Pymol Figure generation Protocol

1. In order to work with the protein structure data and generate useful 3-D images we will need to
download and install some software.

• PyMOL. This software is a very powerful tool for manipulating, measuring and
representing protein structure data. You can get a link to it here (you need to register for
an educational version) . http://pymol.org/edu/?q=educational/

2. Next we need to acquire the Protein DataBase (PDB) files for the proteins we are interested in
studying. These files are the “best fit structural data” based on electron density maps, rather than the
electron density maps themselves.

• There are a number of different websites available to search and download the relevant
files but we will use the RCSB site http://www.rcsb.org/pdb/home/home.do

• We are interested in a mutant form of egg-white lysozyme with the aspartic acid residue
(amino acid) at position 48 changed to an alanine residue (this mutation is abbreviated
as D48A). We are planning to compare the structure of this mutant lysozyme in the
presence and absence of an artificial ligand, (GlcNAc)4, which is further abbreviated as
NAG. Both structures were generated by Y. Kawaguchi in 2014. There is a simple
search field on the RCSB as well as a drop down menu for advanced search options. You
should be able to find the two files we need by entering appropriate search terms and
parameters. Download the PDB (text) versions (you may need to scroll down a bit).

3. Opening the relevant PDB files in any text editor (Word, for example) reveals a lot of information
about the conditions used to generate the data as well as the structural data itself. The beginning section
of the file, from “Header” down to “Atom” contains the details of the experimental set up and data
collection. Things which you can find here which are relevant to us are:

• What species was the protein originally from?

• What organism was the protein used in the crystallography generated in?
• What conditions were used to generate the crystal(s) used in the study? (How does this
compare to the conditions you plan on using?)
4. The section beginning “Atom” contains the structural information. It is organized like this:

A B C D E F G H I J K
ATOM 1 N LYS A 1 2.294 9.220 -8.577 1.00 11.72 N
ATOM 2 CA LYS A 1 1.375 9.623 -9.628 1.00 12.55 C
ATOM 3 C LYS A 1 1.525 11.130 -9.906 1.00 10.33 C
ATOM 4 O LYS A 1 1.522 11.866 -9.007 1.00 11.00 O
ATOM 5 CB LYS A 1 -0.041 9.275 -9.177 1.00 13.90 C
ATOM 6 CG LYS A 1 -1.115 9.686 -10.089 1.00 14.54 C
ATOM 7 CD LYS A 1 -2.479 9.142 -9.683 1.00 14.46 C
ATOM 8 CE LYS A 1 -3.601 9.794 -10.549 1.00 17.95 C
etc...

These columns contain all the data PyMOL needs to construct 3-D models:

Column Data
A Atom number
B Atom designation (eg. N for nitrogen, CA for alpha carbon, CB for beta carbon etc.)
C Three letter code for the amino acid containing the atom in question
D Chain; some structures contain more than one molecule. These are labelled A, B etc.
E Number of the amino acid in the structure
F X co-ordinate of the atom in Angstroms
G Y co-ordinate of the atom in Angstroms
H Z co-ordinate of the atom in Angstroms
I Occupancy; some atoms are not in the same location in every asymetric unit within the
crystal. An occupancy value of 1.0 indicates the atom is found in the same place in all
asymetric units, an atom listed twice with two sets of co-ordinates and an occupancy
value of 0.5 each indicates the atom is evenly split between two positions etc.
J B-factor, a measure related (but not identical to) the radius within which you are likely to
find the atom in question. Smaller numbers indicates greater certainty as to the likely
position of the atom.
K Type of atom (element)

Other information you will find in the PDB text includes ligand information (Designated NAG in our
case!) and the location of the atoms of solvent molecules incorporated in the protein crystal (in our case
water, designated HOH). Note; most solvent molecules which are bound into the crystal won't be
visible as they are mobile or “unstructured”. Those that are shown in the PDB are “structured” and
have definite locations within the protein/solvent crystal.
6. Double clicking on the PDB file should launch PyMOL and immediately open the file. The interface
should be two windows, either separate (windows) or linked (OS X). The larger of the windows should
be displaying a wireframe rendering of the 3-D structure and the locations of the “structured” water
molecules. The second window gives information about currently selected atoms within the rendered
structure or about commands given to PyMOL. The main window should be partitioned into the display
window, a list of items currently available for display within the display window (upper right) and a list
of the mouse commands available for manipulating your structure (lower right).

Text
information
window
List of items
available in display
Display window
window

Mouse gesture
guide
Command line input

Before we begin manipulating your structure:

• Using a mouse is the easiest and fastest way to interact with your PyMOL image; touch screens
and trackpads are usable but difficult. In the menu bar of the text information window you will
find the “Mouse” drop down menu; use this to select the type of mouse you are using to display
the correct mouse commands available in the main window (eg. 2 button vs. 3 button etc.). You
will be able to rotate, centre, zoom in/out, translocate etc. your molecule using the various
mouse buttons.

• Another of the menu items here which you should use is File-Save session. It is good practice to
occasionally save your PyMOL session so that you will not have to repeat all your image
manipulations if you unexpectedly close the session.

