Physical Biochemistry X-Ray Crystallography 3

X-ray Diffraction:
X-rays are electromagnetic waves (λ = 0.1 – 100 Å)
We use wavelengths around 0.5 – 2.5 Å (as we are measuring atomic distances between bonds)
The wavelength used for diffraction should be of the same order of magnitude as the distances
being studied. Interatomic distances are generally a few Å, so wavelength in this range is
required.

When an X-ray hits an electron cloud that surrounds an atom it is scattered

Any object can individually scatter radiation, but the diffraction pattern would be very weak, and
not detectable.
Crystals contain regular arrays of molecules
Every atom of each molecule in the crystal contributes to the diffraction pattern, giving rise to
strong signals in certain directions.
The regularity of the protein crystal gives rise to a visible diffraction pattern by
constructive interference effects.

Waves
Cosine wave 

Y = f cos (x - )

Three properties define a wave

 Wavelength ()
 Amplitude (f)
 Phase () values from 0 - 360 (0 - 2)

When two waves are added together, constructive of destructive interference will occur,
depending on the phases of the waves.
Waves that are in phase with the same wavelength will cause constructive interference
Waves that are out of phase by 180 will result in destructive interference
 If waves vary only by phase, amplitude will be 0

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Physical Biochemistry X-Ray Crystallography 3

If path difference = one

wavelength, the waves will
add together

Bragg’s Law

1. Think of the protein

crystal as a series of atoms
that are in planes
 d = separation
between planes
2. A beam of monochromatic
(single wavelength) parallel
X-rays hits the plane.
3. Some X-rays are

scattered by atoms in the 1st plane.

 Θ (theta) = the angle of direction the X-ray is diffracted from the plane
4. Some X-rays will be scattered by atoms in the 2 nd plane
5. Constructive interference will occur when the distance travelled by the two waves differs my a
multiple of the wavelength (n × λ, where n is an integer)

Length of orange line

sin Θ = opposite / hypotenuse

sin Θ = line / d
line = d sin Θ

The extra distance travelled by the

2nd wave = 2d sin Θ
Bragg’s Law states that constructive
interference will occur when”

2d sin Θ = n
Where ‘n’ is an integer

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Physical Biochemistry X-Ray Crystallography 3

Total scattering from a single molecule (or unit cell) in a given direction can be considered a single
wave emerging from one point in the molecule / unit cell. This will only occur if the path difference
between waves of adjacent molecules is n (waves add constructively).

If this is not the case (e.g. path difference = n(/2)) then the waves will interfere destructively.

If we work out the path difference between certain atoms, we can see what kind of interference
will occur. A number of atoms may cancel out the diffraction of other atoms (if pd =
n(/2)). This results in us getting strong diffraction only in certain directions (in the few cases
where pd = n).

The diffraction pattern of a single molecule will be too weak to detect in practice
In an ordered array however, the diffraction pattern will be bright enough to be detectable. It will be
the same as what was predicted for the single molecule, but only observed in particular directions
(made up of spots – constructive interference from adjacent molecules).

Solving a Structure

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Physical Biochemistry X-Ray Crystallography 3

2. Cryocool the crystal or mount in a capillary

3. Expose crystals to a beam of X-rays
4. Collect the diffraction pattern of diffracted X-rays
5. Reduce the data down to a list of diffraction spots and their intensities
6. Obtain phase information
7. Calculate the electron density distribution throughout the unit cell
8. Build the structure
9. Refine the structure
10. Repeat steps 8 and 9 until the structure is refined

Step 1 Grow protein crystals

Growing crystals is the hardest part of the experiment

Step 2 Preparing crystals for data collection

Crystals can be sealed in quartz capillaries with some stabilising liquid
This allows data collection at room temperature

Alternatively, crystals can be preserved at -173C

Crystals are manipulated and cooled in fibre loops (300 micron diameter)
To prevent ice formation, the crystal is first placed in a cryoprotectant solution
 e.g. 20% glycerol, low MW polyethylene glycol or sucrose
Rapid cooling then takes place in liquid nitrogen
Data is collected at -173C
A lot of data can be collected as the low temperature slows the deterioration of the crystal

Step 3 Mount crystal for data collection

Crystal is placed on a goniometer

Step 4 Test crystal for diffraction

X-rays are generated from in-house X-ray generators
 Copper anode is bombarded with e- from a cathode, resulting in X-rays
 Mirrors and slits channel the X-rays into a focused monochromatic beam
or synchrotrons – most crystals do not defract well in-house, a more powerful X-ray source is
required
1. Electron beam produced and accelerated in linear accelerator
2. Electrons accelerated further in booster
3. Packets are allowed into storage ring
4. Magnets force electrons to change direction

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Physical Biochemistry X-Ray Crystallography 3

5. X-rays are emitted tangentially when electrons change direction in a magnetic field
6. X-ray beam hits crystal

Step 5 Data collection

Rotate crystal by 1 while exposing it to X-rays
Measure intensity of spot on detector
Close shutter
Erase image plate
Repeat to 180
The intensity of diffraction is recorded as an image
on a computer

Backstop (made of lead) behind crystal prevents un-diffracted X-rays from going straight through
the crystal and into the film. This leaves a white dot, known as the backstop shadow.

Depending on the symmetry & space group, it is only required to collect certain wedges of data.
For a high symmetry space group like hexagonal, only 60 data is required

The spots on the film relate to the constructively diffracted X-rays that have come from planes of
atoms / molecules within the crystal lattice.
The diffraction spots have a regular array
There is a difference in intensity between the spots
These differences in intensity provide information about the contents of the unit cell

The diffraction pattern can allow determination of the resolution of the data.
This corresponds to the interplanar spacing
 The distance at which two points in space can be resolved
The data furthest from the backstop shadow has the highest resolution

Low res data gives information about planes separated by ~10 Å

High res data gives information about planes separated by ~2 Å
If there are no spots beyond 4 Å, there will not be enough data to solve the structure
Diffraction pattern gets weaker at higher angles (further out)
If the image has 3 spots, the crystal is a small molecule such as a salt

Step 5 Calculate number of molecules in asymmetric unit

Step 6 Data processing

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For each space group, you can calculate where spots should occur
Spots are indexed; h, k, l.
Intensity of each spot is imaged
Ih, k, l = ?
Intensity (I) is worked out by a computer
Spots that are known to be similar due to symmetry and Friedels Law (F h, k, l = F-h, -k, -l) can be
averaged
A list is obtained of all spots with their indices (h, k, l) and amplitude (F)

I h, k, l = |F h, k, l|2

From the spots…

 The unit cell parameters (a, b, c, , , ) can be determined
 Space group can be determined

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