LMB Bioinformatics workshop
Installing MacPyMOL
). Move this icon to your Applications
folder and put a link to it in your Dock.
To simplify use of this tutorial,
it is assumed that you are using Firefox as a web browser and that your download preferences for
Firefox are set to download files to the Desktop.
Using MacPyMol interactively
In this tutorial, we will make a single
figure interactively. I have also
included a sample script that uses many common commands for generating PyMOL
pictures. The script is annotated
with comments denoted by the # character.
Getting coordinate files
The most common way that
structures are loaded into PyMOL is to read the Protein Data Bank (PDB)
file.
Fetch a PDB file corresponding
to the PH domain from beta-spectrin (PDB id 1e8x) by following our tutorial for
the PDB
database.
A PDB file for the
GLUE domain of Vps36 that will be relevant to the lab workshop that we
will be doing next week can be downloaded by clicking here. This structure will be released upon
publication. Please do not
distribute it.
Quickstart introduction to
MacPyMOL
Start MacPyMOLby clicking on its
icon in the Dock ( 
). This will start MacPyMOL and you will have two windows. One is the so-called "GUI"
where you will type PyMOL commands
Load a PDB file into MacPyMOL
To load the PDB file that you
downloaded from the PDB database, click on the "Open" option under
the "File" menu of the GUI.
Select the file 1BTN.pdb file that
you downloaded from PDB.
By
default, the molecule will be displayed in stick representation and coloured by
atom type.
You can see the name of the object listed in the menu to the
right of the viewer window. By default, the object
will be called the same name as the PDB file loaded.
For example, you can rename the object from the cumbersome
1BTN to something easier to remember such as "spectrin" by using the
"rename object" item.
Manipulate the view of the
molecule
You will need a three-button mouse
to effectively use PyMOL. If
you do not have one, I will provide one for this tutorial.
Most molecular visualization tools use the same
axis convention: X meanss the horizontal axis, Y means vertical
and Z is an axis that is perpendicular to the surface of the screen.
Rotation around X or Y: Left mouse button and drag
Rotation around Z: Place
the cursor toward the left or right edge of the PyMOL Viewer window and then
left button and drag
Translate (move) X or Y:
Middle button and drag (Note: if it gets stuck with the middle button, press
alt and left button to get unstuck)
Zoom (move Z): Right button
and drag up-down
All of these and many more mouse
functions are summarised in a very short manner in the Box to the right bottom
of the viewer window. By default,
the top line of this box reads "Mouse Mode 3-Button Viewing".
Hide the complicated default line
representation by clicking on H (hide)
next the object "spectrin" and choosing "hide lines".
You should now see a lot of dots
representing waters and ligands.
Hide these also by choosing H and "nonbonded" in the submenu.
Changing the representation of the
molecule
Make a cartoon representation of
the "spectrin" object by choosing S (show) and submenu "cartoon".
You can
change the look of the cartoon by choosing Setting 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).
Try various representations of the
molecule by clicking on S (show) next
to spectrin then 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 Mac is.
Under the A, there is also a menu of various "preset" styles that you can apply to an object. Probably the most useful is the preset/publication. You can try
running through these presets to see
what is available. The find action can also be useful for quickly identifying
protein/ligand contacts.
Identifying atoms or residues on the screen
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 of which the selection is a part.
Labelling You can label every residue in the molecule by choosing the
"residues" submenu under the L
menu to the right of the spectrin object.
Clearly, this is not a very useful way to label a molecule. In general, for publication-quality
labels, you will add them later in an application such as Adobe Illustrator.
For the purpose of examining the
molecule interactively, you can easily get only selected residues labelled.
Clear the mess of labels by
choosing the "clear" submenu
of the L menu next to the spectrin
object.
Viewing the sequences of the molecules
You
can see the sequence of the molecule by clicking on Display then Sequence On
in GUI window.
By pulling the
slider under the sequence toward the right, you can see that there is Inositol
1,4,5-tetrakisphosphate (I3P) in the complex and it is labelled residue
100. This is why the I3P residue
is in the sequence viewer before residue 100 of the protein, Often authors try to make the residue
numbers unique so that such ambiguities do not occur.
Selecting parts of the structure
Select the I3P molecule
in the complex by typing "select I3P=( r;I3P ) " in the command line
of the GUI window.
You could have
also selected this residue by just clicking on its name in the Sequence or by clicking on the I3P in the Viewer window.
Although the pull-down menus
allow some common selections to be made without entering commands on the
command line, you may find it faster to make complicated selections via the command
line. The text box 1.0 lists some
examples of commonly used commands for selecting parts of molecules.
Manipulating a
selection
You can choose the various ASHLC options next to I3P and they will
apply only to the I3P atoms.
Make a solid object of the I3P by using the "Spheres" option under the S (show) menu next to the I3P object
You can select the residues near the I3P by choosing the "Around" under the Action menu then choosing a distance from the "Around" submenu.
Making measurements
Measuring distances To measure distances between two atoms, choose the Measurement
option under the Wizard menu in the GUI window, This creates some sub-menus to the right of the view window
corresponding to various measurements that can be made.
Choose Distances. A message
appears in the PyMOL viewer (upper left corner) saying "Please click on
the first atom..." Click on an atom
using left button. A message now
appears in the PyMOL viewer saying "Please click on the second
atom..."
Clipping planes
As
you roll the spectrin object around using the left button, you can see how
elements of the structure are cut away.
This is due to "clipping". Clipping planes are imaginary planes in front of and behind the
object. Things that are closer or
farther away are clipped.
