Experimental

Crystallization

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Protein-DNA Complex

In order to achieve a crystalline structure, we must combine the protein and matching DNA to form a Protein-DNA Complex. This process involves finding the best ratio of protein to DNA to add together and incubating the mix.

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A dimer bound to a DNA duplex forming a protein-DNA complex.

Screening

With the DNA bound by the protein, we can now set up crystallization trials. We use a system called screening to find the best conditions for crystal growth.

The crystal plates that we screen with use a method called Sitting-Drop Vapor Diffusion crystallization. For a thorough explanation of this process, please visit Hampton Research.

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Image of one well in the crystallization plate. (Hampton Research)

Basically, the solution is very concentrated in the top well and it wants to be in equilibrium with the giant puddle of solution in the reservoir. The water will slowly diffuse into the drop in the well until there is equilibrium between the solutions. At various points in this equilibrium process, the protein-DNA complexes floating in solution will order into a crystalline structure like the one shown below.

Our Process

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Example of screening conditions

How does the crystal grow?

There are many factors you must consider. Some include, the salt concentration, the pH, and the drop to reservoir ratio.

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A protein crystal grown in the Snow Lab.

X-ray diffraction

After the crystals are grown, we will shoot the crystals with X-rays to give a diffraction pattern. Through interpretation, the diffraction will give us atomic level detail of our scaffold.

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An example of the resulting pattern given from X-ray diffraction.


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