X-ray diffraction and crystals Flashcards
1
Q
What crystallising agents can be used to grow crystals
A
- Salts
- e.g. Sulphates, phosphates
- Long chain organic polymers
- Polyethylene glycols (PEG) - Organic solvents
- Generally hydrophilic alcohols, ethers or ketones
- e.g. Methyl-pentanediol, isopropanol
2
Q
What are the three methods to precipitate a protein
A
- High salt
- Organic solvents
- Long chain organic polymers
3
Q
Describe the high salt method to precipitate a protein
A
- The salt ions order water molecules around them, leaving less unstructured water to solubilize the protein
4
Q
Describe the organic solvent method to precipitate a protein
A
- These effectively dilute water with a less polar, less H- bond capable solvent with lower dielectric etc.
5
Q
Describe the long chain organic polymer method to precipitate a protein
A
- 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
6
Q
What is PEG
A
- A major role of PEG in the nucleation/crystallization process is to repress the formation of salt-induced, disordered aggregates
7
Q
What other factors influence crystallisation
A
- Protein concentration- Need less precipitant to precipitate the more concentrated the protein
- pH- Changing the pH adds/removes protons from individual residues, possibly creating new salt bridges/H-bonds
- Temperature- As temperature changes, so do the enthalpic and entropic contributions to DGcrystallization
- Presence of ligands- Ligands may lock the protein into one conformation, which can help crystallization
8
Q
What is vapour diffusion
A
- Slowly increases protein and precipitant concentrations
- 12 h to 4 days to equilibrate
- Mix protein solution with precipitant solution (1:1) and equilibrate against excess of the latter
- Need 1 µL of 10 mg/ml protein solution per experiment (well)
9
Q
What are typical crystallisation procedures
A
- Screening
- Start with commercial screening kits derived from extensive practical experience; there are hundreds mixtures covering wide range of conditions
- Optimization
- Once a lead condition is found from the screening process, expansion (pH and concentration of precipitant etc.) will be carried out
10
Q
Describe crystal screening
A
- Combinations of precipitating agents and factors that might lead to a crystal is near infinite
- A typical protein will only crystallize in a small fraction of these conditions
- When screening you look for crystal leads
- Anything that appears crystalline- Unlikely to get big, picture perfect crystals
- Not all proteins crystallize!
- Often you have to go back, purify your protein further, make a new construct…
11
Q
How can you improve the size and diffraction of crystal
A
- Systematic variation of all concentrations and pH
- Additive screens and detergent screens
- Temperature
- Seeding with crushed crystals (micro seeding)
- Dialysis, batch, sitting drop
- check old set-ups for different crystal form
12
Q
What are properties of protein crystals
A
- Protein crystals are about half water, very fragile and grown from super-saturated solutions
13
Q
What should you do if no crystals are produced
A
- Check purity and stability
- Remove cysteins and other trouble makers
- Remove flexible parts
- Try single domains
- Try physiologically relevant complexes
- obtaining diffraction quality crystals is normally rate limiting
14
Q
Why do we use x-rays
A
- When a coherent, monochromatic (focused) beam of X-rays is fired at a crystal, it diffracts from the electrons in all directions
- Many of these diffracted waves cancel each other out as they are out of phase
- In some cases they are in phase and a ‘reflection’ can be detected.
15
Q
What is Bragg’s law
A
- Planes in a crystal are separated by distance (d)
- Incident beam meets the plane at angle q
- Diffraction spots are called reflections, because crystal is composed of lots of “mirrors” that reflect the X-rays
- 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
- The goal of diffraction experiments is to enable constructive diffraction
- Reflections only occur at specific values of theta (whole number values of n)
- For any angle of incidence of the beam, only a subset of planes meet the Bragg law conditions
- Crystal must be rotated to vary the angle of incidence of the beam
- 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)
16
Q
What is diffraction resolution
A
- d is resolution – a important parameter
- How fine and how much detail we can see in the determined structure
- The smallest spacing that will be resolved
- Measured in Å
- Although d is a variable in Bragg’s equation, in reality it is dictated by the crystal
17
Q
Is the distance between the crystal and detector important
A
- Distance between crystal and detector can be readily changed by moving detector
- Capturing diffractions need to know
- Distance between crystal and detector
- Size of the detector
18
Q
When do we see a reflection
A
- We see ‘in-phase’ diffraction from sets of planes at the angle of reflection
- and we only see that reflection/spot if the detector is in the right place to catch it
- At the angle of reflection, ‘individual waves’ are traveling distances which allow the waves to line up
- Depending on the spacing of the points/planes, will depend on the difference in distance travelled by any 2 ‘waves’.
- If this distance equals a whole number of wavelengths, the waves can recombine in phase again as they leave the crystal.
19
Q
What does a diffraction spot of a crystal tell you
A
- What set of planes it came from by its angle (relative to the crystal position/centre of image)
- For a salt crystal with atoms mainly on the lattice points, this is most of a diffraction pattern
- For a protein, we know that the lattice points do not represent the full structure
- But if we see ‘reflections/spots’ resulting from sets of planes, then this sums the scattering of many bits of the protein
- This size of the spot is directly related to the amount of matter on that set of planes
- Can tell how much matter there is on that set of planes by its magnitude/intensity
20
Q
How can you find information about the rest of the structure- not just unit cell edges
A
- The repetitive nature of crystals means that this ‘in phase’ diffraction occurs for many sets of planes at all different angles
- To record all of these ‘reflection/spots’ all we need to do is rotate the crystal such that they can hit the detector.
21
Q
What can we determine by collecting all the ‘reflections/spots’ available for our crystal
A
- The shape of the lattice from the position of the spots on the detector (relating to the sets of planes)
- The amount of matter on those planes from the intensity
22
Q
How do we determine the structure from a bunch of spots?
A
- Need to sum up what we know about the ‘amount of matter’ and how it interacts with planes.
- Planes divide the unit cell at many angles, so most electrons will exist on more than one set of planes in the unit cell
- 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!
- There is a mathematical operation called Fourier analysis which can reversibly interchange this ‘data separated into component bits’ and the ‘bigger picture’
23
Q
What is Fourier analysis
A
- Fourier analysis is a reversible process that can swap between the wave describing our electron density and the intensity of spots diffracted from planes
- The diffraction experiment filters our electron density into contributions from different sets of planes
- To create our electron density map we need to add together and combine our ingredients into a ‘smooth mix’
- E.g. different fruit purees add up to make smoothie