7. In the list of items displayed by PyMOL you will immediately see an item “ALL” and an item
corresponding to the whole of the PDB you opened. Clicking on the name of either item will hide it (try
this out and see what happens). To the right of each name are a number of lettered buttons; A, S, H, L
and C. These correspond to Action, Show, Hide, Label and Colour. Each of these buttons opens to a
submenu of options which, when selected, are applied to the corresponding item in the list (eg.
applying Show-Cartoon to your PDB item will replace the wireframe with a different representation
(try it out!) or applying Colour-Yellow will change the colour of the entire structure). We will be
making use of Action, Show, Hide and Colour.

8. Let's begin some manipulation of our image by hiding the water molecules (they are represented by
the floating “X”-like points. Select the Hide button for the list item which correspond to your PDB (ei.
not “ALL”). Under this submenu you will find many options, by selecting “nonbonded” you will hide
any information about molecules not bonded to our molecule of interest, in this case, the unstructured
water molecules. But what about our ligand? There are bonds within the ligand and so it too is a
“bonded” structure.

9. Regarding the ligand; what if we wanted to select only that molecule for manipulation? At the
bottom of the display window you will find a small command prompt (PyMOL>); there are many
powerful commands that can be used in this space but we will predominantly make use of the “select”
command.

• Open the PDB corresponding to the lysozyme/ligand complex. In the command line type the
following (without quotes) “select ligand, resn NAG” . This command tells PyMOL to select all
residues named (resn) NAG (the three letter code given to our ligand) and name that new
selection “ligand”. (The name is arbitrary, we could call it whatever we want). What should
happen is the atoms corresponding to the NAG molecule appear highlighted in the display
window and a new item appears in the display list “(ligand)”. Items in the list in parentheses are
subselections of another item; they can be manipulated separately from the main object but
there are some limitations. Alternatively, selections can can the made into their own objects by
using the Action button and selecting “copy to object”. This generates a new object which can
be manipulated independent of the larger object it was derived from.

• Another command which may prove useful in “set transparency”. For your protein molecule,
use the submenus to Show-Surface. In the PyMOL command line type (without quotes) “set
transparency, 0.5” and observe the results.

• If we wanted to only select a specific amino acid based on its position within the chain, we
would use a similar combination of commands. “select aa, resi 48” for example, will select the
amino acid residue identified as number 48 and name that new selection “aa”

• A list of just some of the other available commands can be found in the PyMOL wiki here:
http://www.pymolwiki.org/index.php/Category:Commands

10. Some other things which may be useful in analysing this structure and producing good figures;

• You can change the background colour of the display by using the command “bg_color”
followed by the name of the colour your would like; white, black, blue etc. Or from the Display
drop down menu in the Text Information Box

• You can show only the side chain(s) of selected residue(s) by using the Hide-Main chain

• You can colour objects or selected parts of objects according the the atoms which make up the
structure by using Colour-by element and then choosing the scheme you prefer.

• You can identify hydrogen bonding to a particular object by selecting the object in question and
then Action-Find-Polar contacts and selecting the set of atoms you want to consider for the H-
binding calculations. (Note, PyMOL will show H-bonds with empty space; these are H-bonds
with structured water molecules which you may not be displaying)
 • Manually selecting an amino acid with the mouse in the display window will identify the amino
acid in question in the second, text based PyMOL window.

• Distances between atoms in the structure (in Angstroms) can be found using the Wizard-
measurement menu in the text PyMOL window and manually highlighting the atoms in
question.

11. Next, let's superimpose our two structures (mutant lysozyme with and without ligand) and see what
structural differences are induced by ligand binding. Open the second PDB in the same instance of
PyMOL using the File-Open menu. When both structures are displayed use the Action-Align-to
molecule command to superimpose the structures. You should notice that the structures are very similar
but a closer inspection will show some significant changes to the location of some side-chains,
particularly in proximity to the ligand itself.

12. Finally, lets produce a figure. Once you have manipulated, selected, coloured, set transparencies,
rotated and tweaked your displayed structure there are two ways to produce images. The first is simply
to go to File-save image as-PNG. Alternatively you can use the command line to apply the “ray”
command before using these menu commands. “Ray” generates a shadow and will generate an image
of whatever resolution you like. Simply type “ray 1000” to generate an image of 1000 pixels in the
width dimension. Then follow that up with File-save image as-PNG. Be careful not to set the pixel
count too large!

For inclusion in your report I would like you to produce 2 figures:

The first is a representation of the mutant lysozyme bound to the ligand. In the figure you should
clearly distinguish:

• The ligand
• The important catalytic side chains on lysozyme (PUBMED research can help identify these)
• The location of positively charged and negatively charged side chains in lysozyme (this can be
done with colour!).
• The side chains of lysozyme which make hydrogen bonds directly to the ligand
• The location of the tryptophan side chain which “stacks” (in parallel) with a glucose-like ring in
the ligand.

The second figure should show:

• A superposition of the ligand-bound and unbound lysozyme mutants

• The locations of two side chains in proximity to the ligand binding site which move
significantly on ligand binding. In the text of your report you should also provide a list of how
far each of these side chains moves.