X-Ray Crystallography Flashcards
(18 cards)
Step 1 - XRC
grow protein crystals of greatest purity and highest conecentration
Step 2 - XRC
shoot x-rays at the crystals to give diffraction patterns that can determine electron density
Step 3 - XRC
create an electron density map.
Step 4 - XRC
atomic model of the proteins structure is curated using the electron density map.
Why are crystal used?
Crystal are easy to work with and shoot x rays at because proteins in their natural state are not still.
x-ray signals are more greatly amplified because a crystal is the same molecule arranged repeatedly that will all produce the same diffraction pattern.
Why are x-rays used?
the electromagnetic wave of an x-ray is small enough to be affected by electron clouds in the bonds of a protein.
How are the crystals formed?
precipitation
what is precipitation
molecules coming out of solution because the solvent is oversaturated buy solute.
what other factors help precipitation to occur other an protein concentration
- salt
- pH where pH = pI
- organic solvents
- temperature
What zone of concentration will produce the correct size crystals needed and why
The metastable zone. This zone is just above the solubility max and one is able to slowly increase the concentration of proteins to produce neat and appropriately sized crystals
what kind of crystals are produced in the precipitation zone
Amorphous aggregates from here because the concentration was increase too quickly
What kind of crystal are produced in the precipitation
Lots of very small crystal nuclei are produced which are not very useful for analysis.
What method can be used to slow increase the concentration of protein
Hanging drop method
What is the principle of the hanging drop method
Takes advantage of substances being hygroscopic. The droplet cotaining the protein is placed above a reservoir. The Reservoir is more hygroscopic than the drop and will pull water from it decreased the volume of the drop therefore concentrating it and a precipitate will form at the bottom of the drop.
What is the data collected after shooting x-rays at the crystals?
diffraction patterns in different orientations collected by a detector film.
what makes the electron density map relevant?
It makes sense chemically, physically and biologically in predicting the structure of a protein
what does red mean on an EDM
this shows discrepancies that need to removed
what does green mean on an EDM
This shows where density needs to be added. Shows what’s missing in the model that is most likely present in the real structure.
