X Ray 1 Flashcards
For x ray crystallography what does the protein sample need to be
Very pure
What are the characteristics of the sample for XRD
How does if get into the crystal
Has to be pure and homogenous (meaning every molecule is folded the same)
The sample size is usually 10mg
The nucleus of a few molecules come together and others follow
What interactions might be keeping the protien molecules together
HB
The side chains of protien in aqueous solution interact with the water
Side chains can bind favourable with other side chains using HB and polar interaction
What happens if you crystallize an impure sample
Leads to improper/missing protein protein interactions
Disrupts the crystal structure
Inhibits the crystals growth past the unit cell
What is a unit cell
A repeating unit in a crystal
Has the protein in a specific confirmation on a repeating unit
What an example of crystallizaing membrane protiens
Use antibodies that interact with the protein to make crystallizing interactions
What is special about the crystals of protien that form ion channels
What do we need to do to keep it good
The crystal is 50% protien And 50% solvent
Different than regular salt/sugar crystals because this one is softer
Need to keep the crystal hydrated or else it can crack and break
Why do we need crystals
In a crystal form, the x ray gives more scattering than normal because of the repeating units
This gives a higher signal than if it wasn’t in crystal form
What is the Vapor diffusion technique of making crystals
You have your protein solution on a cover slip over top of a precpitant solution
Pipette an equal volume of the prcipitants solution onto the protein solution
Flip over the cover slip so that the solution can’t evaporate out now
What makes the vapour diffusion technique work
The protien solution has been diluted using the precipitant
But the precipitant has also been diluted by the protein solution
So in the droplet there is a lower concentration of precipitant
This makes the water vapour diffuse from the droplet down to the bottom of the container
Makes the concentration in the or protien in the drop high until supersaturation is reached so a crystal forms
What are the advantages to the Vapor diffusion technique
Can use small volumes
The vapour diffuses gradually over time
Some changes can be made ex. if the crystal isn’t formed can change the conditions like adding more precipitant to the well and try again
What are the variables to consider in crystallization of a protein
Concentration of the protien
Concentration and type of the precipitant
PH
Temp
Additives (like metal and small molceules that aid in the folding of the protein)
The method of crystallization
What are the two ways to judge a protein crystal
Through a microscope (visually)
Through the diffraction pattern
What is the diffraction experiment
The x rays direct onto a crystal are diffracted
During the experiment the crystal is rotated to collect all the data
How is x ray diffraction complentary to NMR
In NMR we use the spin states of the protons (so we can’t see the electrons since a proton has no electrons)
But in XRD, you can only see the electrons (no protons) because the diffraction is due to the scattering off the electrons
What are the two sources of x rays
Rotating anode x ray generator
Synchrotron
How does a rotating anode x ray generator work
Electrons hit the anode
The anode rotates to cool down
The collision of electrons with the anode knocks out electrons
The knocked out electron drops on energy, emitting an x ray
How does a synchotron work
The electrons are going around the ring
Whenever an electron goes around a bend, it gives off radiation (x ray)
Can select for a specific wavelength
What are the advantages of a synchrotron
Gives high intensity x rays
It’s tuneable (you can set the x ray wavelength)
Why do we collect data for XRD at low temp?
This is to reduce the decay because when x ray is exposed to crystal, there is some decay of the crystal
How do we store and handle the crystals
Use a cryoprotectants to vitrify them (ex. Glycerol, protect the crystal)
What does the detector in a diffraction experiment collect
The direct beam
And the diffracted beam from the crystal
Can you detect anything in the middle of a diffraction pattern
No because this is the area where there is the direct beam which is blocked by lead to protect the detector
How many units is a diffraction detected composed of
What does this mean
4 units
We can’t measure in between the units when the whole thing comes together
Why do we use a diffraction pattern in. XRD instead of just reconstructing the image
There’s no lens, so you can’t recombine the scattered light
So we use the computer to reconstruct the electron density
