How To Study Proteins By X-ray Crystallography Flashcards Preview

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Flashcards in How To Study Proteins By X-ray Crystallography Deck (8):
0

General workflow

- 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

1

How do crystals form?

When protein molecules come out of solution in a consistent conformation and associate in an identical way with their neighboring molecules

2

Conditions that crystal formation depends on

Precipitant
Precipitant conc
Protein conc
PH
Salt conc
Special ions

3

Braggs law

At one angle, waves interfere constructively and see bright spot
Another angle, waves interfere destructively and see nothing

4

Methods of determining protein structure

- X-ray crystallography
- NMR spectroscopy
- electron microscopy

5

Advantages of xray chromatography

- good accuracy and resolution
- higher MW proteins are no prob
- complexes and oligomers easily studied

6

Weaknesses of X-ray crystallography

- requires a crystalline protein
- need to solve phase problem
- dynamic features not studied
- folding intermediates not identified

7

Disadvantages of NMR spectroscopy

Expensive
Need highly soluble proteins