X-ray diffraction and crystals

Question 1

What crystallising agents can be used to grow crystals

Answer 1

1. Salts
   - e.g. Sulphates, phosphates
   - Long chain organic polymers
   - Polyethylene glycols (PEG)
2. Organic solvents
   - Generally hydrophilic alcohols, ethers or ketones
   - e.g. Methyl-pentanediol, isopropanol

Question 2

What are the three methods to precipitate a protein

Answer 2

1. High salt
2. Organic solvents
3. Long chain organic polymers

Question 3

Describe the high salt method to precipitate a protein

Answer 3

1. The salt ions order water molecules around them, leaving less unstructured water to solubilize the protein

Question 4

Describe the organic solvent method to precipitate a protein

Answer 4

1. These effectively dilute water with a less polar, less H- bond capable solvent with lower dielectric etc.

Question 5

Describe the long chain organic polymer method to precipitate a protein

Answer 5

1. PEG prefers to writhe over a large volume of space

2. Taking the protein out of solution frees up more space for PEG and is energetically favoured

Question 6

What is PEG

Answer 6

1. A major role of PEG in the nucleation/crystallization process is to repress the formation of salt-induced, disordered aggregates

Question 7

What other factors influence crystallisation

Answer 7

1. Protein concentration- Need less precipitant to precipitate the more concentrated the protein
2. pH- Changing the pH adds/removes protons from individual residues, possibly creating new salt bridges/H-bonds
3. Temperature- As temperature changes, so do the enthalpic and entropic contributions to DGcrystallization
4. Presence of ligands- Ligands may lock the protein into one conformation, which can help crystallization

Question 8

What is vapour diffusion

Answer 8

1. Slowly increases protein and precipitant concentrations
2. 12 h to 4 days to equilibrate
3. Mix protein solution with precipitant solution (1:1) and equilibrate against excess of the latter
4. Need 1 µL of 10 mg/ml protein solution per experiment (well)

Question 9

What are typical crystallisation procedures

Answer 9

1. Screening
2. Start with commercial screening kits derived from extensive practical experience; there are hundreds mixtures covering wide range of conditions
3. Optimization
4. Once a lead condition is found from the screening process, expansion (pH and concentration of precipitant etc.) will be carried out

Question 10

Describe crystal screening

Answer 10

1. Combinations of precipitating agents and factors that might lead to a crystal is near infinite
2. A typical protein will only crystallize in a small fraction of these conditions
3. When screening you look for crystal leads
4. Anything that appears crystalline- Unlikely to get big, picture perfect crystals
5. Not all proteins crystallize!
6. Often you have to go back, purify your protein further, make a new construct…

Question 11

How can you improve the size and diffraction of crystal

Answer 11

1. Systematic variation of all concentrations and pH
2. Additive screens and detergent screens
3. Temperature
4. Seeding with crushed crystals (micro seeding)
5. Dialysis, batch, sitting drop
6. check old set-ups for different crystal form

Question 12

What are properties of protein crystals

Answer 12

1. Protein crystals are about half water, very fragile and grown from super-saturated solutions

Question 13

What should you do if no crystals are produced

Answer 13

1. Check purity and stability
2. Remove cysteins and other trouble makers
3. Remove flexible parts
4. Try single domains
5. Try physiologically relevant complexes
6. obtaining diffraction quality crystals is normally rate limiting

Question 14

Why do we use x-rays

Answer 14

1. When a coherent, monochromatic (focused) beam of X-rays is fired at a crystal, it diffracts from the electrons in all directions
2. Many of these diffracted waves cancel each other out as they are out of phase
3. In some cases they are in phase and a ‘reflection’ can be detected.

Question 15

What is Bragg’s law

Answer 15

1. Planes in a crystal are separated by distance (d)
2. Incident beam meets the plane at angle q
3. Diffraction spots are called reflections, because crystal is composed of lots of “mirrors” that reflect the X-rays
4. When light (in our case X-rays) is reflected from a mirror, the angle of incidence is equal to the angle of reflection- a lot of calculations look at notes
5. The goal of diffraction experiments is to enable constructive diffraction
6. Reflections only occur at specific values of theta (whole number values of n)
7. For any angle of incidence of the beam, only a subset of planes meet the Bragg law conditions
8. Crystal must be rotated to vary the angle of incidence of the beam
9. As each system of planes reaches its Bragg angle, a reflection is recorded at a corresponding point in a detection plane (on a detector, used to capture the reflection)

Question 16

What is diffraction resolution

Answer 16

1. d is resolution – a important parameter
2. How fine and how much detail we can see in the determined structure
3. The smallest spacing that will be resolved
4. Measured in Å
5. Although d is a variable in Bragg’s equation, in reality it is dictated by the crystal

Question 17

Is the distance between the crystal and detector important

Answer 17

1. Distance between crystal and detector can be readily changed by moving detector
2. Capturing diffractions need to know
   - Distance between crystal and detector
   - Size of the detector

Question 18

When do we see a reflection

Answer 18

1. We see ‘in-phase’ diffraction from sets of planes at the angle of reflection
2. and we only see that reflection/spot if the detector is in the right place to catch it
3. At the angle of reflection, ‘individual waves’ are traveling distances which allow the waves to line up
4. Depending on the spacing of the points/planes, will depend on the difference in distance travelled by any 2 ‘waves’.
5. If this distance equals a whole number of wavelengths, the waves can recombine in phase again as they leave the crystal.

Question 19

What does a diffraction spot of a crystal tell you

Answer 19

1. What set of planes it came from by its angle (relative to the crystal position/centre of image)
2. For a salt crystal with atoms mainly on the lattice points, this is most of a diffraction pattern
3. For a protein, we know that the lattice points do not represent the full structure
4. But if we see ‘reflections/spots’ resulting from sets of planes, then this sums the scattering of many bits of the protein
5. This size of the spot is directly related to the amount of matter on that set of planes
6. Can tell how much matter there is on that set of planes by its magnitude/intensity

Question 20

How can you find information about the rest of the structure- not just unit cell edges

Answer 20

1. The repetitive nature of crystals means that this ‘in phase’ diffraction occurs for many sets of planes at all different angles
2. To record all of these ‘reflection/spots’ all we need to do is rotate the crystal such that they can hit the detector.

Question 21

What can we determine by collecting all the ‘reflections/spots’ available for our crystal

Answer 21

1. The shape of the lattice from the position of the spots on the detector (relating to the sets of planes)
2. The amount of matter on those planes from the intensity

Question 22

How do we determine the structure from a bunch of spots?

Answer 22

1. Need to sum up what we know about the ‘amount of matter’ and how it interacts with planes.
2. Planes divide the unit cell at many angles, so most electrons will exist on more than one set of planes in the unit cell
3. therefore each electron will contribute to the intensity of many of the spots…and many spots tell you about the matter at any one place in the crystal!
4. There is a mathematical operation called Fourier analysis which can reversibly interchange this ‘data separated into component bits’ and the ‘bigger picture’

Question 23

What is Fourier analysis

Answer 23

1. Fourier analysis is a reversible process that can swap between the wave describing our electron density and the intensity of spots diffracted from planes
2. The diffraction experiment filters our electron density into contributions from different sets of planes
3. To create our electron density map we need to add together and combine our ingredients into a ‘smooth mix’
4. E.g. different fruit purees add up to make smoothie