PyMOL – Introduction
Proteins play essential roles in living organisms.  Their functions are not only restricted to catalytic (e.g., Enzymes) but also structural (e.g., keratin and collagen).  Essential to protein function is its structure, and thus, it is necessary to be able to visualize the structure of a protein.  Several attempts have been made to obtain the structure of several proteins, and over the past several years, several proteins have been characterized and deposited into the Protein Data Bank (PBD).
The files on the PBD are visualized using software tools that enable the data to be read as interactive pictures.  Visualization tools help in the viewing of the 3D structure of the protein, communicate and draw some conclusions on protein interactions both for binding (such as inhibitors.) and catalytic (e.g., substrates).  The visualization software includes SWISS PBD (DeepView), Pymol, and Chimera.  In this lab, we will be focusing on PyMol and learn some basics.
PyMOL – Activity 1 – Lysozyme
Lysozyme is a model enzyme in protein crystallography: is relatively robust, easy to purify, and crystallizes in many crystal forms in a wide range of conditions.
 
In 1965, lysozyme became the first enzyme, and only second protein, to have its structure solved. Prior to structure solution, lysozyme was known to have the ability to destroy bacterial cell walls- the body’s own natural antibiotic. However, the mechanism by which lysozyme actually carried out this reaction was less clear and was finally elucidated upon examination of the lysozyme crystal structure.
 
Today, crystallographers regularly use lysozyme for calibration of X-ray instruments, and professors use it in the lab to train new generations of scientists in structural biology and chemistry. Because of its superior crystallization propensity, lysozyme has even been fused to hard-to-crystallize proteins and enabled the solution of those structures.
 
PYMOL:

1. Start PyMOL. (Two windows will appear if running on a PC vs one on a Mac).
2. File >Open> pdbfrom the Desktop (or double click your file) or in the command line type “fetch 4LZM”
3. In the right panel, you should see tabs “all” and “4LZM”. The buttons next to these (A S H L C) allow you to manipulate your structure.
   1. A- Actions: can rename, delete selection etc
   2. S- Show: change visualization such as ribbon diagram vs. sticks etc.
   3. H- Hide
   4. L- Label- can add atom labels etc.
   5. C- Color – changes colors of the selected structure
4. Play with the S and C controls of “all” to familiarize yourself.
5. If you click on any residue(s) in the structure it will create a new selection that will appear in the panel as “sele”. You can rename this with the A button or click on the residues again to deselect them.

 
Overall structure:

1. First, some practice-changing views:
   1. In all: S>Ribbon. H>Lines
   2. Now show as Sticks. Hide Ribbon. Show as Cartoon. Hide Sticks.
   3. Hide waters. Rotate and zoom in.
   4. Change your background to white. Display> Background> White
2. Choose the view (cartoon, stick, ribbon, colors etc.) you prefer. Make sure you can see the entire structure.
3. Find the command line: PyMOL>.
4. Into the command line box type: set use_shaders. Click Enter/Return
5. Next, type: png image.png, 4000, 3000. Click Enter/Return.
6. DO NOT click on your structure. File >Save Image As…> PNG.

PyMOL – Activity 2 – Exploring the Protein Data Bank (PDB)
We will be exploring the Protein Data Bank (PDB) to understand, the type of information that can be found on the PDB.  Transmembrane alpha-helical proteins, like the dopamine receptor, are very challenging to crystallize. The first human transmembrane alpha-helical protein structure (beta-adrenergic receptor) was solved in 2007. Two reasons they remained elusive for so long is that they are very flexible and as they are inserted into the cell membrane possess large hydrophobic regions, so attempts to crystallize them failed. The structure of the dopamine receptor, as well as the beta-adrenergic receptor and several other membrane proteins, was solved by taking advantage of lysozyme’s propensity to crystallize. Using protein engineering, scientists fused lysozyme to the receptors, purified the fusion protein, and crystallized the fusion protein.
 
You should use the PDB site to answer these questions.

1. Navigate to https://www.rcsb.org/. Find the structure with PDB ID: 3PBL.
2. Explore all the tabs (Summary, Derived Data, Sequence, etc.) to answer the following questions about 3PBL, the dopamine receptor:
   1. What is the organism is the dopamine receptor from (scientific name)?
   2. How many ligands are bound to the receptor? And what are their names?
   3. What is the resolution of the crystal structure?
   4. What method was used for crystallization?
   5. What temperature was it crystallized at?
   6. Was the data collected at a synchrotron?
   7. What software was used to complete refinement?
   8. Is the structure alpha-helical? Made of beta-sheets? or both? (give an estimate)
   9. How many amino acids does the dopamine receptor structure contain?

