Chem 110L: Macromolecular Visualization Laboratory Exercise: PyMOL

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Chymotrypsin

Chymotrypsin is a well-studied proteolytic enzyme that functions in our digestive tract where it assists in the hydrolysis of proteins that we eat. It readily cleaves the peptide bonds adjacent to aromatic amino acids tyrosine, phenylalanine and tryptophan. Its mechanism of action is well-understood and is often taught in undergraduate biochemistry courses as an example of the enzyme catalysis. The active site of chymotrypsin has two interesting structural features:

• A hydrophobic pocket that can accommodate aromatic amino acids. This feature is responsible for the specificity of the enzyme toward peptide bonds that have an aromatic amino acid on the C-terminal side.
• A catalytic triad consisting of side chains of residues Ser195, His57, and Asp102. These three residues form a hydrogen-bonding network that allows deprotonation of Ser195 during the reaction. The nucleophilic alkoxide anion then attacks the carbonyl carbon in the peptide bond.

Introduction to PyMOL

According to the program's author, Dr. Warren DeLano, PyMOL is a molecular graphics system with an embedded Python interpreter designed for real-time visualization and rapid generation of high-quality molecular graphics images and animations. It can also perform many other valuable tasks (such as editing PDB files) to assist you in your research.

Two unique and valuable features of this program over some other visualization program are the use of the powerful programming language (Python) and an emphasis on high-quality graphics. At the present time the program is still undergoing active development, but it is mature enough to be used for learning and research. During the early development, emphasis was placed on functionality, and not on intuitive user interface. As a result, it takes a little time to learn the program. You can interact with the program via four complimentary ways

• Pull-down menus provide access to the file functions, the molecular editor, the program options, and a few more advanced tasks. For example, you can change the display quality settings, the background color, the size of your spheres, or the transparency of the surface from the pull-down menus.
• A task bar on the right side of the screen allows one to select which objects are displayed in which way. For example, you can show your protein as a gray surface and the inhibitor as green sticks if you have separated them into two different objects.
• The molecular display area allows one to view and interactively manipulate molecules. Your three-button mouse allows you to rotate, move, and zoom in/out by holding down the left, middle, or right button, respectively. Furthermore, you can select and perform specific tasks via mouse and keyboard shortcuts. For example, you can select two atoms (first by CTRL-SHIFT/left-clicking, second by CTRL-SHIFT/right-clicking ) and create a covalent by pressing CTRL-T.
• The command line allows you to type in commands that can accomplish almost any of PyMOL's functions. For example, you can create dashed lines (a good way to show hydrogen bonds) between oxygens and nearby amide hydrogens in the alpha-helix by using a command "dist (helix and name o), (helix and (name h and neighbor name n)), 2.5". This is the most powerful way to interact with the program, but requires that you are familiar with the logic and syntax of PyMOL commands. The commands are explained in the PyMOL manual.

This program is available for many computer platforms, but it runs slow on our SGI workstations. For effective use, you should download and use it from your personal computer.

Chymotrypsin Active Site with PyMOL

Your second homework assignment asked you to create an image that illustrates the following aspects of the chymotrypsin mechanism:

1. That a ligand (the substrate or inhibitor) is bound to the active site
2. That the active site has a pocket that accommodates an aromatic amino acid residue from the ligand
3. That the active site contains a catalytic triad, consisting of Ser, His, and Asp residues

A suitable structure of chymotrypsin with a bound inhibitor in the PDB file 1AFQ. Open this file in PyMOL. It looks pretty complex, so let's simplify the view by displaying parts that are important for your assignment. Visual inspection of the PDB file reveals that the chain D is the ligand. We want to show this ligand, and the catalytic residues in some detail while displaying the rest of the protein as a transparent surface. To accomplish this, you can type the following commands into the command line:

hide


select inhi, chain d
show sticks, inhi
show sphere, inhi
color forest, inhi
cmd.disable('inhi')


select active, (resi 57,195,102)
show sticks, active
color marine, active
cmd.disable('active')


create prtn, (chain a,b,c)
show surface, prtn

This gets you the basic elements in place, and now you just need to alter the settings for surface color, transparency, and sphere scale to have an esthetically pleasing image. You can change these settings from Edit All ... under the Settings menu. Next, rotate and zoom the structure into a position that best illustrates what you want to show. Finally, you may raytrace the image with the command ray to provide a more photorealistic look and save it as a PNG file on your hard disk. You can insert PNG image files into your Word documents by selecting Insert -> Picture -> From File ...

Optional Further Reading

The structure and function of chymotrypsin are further described in a Chime-based tutorial from the Imperial College in London. Please note that you need a Chime plug-in to see this structure. The plug-in is not installed on SGI computers.

A gallery of PyMOL images with associated PDB files and scripts (you can run these from File > Run) is available at www.chem.ucsb.edu/~molvisual.

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Tutorial by Dr. Kalju Kahn, Department of Chemistry and Biochemistry, UC Santa Barbara. ©2004