Transcript Crystallography: An introduction

Crystallography: An introduction
Harma Brondijk
Crystal and Structural chemistry, Utrecht University
Crystallography
Phase problem
 
Construct
Pure
protein

Crystal
3D structure
X-ray Diffraction
Electron density
Why X-ray crystallography?
(Light) microscope:
Limitations:
Object needs to be larger than the
wavelength of the light (visible light
400-700 nm, atoms = 0.15 nm apart)
X-rays(0.08-0.6 nm) cannot be
focussed by lenses
Molecules are very weak scatterers
X-ray diffraction of a crystal
A crystal contains many molecules in
identical orientation
Diffracted x-rays of individual molecules
‘add up’ (positive interference) to
produce strong reflections
Computers can simulate a lens and
reconstruc the image (Fourier
transform)
Growing crystals
Hanging drop: 1 μl protein
solution+ 1 μl reservoir solution
Reservoir: precipitant solution eg.
1 M NaCl or 30% PEG-4k
[precipitant] and [protein] slowly
rise as drop equilibrates with
reservoir
[protein]
precipitation
Nucleation & growth
growth
Soluble protein
[precipitant]
Getting your data
X-ray data are measured on frozen crystals (~100K)
Frozen crystal
mounted in loop for Xray data collection
In house X-ray data
collection set up
For high quality X-ray
data collection
extremely intense
synchrotron beam lines
- like here in Grenoble are used
Fourier transform
(phase problem)
Raw data: Thousands of intensities of
reflections
Electron density
Each diffraction spot (reflection) contains
information on the position of every atom!
The degree of order in the crystal determines the quality of the
diffraction data and ultimately the quality of the final atomic model
“low resolution”
“high resolution”
The precision of the atomic model is mainly determined by the
maximal resolution to which the crystal diffracts X-rays
d=4Å
d=3Å
d=2Å
d=1Å
Atomic resolution
Some real life examples
1.8 Å structure: core vs surface loop at 2σ
3.1 Å resolution, well ordered
core of the protein
What’s a crystal structure?
Different representations of an Fab fragment of monoclonal
antibody 82D6A3 bound to the collagen binding domain of
human von Willebrand factor
Things you - as a potential user of crystallographic
data - should know about crystals and crystal
structures
Protein crystals contain a lot of solvent and are held together by a
limited number of weak contacts between protein molecules
Acetylcholinesterase
~68% solvent
2 Glycoprotein I
~90% solvent
(extremely high!)
Typical solvent content 40-60%
Solvent channels allow diffusion of compounds into crystal
Often these compounds can reach the active or binding site
Often enzymes are active in crystalline state
Two types of solvent: ordered and disordered
•
Ordered water molecules show
up as discrete blobs of electron
density in contact with the protein
or with other ordered water
molecules
•
Disordered water regions show
up as featureless (flat) electron
density
PDB files:
• Basically just simple tekst files
• At the top: information about the crystal:
– Which proteins/ligands etc
– Crystalization conditons
– How was the structure solved
– The resolution
– Some usefull statistics to judge the quality of the crystal
– How to get from the structure to the biological unit
– Remarks about missing bits etc.
• Crystal parameters: cell dimensions/space group
• A list of all atoms in the structure
A crystal structure according to the protein data bank
(PDB)
occupancy
x,y,z coordinates (Å)
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
25
26
27
28
29
30
31
32
33
34
N
CA
C
O
CB
CG
OD1
OD2
N
CA
ASP
ASP
ASP
ASP
ASP
ASP
ASP
ASP
VAL
VAL
A
A
A
A
A
A
A
A
A
A
928
928
928
928
928
928
928
928
929
929
19.062
19.770
19.075
19.074
21.259
22.112
21.693
23.239
18.417
17.726
9.157
10.123
9.938
8.824
9.776
10.245
11.114
9.742
10.985
10.864
35.067
34.232
32.899
32.351
34.071
35.233
36.025
35.349
32.405
31.125
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
4.73
4.58
4.56
5.39
3.13
5.52
5.42
7.93
3.68
4.63
Isotropic B-factor or temperature factor is a measure of the
mobility of an atom
B (Å2) = 82<u2>, where <u2> is the mean square atomic
displacement
N
C
C
O
C
C
O
O
N
C
At typical resolutions (1.8 Å or worse)
• The electron density of hydrogen atoms is not resolved
(and no hydrogen atoms are present in the pdb file)