PyMOL – Activity 3 – Dopamine Receptor

Go to your PyMOL console

1. In the PyMOL command line type: fetch 3pbl. This downloads the structure directly from the PDB into PyMOL. The dopamine structure should appear.
2. All> Color > by chain. You will see there are two different colored chains. These are two (identical) copies of the protein present. We will select just one to work with.
3. In the command line, type select Dopamine1, chain Atab called “Dopamine1” will show up in the right-hand column of the viewer window.
4. All > Hide > Everything
5. Dopamine1 >Show > cartoon. Now the cartoon ribbon diagram of just chain A should appear on your screen. To make manipulating this chain easier, in the “Chain A” menu click on A (for actions), then centerand zoom in.
6. Look at the structure. Can you identify the two individual proteins dopamine receptorand lysozyme? Scrutinize the aa sequence by using the “S” button in the lower right-hand corner again to show the aa sequence or click Display>Sequence.  Is there something distinct about the numbering?
7. Once you have identified the residue range for the lysozyme,
   1. Into the command line type select lysozyme, resi 1-10 (note replace 1-10 with the correct residue range for lysozyme).This will select all the residues in the lysozyme only & a tab “lysozyme” will appear.
   2. Make lysozyme a different color from the dopamine receptor
   3. Show lysozyme as spheres (or other different representation than cartoon).
8. Create an image with a white background. Print for your lab notebook and be sure to label each part of the fusion protein in your lab notebook.

9.             PyMOL – Activity 4 – Enzyme – Inhibitor Interactions

10. Visualizing the Enzyme-Inhibitor Interactions 
11. In this next activity, you will have a chance to observe the inhibitor-enzyme interactions that account for the inhibitory effects of the drug Isoniazid.  When introduced into an organism,  isoniazid derivates are metabolized into an isoniazid-NADH complex (shown in Figure 1).  This is the active form of the drug that will bind to and inhibit the enzymatic reactions of InhA, ultimately leading to cellular death.  PyMOL is an interactive computer program that will allow you to visualize the inhibitor-enzyme complex and the intermolecular interactions from a three-dimensional vantage point.
12. Identifying distances between atoms: One helpful piece of information is to know the distance between various atoms. This can help identify bonding or intermolecular interactions. Recall that typical covalent bonds are about 1.5 Å while other interactions such as hydrogen bonds are in the 2 and 3 Å range. To determine the distance between two atoms in a structure,Under A, find polar contacts and label the polar contacts. (this has been done for you)
14. Activity:
15. The structure of the enzyme-inhibitor complex has been provided for you ( This structure has been truncated to show only important interactions).  Analyze the co-crystal of Isoniazid and the InhA complexed together.Identify all of the hydrogen bonding interactions (which residue is interacting with which atom on the substrate) and identify any hydrophobic pockets (both residues forming the pocket and which portion of the molecule is in that pocket.  The structure can be foundhere(I will put the file in the attachment, the file will only open in pyMol), you can open it in PyMOL by downloading it unto your computer).
17. (Hint: Hydrogen bonding is indicated by dotted yellow lines, but also look for pi stacking and hydrophobic pockets)  In the figure the atoms are color-coded Carbon = orange; Nitrogen = blue; Oxygen = red; Phosphorous = green.

PYMOL – Activity 5 – Exploring Other Proteins

For each of the following proteins, perform the following actions to learn more about each protein using PYMOL. and the PDB
1A3N
1LQP
4LDB
4P1X
1HSG

1. Choose the structure from the database (fetch the structure into PYMOL)
2. What protein do you have (Research on its function/use)?
3. How many chains does the protein have, does it have a quaternary structure? 
4. What is apparent about the secondary structural elements of the protein (post pictures of these)? 
5. Are any coenzymes/ligands/substrates present in the structure? If so what are the names?
6. On the PDB, find the resolution. What information does resolution give about the structure? 

 13| PyMol – Lab Report

Imagine finding the best recipe for your favorite dish and not being able to repeat it.  Hours of time wasted because of a lack of documentation. We do not want that to happen so for each lab there will be the submission of a lab report as the proof of our time in the lab. Each lab report should have the following:

• The sections listed in the table below.
• The report should either be typed out or written in Notability.
• The report should be submitted as a pdf before the next lab