Lecture 2: X-ray Crystallography

Question 1

What are the sources of radiation (x-rays, neutrons and electrons)?

Answer 1

The radiation which is used must correspond to the structural features of the macromolecule one is looking at.
X-rays: are the main form of radiation which is used. They can be created in a number of ways.
• Electron bombardment of metal: This is used in the lab. Copper can create x-rays with a 0.15 nm wavelength.
• Synchrotron: Intense narrow beam with a variable wavelength. It uses electron acceleration by magnets. They produce intense radiation.
• XFEL: electron acceleration in a free-electron laser.
Neutrons: use a nuclear reactor.
Electrons: Used in an electron experiment.

Question 2

What kind of samples can we look at?

Answer 2

The sample must be periodic so either a fibre or a crystal.
• Fibres: Fibres must have periodicity in the z-direction. This can occur naturally (DNA or collagen) or induced (shear or magnetic fields).
• Crystals: crystals must be well-ordered. It is not possible for all molecules.

Question 3

How are crystals grown?

Answer 3

Crystals must be grown to a certain size. This varies on the type of x-ray source.
• Synchrotron = around 20 micromolar
• Microfocus beamlines = around 2 micromolar.
• XFEL = sub micromolar
Crystal growth is long and tedious. The protein sample is purified, homogenised and placed in a solution.
• The crystal is in a droplet of around 200 nL. It is placed either as a sitting drop or a hanging drop. It is in a container with another solution, which is known as the mother liquor which is 10 mg/mL.
• Water moves from the droplet to the mother liquor. The protein concentration rises until it reaches the nucleation zone and small crystals form. The protein concentration then drops again as the protein moves out of solution to form crystals when it is in metastable zone.
• Multiple wells are set up using robotics and a number of temperatures, pHs, salt concentrations etc are tested to see which will work best.

Question 4

How do we collect data?

Answer 4

The sample is protected from radiation during data collection by a stream of nitrogen gas. It is rotated by a device called a goniometer. The diffraction pattern is formed over the course of 1-5 minutes.

Question 5

How do scattering and diffraction patterns work?

Answer 5

• Scattering intensity varies with angle due to interference from different regions. Information is coded in the x and y axes of the detector.
• If particles are unordered then all intensities are averaged out.
• However, if there is an ordered array, then info can be retained. Intensities are similar to the independent particle but seen through a grate.
• Diffraction arises from scattering and interference between scattered waves.
• You get a sharper pattern of spots from more slits. The closer the spacing of the slits, the further the apart the diffraction spots (reciprocal relationship).
• The crystal behaves like thousands of slits in 3D. The h,k, l coordinates in reciprocal space relates to x, y, z in real space.