X Ray 1

Question 1

For x ray crystallography what does the protein sample need to be

Answer 1

Very pure

Question 2

What are the characteristics of the sample for XRD

How does if get into the crystal

Answer 2

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

Question 3

What interactions might be keeping the protien molecules together

Answer 3

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

Question 4

What happens if you crystallize an impure sample

Answer 4

Leads to improper/missing protein protein interactions

Disrupts the crystal structure

Inhibits the crystals growth past the unit cell

Question 5

What is a unit cell

Answer 5

A repeating unit in a crystal

Has the protein in a specific confirmation on a repeating unit

Question 6

What an example of crystallizaing membrane protiens

Answer 6

Use antibodies that interact with the protein to make crystallizing interactions

Question 7

What is special about the crystals of protien that form ion channels

What do we need to do to keep it good

Answer 7

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

Question 8

Why do we need crystals

Answer 8

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

Question 9

What is the Vapor diffusion technique of making crystals

Answer 9

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

Question 10

What makes the vapour diffusion technique work

Answer 10

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 vapour diffuse from the droplet down to the bottom of the container

Question 11

What are the advantages to the Vapor diffusion technique

Answer 11

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

Question 12

What are the variables to consider in crystallization of a protein

Answer 12

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

Question 13

What are the two ways to judge a protein crystal

Answer 13

Through a microscope (visually)

Through the diffraction pattern

Question 14

What is the diffraction experiment

Answer 14

The x rays direct onto a crystal are diffracted

During the experiment the crystal is rotated to collect all the data

Question 15

How is x ray diffraction complentary to NMR

Answer 15

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

Question 16

What are the two sources of x rays

Answer 16

Rotating anode x ray generator

Synchrotron

Question 17

How does a rotating anode x ray generator work

Answer 17

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

Question 18

How does a synchotron work

Answer 18

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

Question 19

What are the advantages of a synchrotron

Answer 19

Gives high intensity x rays

It’s tuneable (you can set the x ray wavelength)

Question 20

Why do we collect data for XRD at low temp?

Answer 20

This is to reduce the decay because when x ray is exposed to crystal, there is some decay of the crystal

Question 21

How do we store and handle the crystals

Answer 21

Use a cryoprotectants to vitrify them (ex. Glycerol, protect the crystal)

Question 22

What does the detector in a diffraction experiment collect

Answer 22

The direct beam

And the diffracted beam from the crystal

Question 23

Can you detect anything in the middle of a diffraction pattern

Answer 23

No because this is the area where there is the direct beam which is blocked by lead to protect the detector

Question 24

How many units is a diffraction detected composed of

What does this mean

Answer 24

4 units

We can’t measure in between the units when the whole thing comes together

Question 25

Why do we use a diffraction pattern in. XRD instead of just reconstructing the image

Answer 25

There’s no lens, so you can’t recombine the scattered light

So we use the computer to reconstruct the electron density