Dear Clare et al:
The absolute classic paper in the field is this:
Schultz, S. C., Shields, G. C. & Steitz, T. A. (1990).
Crystallization of Escherichia coli catabolite gene
activator protein with its DNA binding site. J. Mol.
Biol. 213, 159–166.
I've learned more from this than from reading probably any other
single crystallization paper.
It describes many of the things you ask about, especially trying
different lengths/ends.
(Last time I had heard from Steve Schultz he had ditched the rat race
and was teaching on the Navajo res. He is my hero.)
Bill
On Jul 16, 2009, at 7:09 AM, clare stevenson (JIC) wrote:
> I was hoping people could give some tips on the best way to go about
> crystallizing a protein-DNA complex.
>
> I have a large amount of experience in protein crystallisation but
> have
> never tried co-crystallisation with DNA until I started this project.
> If you want to reply to me personally I will then post a summary.
>
> My protein is a dimer and has been shown by several methods to bind to
> DNA with high affinity (KD ~ nM) with a footprint of ~26 bp. I have
> several questions:
>
> 1. Do people routinely try different lengths of DNA?
> 2. Do you start with blunt or sticky ends?
> 3. Would purification of the resultant complex by gel filtration be
> a good idea as the interaction is so tight?
> 4. Which screens would you try first?
> 5. Where do you order the DNA from as there is a large difference
> in price depending on supplier. What scale do you go for and what
> purification?
> 6. We expect 1:1 binding. What ratios of DNA to protein are
> generally used (bearing in mind the inaccuracies of protein
> estimation)?
>
> 7. Any other useful tips?
>
> Thanks in advance for the suggestions and advice
>
> Clare
>
> Dr. Clare E. M. Stevenson
> John Innes Centre,
> Department Biological Chemistry
> Colney Lane
> Norwich
> Norfolk
> NR4 7UH
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