To make
the front plane move closer or farther away, press shift and the right mouse button simultaneously while dragging up or down. By doing the same thing, but by dragging
left or right, the rear clipping plane moves. To move both planes at the same time, drag at a 45 degree
angle.
Setting the background colour
In general, black backgrounds are great on the screen, but
they can make really horrible pictures for publication because they don't photocopy
or print well in black and white.
Under the Display menu, you can
choose Background to change the
background to a few pre-defined colours, including white. You can also change this and many other
settings by using the "setting"
menu of the GUI window.
Resetting the view
If you get lost in details, you can get back to the
original view by clicking Reset in the
menu to the right of the GUI window
Ray tracing
You can make a higher-quality, but static image of the
molecule by clicking on the "Ray"
menu to the right of the GUI window.
This can take a few minutes.
Saving an image
You can save the image as a png
file for incorporation in Illustrator or Powerpoint by choosing File then Save Image in
the GUI window. Type spectrin and it will save the image as spectrin.png.
Saving a pymol session
If you want to quit
pymol then come back to it later, you can choose File then Save session. This will make a file that has all of
the information necessary to restart PyMOL and get back to exactly the same
view/representation/selections as you had when you quit. To use this file to restart PyMOL, use
the File/Open option.
Superimposing two structures
You can work with many molecules
in PyMOL simultaneously. In order
to be able to see regions in which
two related structures agree or disagree, it is convenient to superimpose the structures. The simplest way to do this is with the
align command.
Read in the coordinates of a
second PH domain. Get the
coordinates for the GRP1 PH domain by downloading PDB database entry 1FGY (just
like you did for 1BTN above).
Rename the object 1FGY to GRP1 (using the A menu to the right of the 1FGY selection).
Superimpose the GRP1 PH domain
onto the spectrin PH domain typing: "align GRP1,spectrin" in the GUI
window.
Although PyMOL does this
structural alignment via an initial
sequence alignment, the sequence viewer does not show the sequence alignment. The structural superposition is most readily seen by hiding
the lines for the spectrin and GRP1 (use the H option in the ASHL menu)
and showing the cartoons (under the S
menu).
Superimposing structures of
proteins with divergent sequences
The align command works fine if the two structures have similar sequences, but fails miserably
if the sequences are more divergent, i.e., when the sequence identity is less than about 25%.
The Vps36 GLUE domain also has the
fold of a PH domain, but its sequence identiy is so divergent that the align
command fails. Try it.
- Get the Vps36 coordinates onto your
Desktop by clicking here.
- Use the Open item in the File menu of the GUI window to read the coordinates.
- Rename the object to GLUE.
- Try aligning the GLUE domain onto the spectrin
PH domain by typing "align GLUE,spectrin" in the GUI window.
Hide the lines for the GLUE domain and show its cartoon.
Clearly, the two structures are not corectly superimposed.
DaliLite to align structures
To align structures with little or
no sequence identity, it is necessary to use some tool external to PyMOL. There are many useful options, but one
which is robust and accessible as a web server is the DaliLite server (http://www.ebi.ac.uk/DaliLite/)
at EBI. This uses purely geometric
features of structures for alignment.
Access the DakiLite server by
clicking here.
Download the DaliLite result consisting of a PDB file that has the
GLUE domain rotated and translated to match the spectrin PH domain. It is called "mol2_1.pdb" by
default.
Open this coordinate file
in MacPyMOL using the File/Open
menu in the GUI winbdow.
MacPyMol scripts
One of the strengths of PyMOL is
its powerful scripting language.
Although it can be used as a menu-driven viewing tool, by making
scripts, we can make publication-quality images that can be reproduced exactly
without any further typing. These
scripts are easy to modify and their intuitive structure makes them
self-documenting. Most of us make
one script for every panel of a figure for publication. Delete everything by typing "delete
all" in the command line of the GUI
window.
Download a prepared sample script by clicking here (save into
the same directory that you have the 1BTN.PDB coordinate file.
If you are using the Firefox browser as I suggested, you
will probably see the text of this
file displayed in the browser. If
so, chose "Save as" from the
Firefox menu, and save the file as spectrin.pml in the desired location as an "Ascii text"
file).
Read this script into MacPyMOL by
choosing the "Run" option
under the "File" menu of the
GUI window and selecting the spectrin.pml
file. Files with pymol scripts are
usually created with a text editor such as emacs or TextEdit and named with a ".pml" extension at the
end.
You should now see a spectrin PH domain with some selections
listed to the right. These include
the elements of secondary structure and a set of residues that the authors
thought might be membrane-interacting residues. These selections were defined in the spectrin.pml script.
Download a startupfile that will
make creating some simple movies easier by clicking here.
Open the file with Stuffit
expander.
Initialize the movie
tools by typing " run
~/Desktop/pymolstartup/movie.py" in the GUI window.
Make a simple movie that spins the molecule around a vertical axis (the "y" axis) for 360 degrees in 10 degree steps:
- mvClear
- mvRot 1-36,y,360
- movie
To control viewing the movie in pymol, use the movie controls on the bottom of
the GUI window.
Click on "Play" to play the movie and "Stop" to stop it. Click on the ">" and "<" buttons to move forward or backward a single frame.
The nice thing about pymol movies is that you can change the view and representation while the movie is playing. When you are happy, you can then save the movie as a Quicktime movie suitable for applications such as Powerpoint.
To save a high quality movie, chose the "Save move as/Quicktime (More choices)" under the "File" menu in the GUI window:
In the Compression type: menu, choose Photo – JPEG. In the Depth: menu choose Best. In the Quality: menu, move the slider to Best. Save the movie as spectrin.mov.