http://www.pdb.org/
Experimental approaches for
structural biology
• X-ray crystallography
• NMR
• cryoEM
Nucleic acids
•
•
•
•
four letters (DNA, RNA)
sequence - AACTAACG (5’ → 3’)
DNA – double helix
RNA – “single stranded” helix, folding
(double helical regions, C2’ -OH →
secondary and tertiary motifs)
nucleotide
nucleoside
Proteins
• 20 letters
• primary structure - sequence
AMNTSSTVG (N-end → C-end)
Alberts, Molecular Biology
of the Cell, 5th Ed.
Where to get structural data?
• biological molecules
– PDB – Protein Data Bank
http://www.pdb.org
free
– NDB – Nucleic Data Bank
http://ndbserver.rutgers.edu/
• organic molecules
– CSD – Cambridge Structural Database
paid
PDB History
1957
• Myoglobin structure determined
1970’s
• Discussions how to establish an archive of protein structures
• PDB established at Brookhaven
– Oct 1971, 7 structures
1980’s
• Technology takes off
– molecular biology, instrumentation, computer hardware and software
• Number of structures increases
• Structural biology is able to focus on medical problems
• IUCr requires data deposition to the PDB
1990’s
• Complexity of structures increases
• Structural genomics begins
Current state of the PDB
• ~61,000 structures in the PDB archive
• Over 7,000 new structures deposited in 2008
• Depositions by macromolecule type
– 92% Protein
– 3.4 % Nucleic acid
– 4% Protein-nucleic acid complexes
• Depositions by experimental technique:
– 86% x-ray diffraction
– 13.2% solution NMR
– 0.4% cryo-EM (256 structures)
data as of 9. 11. 2009
http://www.pdb.org/pdb/static.do?p=general_information/pdb_statistics/index.html
PDB ID
• Each structure in the PDB is represented
by a 4 character identifier of the form [09][a-z,0-9][a-z,0-9][a-z,0-9]
• 1B3T
PDB Format
legacy format
 http://www.wwpdb.org/docs.html
 fortran-like 80 column-wide
not structured enough to describe
complicated 3D objects
its limits have been broken several times
 99,999 atoms, 34 (or 58) chains
readable by most programs
model – chain – residue – atom
Introduction to Pymol
http://www.pymol.org
http://pymolwiki.org
• how do I load DPB file?
– Plugin -> PDB Loader Service (2bhg)
– File -> Open (locally stored)
– command: load
• fetch 2bhg, load 2bhg
• command is a keyword, followed optionally
by one or more comma-separated
arguments
• color red, hetatm
• color red
• help show
Using the mouse
• Unmodified controls
– Left - rotate molecule (x, y and, at edges, z)
– Middle - translate molecule (x, y)
– Right - zoom (=MovZ)
– Wheel - slab/clip
Object menus (ASHLC)
Action
Navigation
Quick draw
Manipulation
Show
Hide
• same content as
Show
• Use Show and
Hide to toggle
things on and off
Label
Color
• Rule: Once a selection is shown (S) it
must be selectively hidden (H) as it is not
removed when another selection (S) is
made. Selections are therefore additive,
which allows for the creation of images
with mixed graphical representations.
• S -> Cartoon, H -> Lines
• S -> as – you do not have to click S,H
• Most options can be set within the
“Setting” menu within the top menu bar.
• Settings -> Cartoon
• Changing the background to white:
– Display -> Background -> White
• The “Display” menu within the top menu
bar contains options for most options
pertinent to displaying the image witin the
PyMol viewer.
• “fog” within the back of the molecule –
Display -> Depth Cue
Contextual menus
• Left double click or right single click to
activate
– click on object or part of object you want to
manipulate
– more or less the same menus as ASHLC
Ligand rescue squad
•
•
•
•
•
load 1BIW into Pymol
show as cartoon
recolor by secondary element
ligand is there, but it has disappeared!
rescue ligand:
– S > organic > spheres
Mouse selection
• Click on one of the spheres of the carotenoid
ligand
– pink dots (click anywhere to unselect)
– a new name called (sele)
– The name of the atom that was clicked appears within
the top text window of the “external GUI.”
You clicked /2BIW.pdb2//B/TYR`322/OH
Selector: selection "sele" defined with 12 atoms.
