Introcution to protein crystallography using x-ray and neutron diffraction Flashcards
Why do we use crystals instead of individual proteins for x-ray or neutron visualisation?
Because the scattered light would be too tiny to detect.
What are the problems you may encounter when generating protein crystals?
- Poor protein instability.
- Poor protein solubility.
- Poor protein expression in cells.
Why is it important to reduce salt concentraition in your protein solution?
Because the salt is more prone to crystalisation than proteins.
Explain the experimental setup of generating a protein crystal.
- Pure protein solution is mixed with precipitate (solution which is supposed to induce crystalisation).
- Place the drop on the lid of a reservoir (well), which contains precipitate.
- Incubate at 6-20 degrees.
When interpreting protein crystals, what do you need to keep in mind?
Keep in mind that the protein is outside of its physiological context as well as that the structure is a snapshot of its conformation.
There are three methods that can be used in order to achieve crystalisation, which?
- Vapour diffusion (drop from the lid of a reservoir). [precipitant in drop] is initially lower than [precipiate in reservoir], then, water gets evaporated from the drop, increasing the [precipitate].
- Batch. Mix protein and precipitants and hope for good circumstances.
- Dialysis. Dialysis membrane is used to allow fresh precipitate to reach the protein crystal nucleus over time.
What’s the idea behind a microseed stock?
You got a big protein crystal which you crush, then use for seeding other crystals.
How do you verify that your crystals are in fact protein crystals and not salt crystals?
Check absorption UV280nm.
You can prepare your crystals for data collection in room temperature or frozen. What are the pros and cons?
Pros RT
- No damage from cryopreservatives.
- The protein is visualized at conditions closer to physiological ones.
Pros Cryo
- Easy to do
- Easy to store
- Samples are protected from x-ray damaging.
What is a synchotron and how does it work?
A synchotron is a small particle accelerator. Electrons are accelerated, then, abruptly, the travelling direction is changed. This generates x-ray radiation.
Diffracted x-rays from the crystal which has been targeted are collected.
X-rays scatter of (1) ___.
Neutrons scatter of (2)_____.
(1) Electron clouds.
(2) The nucleus.
Is neutron crystallography as common as x-ray crystallography?
No. There’s 5 reactors.
Neutrons are gathered from the radiactive decay of U-235.
Is neutron crystallography as common as x-ray crystallography?
No. There’s 5 reactors.
Neutrons are gathered from the radiactive decay of U-235.
What can neutrons distinguish that electrons cant?
Isotopes. Ex: C-13, C-14.
Why is it beneficial to exchange hydrogen for deuterium during nuetron crystallography?
Neutrons don’t effectively visualize hydrogen. Deuterium is heavier, by visualising them, you get an idea of the electrostatic static / hydrogen bonds that are found in the protein.
