminal.app.
Please contact James Mcneely immediately [email protected], if you are having any installation
issues with the software.
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Molecular Visualization Worksheet for LDH
WEEK6: Examination of LDH Structure using PYMOL
INTRODUCTION: (Adapted from CH11 from the Lab Manual)
Molecular modeling and database searching have become essential parts of modern
molecular sciences. It is important for anyone working in biochemistry and molecular biology
today to be familiar with the many databases with molecular information. Access to the
tremendous amounts of data compiled is essential before, during, and after any scientific
project. After all, you should know what has already been done before attempting any new
project or experiment. As a biochemistry student, you should become familiar with retrieving and
manipulating molecular data using the computer. In a recent article in USA Today entitled,
"Databases May Put Drugs on Shelves Years Faster" they cited the increasing efforts by
thousands of biotechnology firms to use available DNA sequence and protein structure data to
supplement, or even replace, the high cost trial-and-error work of drug design. Scientists are
manipulating almost every gene in the human body and other organisms, and are developing
huge databases to house the knowledge gleaned from their studies. This information is helping
design new therapeutics at an unforeseen rate. In particular, protein structures, especially likely
targets for therapeutics, as well as molecular modeling have led to a field called "rational drug
design," which is revolutionizing the pharmaceutical industry. This revolution’s consequences
are already being felt throughout medicine and society.
Forty-five years ago at the height of the cold war, the United States Defense Department
sponsored a small computer communication project designed for wartime emergencies. Several
universities such as the University of California, MIT, and University of Michigan further
developed this project. Later, scientists at Stanford University and the European Organization
Nuclear Research (CERN) standardized these network projects for companies like HewlettPackard and Digital. The Internet went worldwide in 1989 and is now a thriving worldwide
commercial platform in the public domain.
The Human Genome Project was independently begun in 1984 with the goal of obtaining
the complete DNA sequence of humans, as well as other organisms. Furthermore, the advent of
more rapid methods to determine the three-dimensional structures of proteins has lead to an
explosion in the database that collects all these structures. This database, The Protein
Database (PDB), was first housed at the Brookhaven National Laboratory in New York, but is
currently curated by Rutgers University in New Jersey. With the growth and popularity of the
Internet, access to all the information in these databases is now readily available.
In this lab, you will study the three-dimensional structure of Lactate dehydrogenase
(LDH) focusing on both its surface characteristics, as well as its binding to its ligands NADH and
oxalate within the active site of the enzyme. In this limited study, you will specifically be looking
at the following characteristics:
 Secondary Structures (alpha helices, beta strands, loops)
 Distribution of the different types of amino acids within the protein structure (Which
amino acids populate the parts of the protein that are exposed to the solution? Which
amino acids are found in the interior of the protein?)
 The environment of a few specific types of amino acids important for ligand binding.
 Specific types of critical interactions (such as H-bonding) underlying molecular
recognition
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Molecular Visualization Worksheet for LDH
PROCEDURE:
PYMOL program can be run viacommand lines as well as clickable buttons. In this tutorial you
will have instructions to do it either way. Command line instructions will be in “black” and
clickable button instructions in blue. You can find a much more extensive tutorial and help at
the PYMOL WIKI page: http://pymolwiki.org/index.php/Main_Page
Start by opening PyMOL and loading your protein of interest (human LDH chain A) by typing in
the PyMOL Molecular Graphics System window command line in lowercase letters the pdb ID
number. The PDB ID we will use is 4OKN.
fetch 4OKN
Alternatively, you can also load your structure through the PDB Loader service found under the
“Plug-in” Tab on the right hand top of your Command/text window next to the tab Wizard –
Please note if you have a Mac and you don’t see this tab please go back to the setup
instructions and install the plug-in). Click: Plugin PDB Loader Service and type 4OKN in the
“code” box while leaving “chain box” empty.
This will bring up the crystal structure with the polypeptide chain represented in lines. Carbon
will be colored green, Nitrogen will be blue, and Oxygen will be red. Hydrogen atoms are not
shown. The red X marks around your protein are water molecules whose electron density was
seen in the crystal structure. Also there are two ligands in this structure. NADH (recorded as
residue 401 and oxalate (recorded as 402)
 Clicking with the left mouse button will select an amino acid residue, clicking anywhere
in the black background around the molecule will de-select it.
