Ecole des Hautes Etudes de Biotechnologie et de Santé -

Casablanca

Modélisation moléculaire et chimie thérapeutique

PyMOL tutorial
Gene to protein course
USP 2022
Marko Hyvönen
 On these instructions

Commands from top menus are in blue

Scene > Store > F1

Commands from graphics menus are in red

Show > Cartoon

Command line instructions are in Courier and shaded

select CDK, 2wih & chain A & polymer

Commands are always of the syntax:

command first_argument, next_argument, next_argument

comma comma
 PyMOL tutorial aims

• learn the basics of using PyMOL

– Retrieve coordinates
– Familiarise with the menus
– Use of the mouse
– Use the command line
• Learn to select specific parts of the molecule
– Chains, atoms, residues, ligands
• Learn to create and save scenes
• Save sessions (for later use or for sharing)
 Command window

Menus for opening and saving files, changing settings etc.

Output of any actions (great for trouble shooting)

Command line for executing commands, be they simple or complex

 The Graphics Window

Object menu

Mouse mode
and guide

Command line Sequence on/off Full screen

 The object menu

Action something
Show a representation
Hide representation
Light gray: Label
Colour
Visible/active
Dark gray:
Hidden/inactive

An object
Current selection
Named selection

Selections are in parenthesis, e.g. (PH_domain)

Mouse control

On an atom:
On canvas:
 Setting defaults

If using PyMOL frequently, you might want to have certain settings to

be valid all the time.

For now, the main thing to change is the background colour from
black to white:
File > Edit pymolrc

Add this line:

bg_color white

Save and close the file.

Restart PyMOL

(More useful starting settings at the very end of the slide deck)
 Searching for coordinates

http://www.ebi.ac.uk/pdbe

http://www.rcsb.org
 Opening coordinates

• There are at least four different ways

– Click on a coordinate file
(assumes you have associated PDB files with PyMOL)
– Open a file that is in your computer
– Open coordinates from PDB using a plugin
– Fetch the coordinates from PDB with a command
Open from your computer

This loads the coordinates (2wih.pdb) into object called 2wih

File > Open...

PDB loading from the command line

load 2wih.pdb
load C:\Users\Marko\Desktop\tutorial\2wih.pdb
load /Users/Marko/Desktop/tutorial/2wih.pdb
load /home/Marko/Desktop/tutorial/2wih.pdb

Directly from Protein Data Bank

fetch 2wih
fetch 2wih, async=0 (sometime needed)
 Basic display of coordinates

Default view is lines for protein and nucleic acids and crosses for
non-bonded atoms (waters, ions).
Colour for carbons changes for each molecule, the other atoms
are coloured by the element
N = blue, O = red, S = yellow, P = orange, H = white
 Changing the view
First: hide everything
all > Hide > everything

Then, show 2wih as cartoon

2wih > Show > cartoon

Colour by chain (there are 4 in 2wih):

2wih > Color > by chain > by chain(elem C)

Using the command line:

hide everything, all
show cartoon, 2wih
colour green, chain A & name C*
colour cyan, chain B & name C*
colour yellow, chain C & name C*
colour magenta, chain D & name C*
 Getting serious…

2wih > Show > nb_spheres (waters and ions as small spheres)
2wih > Show > organic > spheres (ligands as CPK spheres)
 Hierarchical structure of macromolecular coordinates
ATOM: protein/DNA/RNA
Occupancy
HETATM: ligands
X coord Y coord Z coord B-factor Element
ATOM no.

ATOM 330 N ALA A 141 33.444 27.094 8.380 1.00 8.10 N

ATOM 331 CA ALA A 141 33.397 28.479 8.840 1.00 7.81 C
....
ATOM 1666 N THR B 520 23.744 48.961 3.142 1.00 35.07 N
ATOM 1667 CA THR B 520 24.098 47.838 4.013 1.00 33.12 C
ATOM 1668 C THR B 520 23.952 46.477 3.357 1.00 31.94 C
ATOM 1669 O THR B 520 24.698 45.547 3.676 1.00 31.26 O
ATOM 1670 CB THR B 520 23.242 47.855 5.296 1.00 33.72 C
ATOM 1671 OG1 THR B 520 21.871 47.557 4.982 1.00 33.68 O
ATOM 1672 CG2 THR B 520 23.307 49.243 5.942 1.00 34.95 C

