The Link to X-Ray Protein Crystallography

A. Crystal Production

(wet lab)

Production Purification Crystallisation

B. Data Collection

(x-ray lab)

Mounting Shooting Detection

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C. Structure Solution

(computer lab)

Indexing Integration Scaling Phasing Building Refinement Validation Publication

Protein Production

Goal of this step is to get a lot of protein and soluble protein so that crystallisation trials can start. This sounds easier than it often is. Membrane proteins for example are very difficult to solubilse.

The protein for the structure solution, myoglobin, was extracted from the muscle of sperm whale. This worked because there was abundant material available to extract the protein from. The structures of scarce proteins can only be determined thanks to cloning, allowing the overexpression of the protein. Mostly, this is done by introducing a piece of circular DNA containing the gene of interest into Escherichia coli bacteria cells. There is a variety of other host, such as yeast cells or insect cells available. In other cases the only way to get hold of the protein is extracting it from its natural source.

Cloning a protein in a clever way can help the purification (through the addition of a His-tags for example), the solubilisation (through the addtition of a GST-tag for example) or the crystallisation (through chosing stable domains of the protein for example). By incorporating an unnatural amino acid (selenomethionine) during the production, the phase problem that will arise once diffraction data has been obtained can be solved through anomalous dispersion (SAD or MAD).

Protein expression vector and electrophoresis gel to visualise expression

LINKS

Proteincrystallography.org
The Wolfson Centre for Applied Structural Biology (Danieli & Doron)
P4EU (Protein Production and Purification Partnership in Europe) network initiative