 Holding down the left mouse button and moving the mouse will rotate the structure.
 Holding down the right mouse button and moving the mouse will zoom in and out on the
structure.
 Holding down the middle button and moving the mouse will shift the whole structure to a
new center point (translate).
 At any point you can “save” the current state of your work as a session by go to:
File Save session as name. This will create a PYMOL specific file with an extension
.pse which you can always reopen (Fileopen) within PYMOL, or by double clicking it
within a folder. Please save your work often as going back (UNDO) doesn’t work very
well in PYMOL.
 To save a figure you have created in a format that you can use to insert in the word
document, go to: File Save image as png and save on your computer. Then insert
this picture in the requested space for any Question on the worksheet following this
tutorial.
1. With the line representation it is difficult to see structural features of the protein. It helps to
switch to another representation. On the right-hand panel of your display window, you should
now be seeing an object name “4OKN”. You can use the menu items ASHLC next to the any
object in the right-hand part of the viewer window to generate various actions (A), show various
representations (S), hide various representations (H), manipulate labels (L) or change the colors
(C).
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Molecular Visualization Worksheet for LDH
Hide the complicated default line representation by clicking on H (hide) next to your
object name and choosing "hide everything". Make a cartoon representation of your protein by
choosing S (show) and submenu "cartoon". You should now see the molecule represented as a
cartoon of secondary structure elements (helices, strands, and coil). You can change the look of
the cartoon by choosing Setting (top menu in the small text window) and then Cartoon. For
example, you can select the Highlight Color. This makes the edges of the strands and the inside
surface of the helices a second color (grey by default).
Click on C (color) and then select “by chain” and then again “by chain”. Rotate your
molecule around to get a better feel for what you are viewing.
Finally if you prefer a different background color for your display screen click on the
“Display” tab at the top, select “Background” and your desired color.
Answer Question 1 on your PYMOL worksheet (following this tutorial).
After completing Q1, type:
split_chain
delete 4OKN_B
delete 4OKN_C
delete 4OKN_D
delete 4OKN_E
delete 4OKN_F
delete 4OKN_G
delete 4OKN_H
delete 4OKN
OR click (A) and “delete the object”. Then reload the pdb file using the plug-in PDB
Loader service and typing in 4OKN for “code and A for chain.
Now you should only have the structure for the first polypeptide chain of human LDH to view.
2. Often when working in PyMOL, you’ll want to make some comparisons between different
structures. To keep everything easy to understand, it’s useful to re-name your structure. Type
in the PyMOL window:
copy hLDH, 4OKN_A (or 4OKN if you loaded through PDB loader )
delete 4OKN_A (or 4OKN if you loaded through PDB loader )
OR click (A)  rename object
Now your protein structure will be displayed as the object hLDH (for human LDH) in the
list to the right of the PyMOL Viewer window. PyMOL is picky about file names—keep things
simple and short with no spaces or characters other than letters and numbers.
3. To display the primary structure (sequence) of your loaded structure above the structure
type:
set seq_view, 1
(to turn it off set seq_view, 0)
Or click (S) found at the bottom of the right hand panel to the right of the arrow buttons (not the
S next to the object name)
You can also display the sequence by clicking on Display then Sequence in the top PyMOL
window.
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Molecular Visualization Worksheet for LDH
By pulling the slider under the sequence, you can see the sequence for the entire
structure including water molecules (all the 0’s at the end), and ligands (if present they are listed
right before or after the protein chain before the waters).
The sequence viewer is a very handy tool. When the sequence viewer is turned on, you can
select individual residues or multiple residues by selecting residues, using the mouse, on the
sequence viewer.
4. Hide the complicated default line representation by clicking on H (hide) next to your object
name and choosing "hide everything". Make a cartoon representation of your protein by
choosing S (show) and submenu "cartoon". Try various representations of the molecule by
clicking on S (show) choosing a representation (lines, sticks, ribbons, surface etc). Note that
one representation does not replace another but is added on top of another. Representations
are only removed by using the corresponding entry in the H (hide) menu. For the surface
representation it can take a few seconds to display, depending on how fast your machine is. To
color the protein by secondary structure, click the C (color) menu and choose by ss (secondary
structure) and any of the coloring options.