• The electron densities of C, N, and O atoms are all
rather similar
Position of N and O atoms in Gln (and Asn) side chain
must be inferred from hydrogenbonding network
Main chain
carbonyl
Main chain
amide
?
?
H
H
H
Asp
The same holds for the orientation and protonation of
the imidazole ring of histidines
?
A pdb file may contain residues for which no, or only
limited electron density is visible
?
?
No density for amide
N of glutamine
Break in side chain
density of glutamate
Sometimes the electron density suggests two side chain
conformations but often only one is modeled in the pdb file
Threonine side chain
conformation present in
pdb file
Alternative conformation
that is also compatible
with electron density
The interpretation of dynamic loops in the pdb file may
be tentative
Well defined -strand in the
core of a protein: atomic
positions are reliable
Flexible loop at the surface of
a protein: atomic positions are
not well defined
Look at B-factor distribution!
Protein coloured by B-factor:
Well defined regions have low
B-factors (blue/green)
Poorly defined/more mobile
regions have high B-factors (
yellow/orange/red)
A protein molecule is dynamic
• The electron density is a spatial average over all molecules in
the crystal and a time average over the duration of the X-ray
data measurement
• Multiple discrete conformations of a residue in different
molecules are superimposed.
• Damage caused by X-rays may change the protein (mainly
breaking of disulfide bonds)
• A crude description of dynamics is provided in the pdb file as the
isotropic B-factor
• Some dynamical aspects evident in the electron density are lost
in the pdb file
Reading a crystallography paper:
Judge the quality of the data:
 Rmerge: 0.05-0.10 good, 0.1-0.15 acceptable
 I/σ = signal/noise >2.0
 Completeness
 Redundancy
 Rwork/Rfree:
 difference < 0.05,
 Rwork≈ resolution/10
 Deviations of known geometry
 waters: at 2 Å ≈ 1 water/residue, at > 3Å
usually none
More and more structures: learn how to use them!
Crystallography and drug design and lead
optimization
The crystal structure of a protein-substrate complex can serve
as starting point for structure-based drug design
Guanidino group
provides additional
interactions
Relenza was developed starting from the crystal structure
of influenza virus neuraminidase with bound sialic acid
Structures of bird-flu neuraminidase reveal new cavity
that could be exploited in drug design
Russell et al., Nature 443, 45-49 (2006)
Can X-ray crystallography contribute to lead
discovery?
-
-
Development of high through-put (HTP) methods in
crystallography has considerably reduced the time needed to
solve a crystal structure while minimizing the need for human
intervention
This now allows for screening of medium sized compound
libraries (~1000 compounds)
• Library used in traditional
HTP screen
– 106 compounds
– Mw 300-500 Da
• Library used in X-ray based
screen
– 103 compounds
– Mw 100-250 Da
Technical advances enabling HTP crystallography
Automated set-up
of crystallization in
96-well format (100
+ 100 nl drops
Automated imaging of 96well crystallization plates
Automated crystal
transfer from liquid
nitrogen to X-ray
beam
Stronger in house X-ray sources
Automated beam lines at synchrotrons
Improved software for automated interpretation of ligand density
Expanding hits into larger and higher affinity
compounds
-Joining fragments by an
appropriate scaffold (C)
-Grow a fragment to fill
neighboring pockets (d)
Electron densities of initial
fragment (left) and expanded
fragment (right)
Simultaneous binding of two compounds from a
mixture can be detected
Top: Electron density of two
compounds bound simultaneously
together with the two automatically
built compounds.
Bottom: Electron densities from
individual soaks shown in different
colors
Drug design cycle
Crystallise complex
Test inhibitor
Design better
compound in silico
Synthesize
inhibitors
Contribution of X-ray crystallography to drug design
and discovery
• Lead optimization
– Well established
– All major pharmaceutical companies do it
– Numerous drugs on the market and in pipeline
• Lead discovery
– Promising, but quite recent
– Performed in small companies (that have
collaboration agreements with large pharmaceutical
companies
– Must still prove its value