Introduction to X-ray Crystallography
Alex Cameron
[email protected]
Office B127
Tools
• Spectroscopy
• Biophysical techniques to identify and measure
biological interactions
• Structural biology techniques
Protein Structure
• Want to know the structure of the protein and how
this relates to its function
– X-ray Crystallography
– Electron Microscopy
– NMR Spectroscopy
– Molecular Dynamics
– Structure Prediction
• A biological question often requires a combination of
these
The experimental result
or
Does what is presented come from?
X-RAY CRYSTALLOGRAPHY
Why X-rays
• Interatomic distances are of the order of a few Ångstrom (10-
10
meters)
– C-C bond 1.54Å
• The wavelength used for diffraction experiments should be of
the same order of magnitude as the distances being studied.
• In X-ray diffraction experiments we generally use X-rays of
wavelength 0.5 to 2 Å
Why Crystals?
• When an X-ray hits an electron cloud around an
atom it is scattered.
Scattered
X-ray
X-ray
Electron cloud
around atomic nucleus
• Scattering from a single atom or molecule is very
weak
• The regularity of the protein crystal gives rise to a
visible diffraction pattern by constructive
interference effects.
Microscopy vs Diffraction
Lens Lens
Light
Eye Microscope
Object
Xrays
X-ray Diffraction
Crystal
Detector
X-ray crystallography
X-ray
Gene Structure
Protein
Crystals
Computation
Molecular biology Physics Biochemistry
X-ray crystallography
X-ray
Gene Structure
Protein
Crystals
Computation
Molecular biology
What is a “protein crystal”?
Protein
Repeat “infinitely” in 3D
Large water-filled spaces
How do we achieve this?
• Clone / identify protein of interest
For crystallography want a protein that takes a single conformation so
as to make a crystal lattice
• May be a single domain
• Purify protein
Chromatography techniques (Enzymology modules)
• Crystallise
Vapour Diffusion Method of
Crystallisation
Polyethylene
glycol/ammonium sulphate
/ salts /pH
Volume = 2ml
[Protein] = X
[Precipitant] = 0
Protein(2ml) Volume = 4ml Volume = 2ml
Well solution (2ml) [Protein] = X/2 [Protein] = X
[Precipitant] = W/2 [Precipitant] = W
Time
(Hours->months)
[Precipitant] = W
Robots: High throughput crystallisation
96 well sitting drop trays
• require only 50 – 200 nl
of protein per well
Screen lots of different conditions
• pH, precipitant, salt
X-ray crystallography
X-ray
Gene Structure
Protein
Crystals
Computation
Physics
Data collection
In-house detector
Anode Detector
Synchrotrons
Some information for the general public: https://www.diamond.ac.uk/Public.html
Why the diffraction Pattern
Different Intensities
Regular
Discrete spots
Concentric circles
Diffraction
Electron
Waves
F
1.5
Phase (f)
1
0.5
Amplitude |F|
0
-180 0 180 360 540 720 900 °
-0.5
-1
Wavelength (l)
-1.5
This is a cosine wave F = |F| cos( x - f )
Three properties that define the wave, the wavelength , amplitude |F| and
the phase f, which takes values from 0º to 360º ( 0 to 2π).