The different types of secondary structure are stabilized by different hydrogen-bonding networks
between the peptide bond amine and carboxyl groups in the backbone of the protein as well as
interactions between amino acid side chains. Alpha helices have the following hydrogenbonding pattern:
Image downloaded from:
TutorVista.com
Beta sheets have a different hydrogen bonding pattern:
Image from: http://www.vanderbilt.edu/chemistry/Rizzo/Chem220b/Q_A.html
The arrow on the beta strand cartoon shows the orientation of the polypeptide chain from N to C
terminus. Traditionally helices are labeled by letters (A through Z) and beta strands by numbers
(1 through 10 or more)
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Molecular Visualization Worksheet for LDH
To show the identity of an atom, double click with the left button on any point in the molecule.
This brings up a menu that allows you to do various actions to the atom, residue, chain or
molecule. Anytime you click on a residue to select it, the text window will tell you what you have
selected, e.g. “You clicked /4OKNA//A/HIS`181/CB” means that you have clicked on the beta
carbon of the Histidine residue at position 181 on polypeptide chain A of LDH.
When you have familiarized yourself with your protein's basic structure, it is time to start
describing it.
Go through steps 4-6 and then answer Question 2 and Question 3 at the end of this worksheet.
4. To make descriptions of a protein clearer, people often number the β-strands and letter the
-helices in order, beginning at the N-terminus. Changing the coloring to "by secondary
structure" “ss” from the Color menu may help you to identify the number and orientation of the
secondary structural elements. Additionally turning on the Sequence viewer will help you to
identify the start and end of each of the secondary structures in your structure.
5. If your protein contains a cofactor or bound ligand in the crystal structure, you can display it
by choosing “organic” from the show menu (S) and then selecting the stick or sphere
representation for the ligand or cofactor. This LDH structure contains two LDH ligands: NADH
and oxaloacetate. (In the sequence they are given residue numbers 401 and 402 respectively)
To analyze or manipulate selected regions of the displayed molecule you need to first select
your region of interest and give it a name (if you don’t give a name via the command line, the
selections will appear with the name sele – shown in parantesis- on the right hand menu).
You can select any residue either by clicking the mouse on the desired residue in the shown 3D
structure or on the displayed sequence. You can also type in the command line:
select name you want to give, description
To select all the atoms making the backbone of LDH and name it bb
type: select bb, (name ca,n,c,o)
OR click (S)  main chain
6. Disulfide bonds often hold parts of the protein together. To identify all of the disulfide bonds in
your protein, select all cysteine residues by typing in the top PyMOL window:
select cys, resn cys
This will create a new object that you can use to change the representation of just the cysteine
residues (lines or sticks will help you identify the disulfide bonds).
show sticks, cys
OR find the cysteines in the sequence display and select them by clicking on them (you can do
multiple selection by holding down the control key). Then click (A) copy to object and then (A)
rename obj01  cys then (S) sticks
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Molecular Visualization Worksheet for LDH
7. Now it’s time to get a feel for where different types of residues are in your protein. Describe
the location of the following amino acids and use the included questions to guide your analysis.
You are NOT asked to provide answers to these questions in your worksheet, but you are
expected to think about them to decide how to make the requested figures most effective in
terms of communicating the role of the different types of amino acids in LDH.
A. Hydrophobic (note: to select multiple amino acid types, include a + sign in between the
residue codes; e.g. “select hydrophobic, resn ala+val+ile+leu+met+phe+tyr+trp”)
OR you can individually select the desired amino acids by clicking on the sequence and
then copying the selection to an object ( click (A) copy to an object –creates obj01) and
renaming obj01 (click (A) rename object  hydrophobic)
1. Are these found predominantly on the surface or in the interior?
2. How extensive of a hydrophobic core (dense packing of hydrophobic residues)
does your protein have? (look at the sphere representation of the hydrophobic
residues). Take a look at how the core lines up with the primary structure. Is the core
composed of stretches of amino acids that are adjacent in primary structure or mostly
of hydrophobic residues that are separated in primary structure? Are there multiple
“cores” in your protein? What type of secondary structure is the core predominantly
made up of?