The most important identifiers for

selecting parts of a structure for display:
Residue number is unique to each chain
Chain is made of residues (not necessarily covalently linked)
Residues make up a chain (always three letter code)
Atoms are part of a residue (each atom in a residues has a unique name)

In PyMOL when clicking an atom:

/2wih/A/A/THR`137/CA
/object/chain/conformation/resname`resno/atom
 Selecting parts of the molecules
Selections can be done in many ways:
• From the graphics window
– Left click on an atom selects are residue
• This will also add to current active selection
• You can change what is selected from the red “Residues” text in the
bottom menu
– Right click on an atom, follow the menu to
atom/residue/chain… and “select”
• From the sequence view
– To show the sequence, click the little S button at the very
bottom

You can select residues from the sequence

- one by one
- by normal text selection method
- great also for locating selected residues in sequence.

The default selection will be called (sele)

To rename that to something you’ll remember:
(sele) > Action > rename selection
 Selecting parts of the molecules
From the command line
Syntax: select selection_name, selection
For selection you can use (among many others):
• object name: 2wih
• resn for residue name: resn ASP
• resi for residue numbers: resi 100-200
• chain for chain ID: chain A+D
• name for atom name: name C* (all atoms starting with C :
carbon, Ca, Cd, )
And combination of these, linked with “&”
You can negate the selection with a “!” (“no!”)
Example:
select all glycines in residues 1-100 in object 2wih but NOT in chain
A and call that section GlyNotA
select GlyNotA, resn GLY & resi 1-100 & 2wih &
!chain A

select CDK2, chain A

select cyclin, chain B
select inhibitor, resn p48 & chain A

Now you can manipulate all those selections independently:

• color
• style
• transparency…
 Creating and saving different views

2wih > Hide > everything

(CKD2) > Show > cartoon
(cyclin) > Show > cartoon
Orient the structure as you like
Save this view, or scene:
Scene > store > F1
Now the scene can be recovered by pressing F1
Setting “Buttons” on:
Scene > Buttons
Will show the scene buttons on the screen

Related command line options:

scene F1, store
scene F1, recall
scene F1, clear
set scene_buttons, 1
scene CDK2_cyclin, store
 Saving the work

• PyMOL session file (.pse) will preserve all the molecules,

selections, scenes, colourings
• Keep saving regularly!

• From the commend line:

save my_session.pse

Note: annoyingly, Ctrl-S does not work for this!

 Colouring

Command line: color name_of_the_color, selection

color forest, CDK2 & name C*

Atom type Predefined Helices, sheets Colour by

color colors for and loops property
The first chains All in different Rainbow: N-term
option does chainbows: colour blue to red C-term
NOT change each chain as (urgh…) B-factor: indicates
carbons rainbow mobility of the
residue

All named colours in PyMOL

https://pymolwiki.org/index.php/Color_Values
 Some fancier representations

Cylindrical helices:
set cartoon_cylindrical_helices, 1
(“1” is a Boolean “on”, “0” = “off”)

Backbone as tube:
cartoon tube
Tube with thickness from
B-factor:
cartoon putty

Prefer the “normal” look? Return back to that with:

cartoon automatic
 Surfaces

• Great at showing cavities, channels, binding sites and

for analysis of interfaces
• PyMOL draws surface for the whole object
• An issue: surface of an object will cover all of the
content of that object, not individual molecules

Solution: create an object from a chain

(CDK2) > Action > copy to object (creates obj01)
(cyclin) > Action > copy to object (creates obj02)
(you might want to rename the new objects)
obj01 > Action > Rename (give whatever name you want)
obj02 > Action > Rename (give whatever name you want)
set_name old_name, new_name
Draw the surfaces
obj01 > Show > surface
obj02 > Show > surface
show surface, obj01

Note also that non-polymer atoms (waters, ligands...)

are not covered by surface by default.

