How To Study Proteins By X-ray Crystallography Flashcards
0
Q
How do crystals form?
A
When protein molecules come out of solution in a consistent conformation and associate in an identical way with their neighboring molecules
1
Q
General workflow
A
- create expression plasmid that gives rise to protein of interest in soluble form
- purify soluble protein
- crystallize pure protein
- test whether crystals diffract
- put crystal in Xray beam and measure intensity if diffracted beams
2
Q
Conditions that crystal formation depends on
A
Precipitant Precipitant conc Protein conc PH Salt conc Special ions
3
Q
Braggs law
A
At one angle, waves interfere constructively and see bright spot
Another angle, waves interfere destructively and see nothing
4
Q
Methods of determining protein structure
A
- X-ray crystallography
- NMR spectroscopy
- electron microscopy
5
Q
Advantages of xray chromatography
A
- good accuracy and resolution
- higher MW proteins are no prob
- complexes and oligomers easily studied
6
Q
Weaknesses of X-ray crystallography
A
- requires a crystalline protein
- need to solve phase problem
- dynamic features not studied
- folding intermediates not identified
7
Q
Disadvantages of NMR spectroscopy
A
Expensive
Need highly soluble proteins