3. What types of secondary structure are hydrophobic residues found in? If they are
in helices, how are they distributed in the helix (e.g. all over or more on one side than
the other)? Are there hydrophobic “faces” on one side of any helix or sheet?
4. Are there any hydrophobic residues around the nucleotide binding site? Are they
uniformly distributed along the NADH molecule or concentrated on one side? Why? If
these amino acids found in clusters or patches on the surface of your protein? Is there
any stretch of hydrophobic amino acids contiguous in the primary structure that is
found on the surface of the protein?
For Question 5 in your worksheet, make a PyMOL figure to highlight the hydrophobic
characteristics of your protein. Play around with how you represent and color the backbone and
side chains—decide what is important to display and what should be left out so that it doesn’t
distract from what you want to point out. Paste this into your PyMOL worksheet.
B. Positively and negatively charged (at neutral pH). Arginine and Lysine residues are
positively charged and Glutamate and Aspartate residues are negatively charged. The
coloring convention is for positive to be blue and negative to be red. note: to select multiple
amino acid types, include a + sign in between the residue codes; e.g. “select positive, resn
lys+arg+his”)
OR you can individually select the desired amino acids by clicking on the sequence and
then copying the selection to an object ( click (A) copy to an object –creates obj01) and
renaming obj01 (click (A) rename object  positive)
1. Are these found predominantly on the surface or in the interior?
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Molecular Visualization Worksheet for LDH
2. If they are found in the interior of the protein, what else is around that region that might
explain their function (e.g. another side chain or cofactor close by that could take part in an
electrostatic interaction)?
3. What types of secondary structure are they found in? If they are in helices, how are they
distributed in the helix? Are there positive or negative “faces” on one side of any helix or
sheet?
4. Are these amino acids found in clusters or patches on the surface of your protein? Is the
surface more positive or negative? Are the types of charges mixed on the surface or does
the protein have distinct positive regions and negative regions composed of multiple amino
acids of the same charge grouped together?
5. Are there pairs of negative and positive charged side chains pointed toward each other
that can form salt bridges to stabilize the structure? (not adjacent in primary structure)
For Q6, make a PyMOL figure (Figure 3) to show the charge characteristics of your protein.
Paste this into your PyMOL worksheet.
After you focus in on a characteristic that you think is particularly important for the structure or
function of your protein, it may also be useful to measure distances (in Ångstroms) between
atoms in the protein. To do this type
distance atom#1, atom#2
Ex: distance 193/CA, 99/CA (distance between residue 193 alpha carbon and residue 99 alpha
carbon)
OR select “Measurement” from the Wizard drop down menu on the top PyMOL screen and then
identify the first and second atom for the distance measurement by clicking on the atoms in the
structure.
C. Now let’s turn our attention to LDH active site and more specifically to the pocket
where LDH binds NADH and examine the local environment of NADH and two residues
R99 and H193 that have been shown to be critical for NADH binding.
To see what is within 5 Ångstroms of NADH in this structure type:
select close_NADH, resi 401 around 5
This creates a new object called “close_NADH” that includes all of the atoms near the
residue 401 – which in this structure corresponds to NADH). You can then play around
with the representation of that selection (lines, sticks, color schemes, etc) to examine
what’s around NADH. You can choose any distance you’d like for the area you
examine.
At this point, you might want to hide other objects such as the original complete
structure,to be just viewing the binding site. Remember when you hide objects in
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Molecular Visualization Worksheet for LDH
PYMOL they are not deleted, just not shown. So you can always display them back
using the S(show) options.
Locate R99 and H193 in your structure (either on the 3D structure or on the sequence).
Select them individually and create an object out of your selection.
Create R99, sele
Then you can choose to show it any representation
Show spheres, residue 99
Select NADH (res 401) and create the object NADH from that selection. Try a representation
where you use spheres for NADH and sticks for the two residues. Experiment with different
colors and representations.