Waves through slits
Water waves Light
Constructive interference
• Add together two waves with the same amplitude
that are in phase and you get twice the amplitude
Destructive interference
• Add together two waves that are 180º out of phase
and you get zero amplitude
Argand diagram representing waves
90° (imaginary axis)
“sohcahtoa”
F
B=|F|sinf
f
180° (p) 0° (2p) (real)
A=|F|cosf
270°
F = A+iB = |F|(cosf + isinf) where i = -11/2
Using Euler’s formula eif = cosf + isinf then
F = |F|eif
• Ie waves can be represented on an Argand
Diagram
• Wave can be described by F = |F|eif
1.5
0.5
0
-180 0 180 360 540 720 900
-0.5
-1
-1.5
4
f=0
Wave Summation
3
2
1
0
0 180 360 540 720 900
-1
-2
f=30 f=120
-3 f=120
-4
1.5 F3
1
F2
0.5
F1
0
0 180 360 540 720 900
-0.5
f=0
-1
-1.5 f=30
3
0
0 180 360 540 720 900
-1
-2
-3
Concept
• X-rays are waves
• Waves have an amplitude and a (relative)
phase
• Waves can be represented on an Argand
Diagram
Why the diffraction Pattern
Different Intensities
Regular
Discrete spots
Concentric circles
Bragg’s Law
https://www.youtube.com/watch?v=gBxZVF3s4cU
Stephen Curry, Imperial College, RI, https://www.youtube.com/watch?v=gBxZVF3s4cU
Stephen Curry, Imperial College, RI, https://www.youtube.com/watch?v=gBxZVF3s4cU
Stephen Curry, Imperial College, RI, https://www.youtube.com/watch?v=gBxZVF3s4cU
Stephen Curry, Imperial College, RI, https://www.youtube.com/watch?v=gBxZVF3s4cU
Stephen Curry, Imperial College, RI, https://www.youtube.com/watch?v=gBxZVF3s4cU
Stephen Curry, Imperial College, RI, https://www.youtube.com/watch?v=gBxZVF3s4cU
q
d p = dsinq
p
Stephen Curry, Imperial College, RI, https://www.youtube.com/watch?v=gBxZVF3s4cU
Stephen Curry, Imperial College, RI, https://www.youtube.com/watch?v=gBxZVF3s4cU
Stephen Curry
Stephen Curry
Stephen Curry
Stephen Curry
Stephen Curry
Stephen Curry
Concept
• X-ray diffraction can be described in the same
way as for reflection from a mirror
• Bragg’s Law nl =2dsinq
• Diffraction from a crystal only when Bragg’s
law holds
Stephen Curry, Imperial College, RI, https://www.youtube.com/watch?v=gBxZVF3s4cU
Stephen Curry, Imperial College, RI, https://www.youtube.com/watch?v=gBxZVF3s4cU
Electron density Wave with phase f=2pr.S
F = |F|e if
Stephen Curry, Imperial College, RI, https://www.youtube.com/watch?v=gBxZVF3s4cU
Digression
• Phase difference can be written in terms of a
scattering vector
Phase difference expressed in terms of scattering vector
(S)
Projection of r on s0 Scalar (or dot) product = s0.r
S1
vector in direction of S
S0
wave with length of 1/l
l an e S0
gp
fl e ctin
re
Fig 4.5, Drenth
Figure 6.11, Rupp
Projection of r on s1
dp = (lr.s1 –lr.s0) = (s1-s0).rl = S.rl
Multiply by 2p/l to get the phase difference
df = 2pS.r ie phase difference can be written in terms of scattering vector
Phase difference expressed in terms of scattering vector
(S)
Projection of r on s0 Scalar (or dot) product = s0.r
S1
vector in direction of S
S0
wave with length of 1/l
l an e S0
gp
fl e ctin
re
Fig 4.5, Drenth
Figure 6.11, Rupp
Projection of r on s1
dp = (lr.s1 –lr.s0) = (s1-s0).rl = S.rl
Multiply by 2p/l to get the phase difference
df = 2pS.r ie phase difference can be written in terms of scattering vector
Electron density Wave with phase f=2pr.S
F = |F|e if
Stephen Curry, Imperial College, RI, https://www.youtube.com/watch?v=gBxZVF3s4cU
Stephen Curry, Imperial College, RI, https://www.youtube.com/watch?v=gBxZVF3s4cU
Stephen Curry, Imperial College, RI, https://www.youtube.com/watch?v=gBxZVF3s4cU
Stephen Curry, Imperial College, RI, https://www.youtube.com/watch?v=gBxZVF3s4cU
Stephen Curry, Imperial College, RI, https://www.youtube.com/watch?v=gBxZVF3s4cU
Concept
• All atoms from a molecule contribute to the
total scattering in each direction
• Only see diffraction from a crystal if Bragg’s
Law applies