• This could be read as “you clicked atom OH which
is on the 322nd atom in the file, and belongs to
Tyrosine of chain B in the object created when
opening file 2BIW.pdb2. The complete selection
contains 12 atoms
• (sele) -> C -> by element -> CHNOS
Making images
• File -> Save Image As -> PNG
• Raytracing (Ray button, top right), then
Save Image
• even better quality, use command
– ray x, y (e.g. ray 2000, 2000 - dimension)
Action presets
• A -> Preset -> Default is similar to hide
everything, show lines
• preset options will set some variables that
are specific to these views and may
change further drawings. To remove the
effect of these presets affecting an object
representation, use the A -> preset ->
default.
• Let’s explore other presets.
• simple
• b factor putty
– The segments with the highest temperature factor are
shown as thicker cylinders. Regions of better
resolution have thinner diameter and are usually
found at the core of the protein. Mostly loops in the
outside of the protein wobble: the core portions of the
proteins usually appear more stable than the external
loops. This is mostly useful for crystallographers but
is a cool representation.
• technical
– Color domains in separate rainbow colors and shows
backbone and side chains.
• pretty and publication
• ligands
– zoom in on the ligand site and show the protein as
backbone except in the near vicinity of the ligand
where side chains are shown. The ligand is depicted
as a thicker cylinder
– to zoom out, simply click on the A > Zoom
• ligand sites
– pertinent to looking closely at the ligand in it’s binding
pocket
– solid surface, 2biw > C > Yellows > Sand, select
ligand stick, sele > C > Blues > slate
– Note: If you are preparing a figure for a black and
white print publication, it might be advantageous to
use the various gray scales, black, and white within
the “grays” option.
– explore others by yourself
Measuring distances
• from menu: Wizard -> Masurement
• click on the first atom in ligand, click on the
second atom in ligand
• change the color to white (ASHLC)
• When you are done using the “Measurement”
panel on the bottom right click Done.
• If you no longer need to display the distance
object, click Delete All Measurements.
Alternatively you can use the corresponding “A”
menu and select the delete option.
Clipping
• Clipping planes are imaginary
planes in the front and back of
the molecule. Parts of the
molecule that are outside the
planes are “clipped” and
therefore invisible. This is very
useful for complex or large
structures.
• To move clipping planes press
shift and the right mouse
button simultaneously while
dragging up and down. As an
exercise, try to remove some
of the molecular surface
Save your work
• File -> Save session
– *.pse, binary file
– save your objects, settings
• File -> Open
Pymol command language
• most interaction with PyMOL is via a
scripting language, not all functions are
available from menus
• keyword followed optionally by one or
more comma-separated arguments, e.g.
color red, hetatm – colors all nonprotein atoms red, color red – all
atoms are red
• in most cases 1st argument is specific to
command used, while the 2nd argument is
the name of the object/selection
commonly used commands
more in PyMol docs (paid, obsolete at http://pymol.sourceforge.net/html ) or
at wiki http://www.pymolwiki.org/index.php/Category:Commands
PyMOL Reference card: http://pymolwiki.org/images/7/77/PymolRef.pdf
• download 1GBV in PDB format
• load it into Pymol as 1gbv object
help show
load 1gbv.pdb, 1gbv
hide
show cartoon
hide cartoon, chain A
show ribbon, chain A
set ribbon_width, 5
hide cartoon, chain B
show spheres, chain B
hide cartoon, chain C
show sticks, chain C
color red, chain A
color blue, chain B
color orange, chain C
Save your work
• PyMOL script – text file with commands, each at
one line (see example.pml at the website)
• *.pml
• comments begin with # end_of_line
• run script from PyMOL: @example.pml
• or from command line as
pymol example.pml
• save the image
– ray
– png
Selections
• chain A is an example of a selection
• two selection syntaxes: hierarchical and
algebraic
• hierarchical
/model/segment/chain/residue/atom
– if patterns start with slash, the selection is
worked out left-to-right, if the first slash is
missing, the patterns are worked out right-toleft
– select ///A/10 #residue
color red, sele
color atomic
– select ////10-20/CA #
10 in chain A
atoms CA in residues 10-20
# (any chain)
– select 42/C+N (C,N?)