E. Both of these residues are close enough to NADH to make H-bonds with the polar groups on
NADH. To find out what hydrogen bonds exist between the H193 and NADH (or any other
nearby residues):
First you need to put protons on all the residues (protein and ligands). Since this is a
crystal structure, the pdb file does not contain atomic coordinates for any of the residues.
h_add hLDH
Define hydrogen bond donors and acceptors:
select don, (elem n,o and (neighbor hydro))
select acc, (elem o or (elem n and not (neighbor hydro)))
Show distances between donors and acceptors within 3.2 angstroms of your HIS193 residue:
dist HBA, (HIS193 and acc), (NADH and don), 3.2
dist HBD, (HIS193 and don), (NADH and acc), 3.2
delete don
delete acc
hide (hydro)
Repeat with R99
dist HBA, (Arg99 and acc), (NADH and don), 3.2
dist HBD, (Arg99 and dom), (NADH and acc), 3.2
Write down the hydrogen bonding between H193 rings and functional groups on
NADH as well as R99 and NADH.
For Q7 make a PyMOL figure focusing on the NADH and the residues from LDH that
surround it including the two important active site residues, R 99 and H193 that make
specific interactions with NADH.
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Molecular Visualization Worksheet for LDH
Name:_____________________
Lab Section:________________
PYMOL WORKSHEET QUESTIONS
Q1.
a. How many individual polypeptide chains do you see in the displayed crystal structure?
b. Does LDH have a quaternary structure? If yes what do you think its quaternary
structure is? Does your answer take into account all the chains you are seeing? If not, what
might be the source of the discrepancy?
Q2. a. Make a PyMOL figure of the LDH highlighting its secondary structural elements (cartoon
representation) as well as its two bound ligands (stick representation) (Figure 1).
b. Identify the secondary structural elements within the first 52 residues in the LDH crystal
structure and fill the following table. To determine the residue range, click on the first and last
residue of each element.
Residue Range
Sequence
Secondary Structure
c. How does this table compare to the prediction table you constructed in your bioinformatics
sheet based on the Jpred results?
Q3. Which two beta strands make hydrogen bond with beta strand 2 and how many other strands
are in the beta sheet they belong to? Is this a parallel, antiparallel, or mixed beta sheet?
Q4. Structural information about your protein:
a. Does LDH monomer (LDH_A) has any cysteines? Do they form disulfide bonds in
this structure? (If yes, list residue numbers for each pair of bonded cystines)
b. Does the structure depict any co-crystalized ligand? If yes, what is/are the ligand(s)?
Q5. Make Figure 2 with legend characterizing the distribution of hydrophobic residues in your
protein. Use your answers to the questions included in the tutorial to determine the important
characteristics of the hydrophobic residues in LDH to highlight in your figure and include in your
figure annotation and legend.
Q6. Make Figure 3 with legend characterizing the charge distribution in your protein. Use your
answers to the questions included in the tutorial to determine the important characteristics of the
charged residues in LDH to highlight in your figure and include in your figure legend.
Q7. Make Figure 4 with legend focusing on the NADH and the residues from LDH that surround
it including the two important active site residues, R 99 and H193 that make specific interactions
with NADH. Make sure that your figure shows the hydrogen bonding (with distance labels)
between R99 and NADH as well as H193 and NADH. Then identify three other distances less
than 5 angstroms between a residue on LDH and an atom on NADH and include those in your
figure.
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Molecular Visualization Worksheet for LDH
OPTIONAL:
Q8. Based on your work on visualizing the crystal structure of human LDH active site bound to
NADH and oxaloacetate (PDB ID#: 4OKN) you hypothesize that Arg99 should play a key role in
ensuring high enzyme activity. To test this hypothesis, you set out to engineer a mutant form of
LDH, by making a single amino acid substitution replacing Arg99 with Glu. You express this
mutant protein in bacteria and then set out to purify it.
Can you use the same purification scheme you performed in the lab as described in Ch3 in its
entirety to purify your mutant protein, or would you need to make any modifications to any of the
purification steps? Briefly justify your answer. Use PYMOL to look more closely around Arg99
and consider what impact (if any) would changing that residue to Glu99 would be expected to
have at a molecular level.