X-ray crystallography

Question 1

Why does the protein need to be in a crystal?

Answer 1

The signal gets amplified so it can be detected, and so it isn’t all scrambled from protein movement

Question 2

What part of a protein will scatter x-rays?

Answer 2

Electrons

Question 3

How long does the wavelength of radiation need to be in order for it to hit an object?

Answer 3

Comparable to or smaller than the object

Question 4

Why do we use X-rays for crystallography?

Answer 4

The wavelength is about 1 angstrom. Bonds in a protein are 1 - 1.5 angstrom

Question 5

What is the phase of a wave?

Answer 5

Where the wave starts relative to an origin

Question 6

How do we focus the image produced by the scattered x-rays?

Answer 6

Computers

Question 7

What image do we get from x-ray crystallography?

Answer 7

An electron density map

Question 8

A single protein molecule only diffracts x-rays weakly. How do we get around that?

Answer 8

Crystallize the protein, so it forms a regular array and amplifies the signal

Question 9

Does the crystal interfere with the X-rays?

Answer 9

Yes, it diffracts the waves and interferes with them

Question 10

Why does the diffraction image only have a few spots?

Answer 10

They are the places where the crystal isn’t interfering with the signal from the protein, so we see them and everything else gets interfered with

Question 11

What is each spot on the diffraction image?

Answer 11

A diffracted wave

Question 12

What is the Phase Problem?

Answer 12

We don’t know the phase of the diffraction spots, and we need it to be able to focus the image with computers

Question 13

What are two ways we determine the phase of the diffraction spots?

Answer 13

Perturb the structure in a predictable way and use that diffraction image
Guess if you know a very similar structure

Question 14

How would we perturb the structure for determination of the phase of the diffraction spots?

Answer 14

Incorporate selenomethionine into the protein. Selenium has more electrons than sulfur and it messes with the diffraction

Question 15

How do we guess the phases of diffraction spots?

Answer 15

If we have a similar structure, can compare the two diffraction patterns

Question 16

What are the steps for doing x-ray crystallography?

Answer 16

Purify the protein
Crystallize the protein
Collection diffraction data
Determine phases
Compute electron density map
Build protein model 
Refine model
Validate model

Question 17

Which step in x-ray crystallography is the hardest and most time-consuming?

Answer 17

Crystallizing the protein

Question 18

What is protein crystallization?

Answer 18

Driving the protein out of a supersaturated solution in just the right conditions so that it comes out as a crystal instead of a precipitate

Question 19

What does a protein need to be in order to possibly crystallize?

Answer 19

Pure
Well-folded - no flexible parts hanging around
Fresh

Question 20

What conditions would we vary to try and crystallize a protein?

Answer 20

pH, buffer composition, salt concentrations, precipitant, protein concentration, additives

Question 21

Why do we need to freeze the crystals in liquid nitrogen before hitting them with the x-rays?

Answer 21

X-rays can damage the protein structure, and freezing the crystals will stop that

Question 22

What do we need to do to the crystals before dunking them into liquid nitrogen?

Answer 22

Cryo protect them. Have glycerol in the water to prevent the water from expanding when it freezes

Question 23

How do we validate our structure from the image that we get from crystallography?

Answer 23

Look at bond lengths and angles and calculate Ramachandran plots

Question 24

What is resolution?

Answer 24

The level of detail on the electron density map, measured in angstroms

Question 25

Does more diffraction spots farther away from the centre mean a high resolution or low resolution?

Answer 25

High

Question 26

How do we get a high resolution?

Answer 26

Better ordered crystals and a shorter wavelength

Question 27

How could we get a better resolution?

Answer 27

Get better crystals

Use a shorter wavelength

Question 28

How could we get better crystals?

Answer 28

Try different conditions, test crystallization with different binding partners, try the protein with different parts cut out, try an ortholog

Question 29

How could binding partners create better crystals?

Answer 29

Can stabilize the structure and cause better folding

Question 30

How could we get a shorter wavelength?

Answer 30

Use a synchrotron facility

Question 31

What are the pros of x-ray crystallography?

Answer 31

Detailed structures, a lot of automation now so can be done quickly, no size limit to the protein

Question 32

What are the cons of x-ray crystallography?

Answer 32

Requires a crystal, structures are static and don’t show flexibility, can’t see hydrogens (but can usually deduce where they are)

Question 33

What info on flexibility can we get from x-ray crystallography?

Answer 33

We know disordered parts are present even though we can’t see them, and can tell how many conformations a protein has