This includes also non-natural amino acids like

seleno-Methionine
To include this in surface rendering you need to:
set surface_mode, 1
 Surfaces & interfaces

Colour surface differently:

set surface_color, white, obj01
(to revert to atom colours, set surface_color, default)

Colour interface contacts between CDK2 and cyclin

select contact1, (obj01 within 4.0 of obj02)
select contact2, (obj02 within 4.0 of obj01)
(these select atoms in each molecules within 4.0 A from the other one)
set surface_color, limegreen, contact1
set surface_color, skyblue, contact2

Show water molecules between the two proteins

select interface_waters, (2wih within 4 of obj01)
& (2wih within 4 of obj02) & resn HOH

Like that? Best save it:

scene interfaces, store
 More complicated structures:
handling symmetry
fetch 3ry2, biotinSA
(load coordinates and call them biotinSA)
hide everything, all
(hide all the objects)
show cartoon, biotinSA
Two protein molecules, but streptavidin is a tetramer. The missing half must come
from crystallographic symmetry
Let’s generate this by generating symmetry related molecules of 3ry2
symexp sym, biotinSA, biotinSA, 4
Or use the object menu:
Action > generate > symmetry mates > within 4 Å
• Choose in the graphics window which of the symmetry
molecules corresponds to the missing part of the tetramer.
• Rename the other half to biotinSA2
• Delete the other symmetry mates:
delete 3ry2* or delete sym*
show sticks, resn BTN
(show biotin (residue name BTN) as sticks)
 Aligning structures

Continue with the same PyMOL session:

fetch 3ry1, apoSA
(load apo coordinates and call them apoSA)
hide everything, apoSA
(hide everything from the apoSA coords)
show cartoon, apoSA
(show it as cartoon, and perhaps color differently)
align apoSA & chain A, biotinSA
(align the apo coords to the complex)
colour apoSA & name C*, density
(colour apo streptavidin in dark blue, but carbons only)

Can you see what the differences are between biotin bound
streptavidin and the apo form?
 Electron density

First, load electron density map. Needs to be in so-called ccp4

format. You can get the density file from the Downloads menu at
PDB Europe https://www.ebi.ac.uk/pdbe/ for (almost) any PDB
entry.
We’ll load map for the biotin-bound streptavidin and show the
density for biotin
First we load the map file and call it 3ry2_map:
load 3ry2.ccp4, 3ry2_map
Then we make selection for biotin in chain A:
select biotin, resn BTN & chain A & biotinSA
The we show the map as a mesh at 1.0 sigma/noise level within 1.5
Å of biotin:
isomesh 3ry2_mesh, 3ry2_map, 1.0, biotin,
carve=1.5
color blue, 3ry2_mesh (colour the mesh in
blue)
set mesh_width, 0.5 (make the mesh a bit
thinner)

A handy plugin to have is “Isocontour slider”

https://pymolwiki.org/index.php/Isoslider
 NMR ensembles

• NMR structures typically submitted as ensemble of 20-50

structures that all satisfy experimental constraints.
• By default PyMOL shows only the first molecule of the coordinate
set
• To see them all at once:
fetch 1mph.pdb, async=1
set all_states
• Or if you want them separated into individual objects:
unset all_states (or: set all_states, 0)
split_states 1mph
delete 1mph (to remove the original ensamble)
• The ensemble is best shows a Ca-trace, aka “ribbon” in PyMOL
show ribbon, 1mph*
 One state or all states

set all_states, 0 set all_states, 1

set state, 1
 Making figures

Ray trace for best quality images:

ray 2000 (ray trace image as 2000 pixels wide)
png myimage.png (to save the image as PNG)

Educational version of PyMOL does not allow ray tracing, but

using:
set use_shaders
png myimage.png, 2000
Will result in quite decent images.

If ray tracing , you might want to:

Take the shadows off, as they can make the figures too busy:
Setting > Rendering > Shadows > none

Set transparent background

set opaque_background, 0
set ray_opaque_background, 0
Nice(?) mode with black outlines for the molecule:
set ray_trace_mode, 1
 More PyMOL material

• PyMOL wiki at https://pymolwiki.org

• Great PyMOL tutorials:
– Intro:
https://www.researchgate.net/publication/31387700
4_Introduction_to_PyMOL
– Intermediate:
https://www.researchgate.net/publication/31387700
9_Intermediate_PyMOL
– Advanced
https://www.researchgate.net/publication/31387707
5_Advanced_PyMOL
 Some useful default settings

File > Edit pymolrc

bg_color white
set ray_opaque_background, 1
set opaque_background, 1

set mesh_width, 0.5

set mesh_quality, 3
set mesh_colour, blue

set dash_color, grey30

set dash_length, 0.25
set dash_gap, 0.15
set dash_round_ends, off

set ray_shadow, off

set ray_trace_mode, 1
set use_shaders
set pse_export_version, 1.74