X-ray crystallography Flashcards
Why does the protein need to be in a crystal?
The signal gets amplified so it can be detected, and so it isn’t all scrambled from protein movement
What part of a protein will scatter x-rays?
Electrons
How long does the wavelength of radiation need to be in order for it to hit an object?
Comparable to or smaller than the object
Why do we use X-rays for crystallography?
The wavelength is about 1 angstrom. Bonds in a protein are 1 - 1.5 angstrom
What is the phase of a wave?
Where the wave starts relative to an origin
How do we focus the image produced by the scattered x-rays?
Computers
What image do we get from x-ray crystallography?
An electron density map
A single protein molecule only diffracts x-rays weakly. How do we get around that?
Crystallize the protein, so it forms a regular array and amplifies the signal
Does the crystal interfere with the X-rays?
Yes, it diffracts the waves and interferes with them
Why does the diffraction image only have a few spots?
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
What is each spot on the diffraction image?
A diffracted wave
What is the Phase Problem?
We don’t know the phase of the diffraction spots, and we need it to be able to focus the image with computers
What are two ways we determine the phase of the diffraction spots?
Perturb the structure in a predictable way and use that diffraction image
Guess if you know a very similar structure
How would we perturb the structure for determination of the phase of the diffraction spots?
Incorporate selenomethionine into the protein. Selenium has more electrons than sulfur and it messes with the diffraction
How do we guess the phases of diffraction spots?
If we have a similar structure, can compare the two diffraction patterns
What are the steps for doing x-ray crystallography?
Purify the protein Crystallize the protein Collection diffraction data Determine phases Compute electron density map Build protein model Refine model Validate model
Which step in x-ray crystallography is the hardest and most time-consuming?
Crystallizing the protein
What is protein crystallization?
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
What does a protein need to be in order to possibly crystallize?
Pure
Well-folded - no flexible parts hanging around
Fresh
What conditions would we vary to try and crystallize a protein?
pH, buffer composition, salt concentrations, precipitant, protein concentration, additives
Why do we need to freeze the crystals in liquid nitrogen before hitting them with the x-rays?
X-rays can damage the protein structure, and freezing the crystals will stop that
What do we need to do to the crystals before dunking them into liquid nitrogen?
Cryo protect them. Have glycerol in the water to prevent the water from expanding when it freezes
How do we validate our structure from the image that we get from crystallography?
Look at bond lengths and angles and calculate Ramachandran plots
What is resolution?
The level of detail on the electron density map, measured in angstroms
Does more diffraction spots farther away from the centre mean a high resolution or low resolution?
High
How do we get a high resolution?
Better ordered crystals and a shorter wavelength
How could we get a better resolution?
Get better crystals
Use a shorter wavelength
How could we get better crystals?
Try different conditions, test crystallization with different binding partners, try the protein with different parts cut out, try an ortholog
How could binding partners create better crystals?
Can stabilize the structure and cause better folding
How could we get a shorter wavelength?
Use a synchrotron facility
What are the pros of x-ray crystallography?
Detailed structures, a lot of automation now so can be done quickly, no size limit to the protein
What are the cons of x-ray crystallography?
Requires a crystal, structures are static and don’t show flexibility, can’t see hydrogens (but can usually deduce where they are)
What info on flexibility can we get from x-ray crystallography?
We know disordered parts are present even though we can’t see them, and can tell how many conformations a protein has
