CHEM 422
Fall 2025
Name:
CURE Module 2 – PyMOL
Enzyme: Glutamine-dependent NAD(+) synthetase
PyMOL is a useful software to visualize biomolecules. It can also be challenging to learn. Don’t get discouraged – keep
practicing. Always save your session; PyMOL is infamous for users accidentally quitting and losing all of their
progress (File Save Session As…). PyMOL session files are saved [Link].
** You can find a much more extensive tutorial and help at the PyMOL WIKI page:
[Link] <--- VERY helpful!
1. Go to [Link] and download the educational version of PyMOL. Once downloaded, open the program.
2. The command line can be used for multiple purposes. One way is to quickly download structures from the Protein Data
Bank (PDB). This can be done by typing “fetch 6OFB” and hitting enter. This will rapidly allow you to visualize the
protein structure with the code 6OFB. Do this for the protein structure you visualized last week using the 4-letter code
given to you.
3. The bar on the right is used to visualize your structure:
A – action
S – show
H – hide
L – label
C - color
4. The red dots you see scattered around your structure are water molecules. Why is this, do you think?
5. Let’s clean it up. Go to action (“A”) and remove water molecules. check
6. On the bottom right, select “S” (or “SEQ”). What changed? *Note: To clear the selection, just click into black space on
the screen. check
7. Color your protein by chain and elemental colors (oxygen atoms = red, nitrogen atoms = blue). Describe what each
color represents in the cartoon representation.
Oxygen = red ball; Carbon = green, Blue = Phosphate
2 protein colors: 1 is green and another is blue 2 different protein structures
8. Let’s look at the enzyme’s active site. First, identify which amino acids are catalytic. This will require you to do some
literature search. You should start with the scientific article that is associated with this structure (find it on the PDB).
Catalytic lysine, catalytic cysteine, catalytic triad (Cys175 HS–Glu45HS–Lys114HS)
9. Before altering anything, can you see these amino acids in the current representation? Be descriptive.
CHEM 422
Fall 2025
Cannot see anything because there are too many interferences for the triad to be visible need to set
transparency to 0.8, hide all lines, select the 1-letter code for the correct triad in the sequence for the triad to be visible
10. Now that you have an idea of some catalytic residues, let’s visualize them. Find these amino acids in the sequence and
click on them. As you click on them, this will make a new selection called “sele” on the right side. You can modify the
colors of these “sele” and it will only alter these amino acids. check
11. Once you have identified these amino acids, change the representation into sticks (sele S sticks). List the amino
acids below (in format Gly1, Ala2, etc.) and include a snapshot of the active site.
=> To take a screenshot, go to the top right and select “Draw/Ray” and click on “Ray (slow)”.
Cys175–Glu45–Lys114
12. Let’s look at one chain of your protein. Hide everything (H everything) and select only one chain. You can do this
by highlighting ALL of the amino acids in the sequence. Sele S cartoon
Show this chain as cartoon. Hide everything again and show the same chain as a surface (select amino acids S
surface). What information can you get from each of these representations? Provie pictures of each of these
representations.
CHEM 422
Fall 2025
Label: amino acid in sequence: showing the secondary + tertiary structure with alpha helix and beta sheet
Label: Surface – showing electron density and Van der Waals interaction between molecules
13. Look for the domain identified in Module 1 for your enzyme. What is the name of the conserved domain? Try to find
this domain and take a picture of it (in cartoon representation).
Rossman – fold:
Near NAD site, parallel beta sheet
CHEM 422
Fall 2025
14. PyMOL can also be used to visualize surface electrostatics. This lets us see positive and negative charges on the
surface of the protein. Before starting, how could we use this information to describe structure and function of your
protein?
Identifying active sites with positive/negative charge
Substrate specificity and binding site
Protein interaction with each other
15. Select Plugin APBS Electrostatic. Run the program on your protein (will take a minute). Visually inspect the output
and describe what you see. Include two snapshots (different sides/orientations of your protein). What do the red/blue
regions represent?
CHEM 422
Fall 2025
Similar to ‘Surface’ setting, but color is more concentrated in certain parts of the protein.
There is opening to the NAD molecule
Red = negative charge potential (high acidic residue like glutamate)
Blue = positive charge potential (high in basic residue like lysine)
16. Can you see where the binding pocket is for your enzyme? This may not be possible for all enzymes – why not?
Yes, it is at the hollow area at the NAD site, near the Rossman
Not all is visible because not all enzymes are dealing with NADH, so they do not the NADH binding site could
cause the enzyme to undergo conformation change close the site
17. You can make the surface transparent by typing into the command line “surface_transparency, 0.5”. Try showing the
cartoon representation of your molecule – this way, you can see both the cartoon and a “molecular blob” of your enzyme
simultaneously. You can change the transparency level by making it more (ie. 0.8) or less (ie. 0.2) transparent.
CHEM 422
Fall 2025
Cartoon Surface
20. Provide a summary of what you learned about your protein today using PyMOL. What questions can you think of next
for your enzyme that you would like to study further using PyMOL?
Catalytic triad is far away from the NAD binding site,
Rossman is very close to NAD,
The enzyme consists of 2 protein subunits mirroring each other,
Charged regions and their concentration
Binding site is visible
Questions: How would the site open/close resulting in conformation change? Substrate specificity of 6OFB vs
other LDH enzymes?