3.2: X-ray Crystallography

Question 1

why aren’t x-ray lenses suited for imaging?

Answer 1

• refractive index for x-rays of most materials is close to 1.
  > x-ray lenses depend on diffraction
• not suited for high res imaging

Question 2

X-ray crystallography step by step

Answer 2

1. protein production
2. crystallisation
3. diffraction
4. structure determination

Question 3

X-ray crystallography diffraction method

Answer 3

1. monochromatic x-ray beamed through sample
2. block direct beam
3. record intensity of weaker diffracted beams with area detector
4. in silico translation of intensities into e- density map

Question 4

Bio macromolecular crystals arrangement and building blocks

Answer 4

• regular 3d arrangement
• built by translations of unit cells
  > may comprise of rotational elements
• smallest element representing both translations and rotations = asymmetric unit
  > used in PDB

Question 5

biomolecular crystal info

Answer 5

small - 20-500 microm
sensitive
50% solvent approx

Question 6

crystallisation: process, one example
and what is important to know about crystallisation

Answer 6

• crystals grown from supersaturated proteins
  > kinetically trapped, prevents decomposition
• can decompose into
  > precipitate, phase separation or crystals
• 2 step process
  > stochastic nucleation and crystal growth
• e.g. vapor diffusion crystallisation

important !
> not predictable for both when and conditions !

Question 7

crystallisation artifacts ?

Answer 7

typically grown under native conditions (neutral pH)
protein concentrations similar to inside cells
stabilised by weak forces

Question 8

Classic X-ray generation and detection

Answer 8

1. cathode ray tubes
   > thermally extracted electrons, photoabs. leads to emission of x-rays
2. rotating anode
   > heating problem overcome

detected by phosphorescent screens

Question 9

Modern X-ray generation, detection, collection times

Answer 9

using particle accelerators/synchrotrons
allows tunable wavelength using synchrotron effect

detected by: low temperature 100k in Nitrogen gas to reduce radiation damage

collection times: seconds to minutes

Question 10

X-ray interactions: which is relevant to X-ray crystallography, chance, aka

Answer 10

Elastic scattering at electrons, when E(scattered) = Ei
has 0.2% chance
aka rayleigh scattering

Question 11

single particle diffraction thought experiment

and why can’t it be done in reality

Answer 11

partial waves scattered at individual atoms travel different distances - path difference
> depends on scattering angle

interference of waves - intensity changes !!
> superposition all waves to generate pattern

not realistic
> intensity too low, radiation dmg, difficult to position particle

Question 12

grating experiment

Answer 12

only obtains maxima at certain points, constructive interference

Question 13

single crystal diffraction thought experiment

Answer 13

INTERmolecular interference
> equivalent to grating diffraction
> position of reflections
> provides unit cell parameters, orientation
due to regular nature of crystals, made of unit cells

INTRAmolecular interference
> intensity of reflections
> provides structural info

Question 14

Phase problem of crystallography

Answer 14

detectors are only sensitive for intensities
missing individual phases which contain info for e- density, which is not obtained

Question 15

Iterative Refinement

Answer 15

method to improve electron density calculation by indirectly calculating phases of reflections and refining it according to phase estimate and so on

iterate until convergence

Question 16

super resolutions

Answer 16

combination of prior knowledge and exp data results in super-resolution

Question 17

X-ray crystallography summary

Answer 17

Interaction (scattering) of X-rays on the electron layers of atoms

+: 3D structure with atomic resolution
-: crystallisation, hydrogen atoms not visible, crystal packing effects, requires X-ray source

> electron density in crystal, internal structure of crystal