show spheres, sele
#atoms C and N in residue 42
• algebraic
– combine terms (e.g. chain A) using
logical operators (AND, OR, NOT)
– select wc, chain A # wc is the name of selection
– select chain A and resi 10:20 # residue number
– select resn ALA and name N # backbone nitrogens
# from ala residues
– select elem O and not name OH
# oxygen atoms
# except hydroxyls
• The items in a list of identifiers are
separated by plus signs (+) only. Do not
add spaces within a list of identifiers. The
selector resi takes (+)-separated lists of
identifiers:
– select nt, resi 1+2+3
– select nt, resi 1-3
– don’t combine these two (1-3+6 is wrong)
property
example
symbol
e. chemical symbol, select polar, symbol (e.) o+n
name
n. atom names, select carbons, n. ca+cb+cg+cd
resn
r.
residue names, select aas, r. asp+glu+asn+gln
residue numbers, select mults10, i. 1+10+100+1000
resi
i.
chain
c.
chain ID
ss
-
secondary structure type, select allstrs, ss h+s+l+""
select one, resi 10
zoom one
Selection algebra
• and, or, not etc.
• how would you select resid 27 and resid
28?
property
short
example
not s1
and
or
! s1
&
|
atoms that are not included in s1
atoms included both in s1 and s2
atoms included in either s1 or s2
in
-
s1 atoms whose name, resi, resn,
chain match s2 atoms
like
l.
s1 atoms whose name, resi match s2
atoms
property
short
example
s1 around X
s1 a. X
atoms with centers within X Angstroms of the
center of any atom in s1
select near10, resi 10 around 5
s1 expand X
s1 e. X
Expands s1 by all atoms within X Angstroms of
the center of any atom in s1
select near10x, near10 expand 3
s1 within X of s2 s1 w. X
of s2
Selects atoms in s1 that are within X Angstroms
of the s2
byres s1
Expands selection to complete residues
br. s1
select cmplt, byres near10
neighbor s1
nbr. s1
atoms directly bonded to s1
select vic, nbr. resi 10
s1 extend X
s1 ? X
Extends s1 by X bonds connected to atom in s1
select connect_x, near10 extend 3
# selects atoms that are part of chain a,
but not # residue number 125.
select chain a and (not resi
125)
# The following two selections are
equivalent
select (name cb or name cg1 or
name cg2) and chain A
select name cb+cg1+cg2 and
chain A
• select residue 10 in chain B (name it
cpk10)
• show it as cpk, zoom at it
• hide cpk10 selection
• make a selection called bb containing C, O
and N atoms
• count atoms in selection (count_atoms)
• remove residue no. 5 (remove)
• count atoms again
objects vs. named-selections
• PyMOL creates an object-name to locate data when you
load a data file.
• Making selections is a way of pointing to a subset of that
data.
• Selections are in parentheses in control panel.
• When you delete a selection-name, the data are still
found under the object-name, but the data are no longer
organized as the selection. In contrast, after you delete
an object, you must reload the data to have access to it
again.
delete
• PyMOL can hold several objects at the same time (load
1gbv and 1bna)
disable, enable
TIP: get rid of pink dots
select bb, name c+o+n+ca, disable bb,
color red, bb
• Named selections are static. Only atoms that
exist at the time the selection is defined are
included in the selection, even if atoms which
are loaded subsequently fall within the selection
criterion
• clear your workplace, load 1gbv
• make selection called static containing all
1gbv atoms
• count atoms in static
• add hydrogens (h_add)
• count atoms in static, compare with count atoms
in the whole structure
Single word selectors
Selector Short form Description
all
*
All atoms currently loaded into
PyMOL
none
No atoms (empty selection)
hydro
h.
All hydrogen atoms currently loaded
into PyMOL
hetatm
het
All atoms loaded from Protein Data
Bank HETATM records
visible
v.
All atoms in enabled objects with at
least one visible representation
polymer
All atoms on the polymer (not het).
Point of view
• zoom
• orient
– aligns the object or selection so its largest
dimension is shown horizontally, and its
second largest dimension is shown vertically
• view
– store orientations and recall them later
– view v1, store; view v1,
recall; view v1