Dear CCP4
I wish to crystalise a transcription factor in complex with DNA. Are there any good papers you can recommend on protein-DNA crystallisation?
Also any helpful tips on protein:DNA ratios, protein concentrations or buffer conditions that have worked for any of you would be most helpful
Thanks
Careina
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1) Adjust DNA parameters before protein
DNA plays a critical role in crystallization since it is often the source of crystal contacts. Use a ratio for protein to DNA of 1:1.2-5
2) Start with 10-11 base pairs of DNA
Increase 1-2 bases at a time, usually stopping at 21 bp. The idea is that a helix forms every 10.5 base pairs and forms a pseudo-continuous helix.
3) Sticky ends
The sticky ends do not have to be complementary. Blunt ends can work, but this would not be one of the first variables to change.
4) Screening
Expect all the normal fun of protein screening. I would favor PEG or MPD in screens (Natrix) rather than high salt screens. The high salt can disrupt the charged interaction between the protein and DNA.
5) Run a gel
If you are unsure if you have protein-DNA complex formation
6) Concentrate your protein-DNA before crystallization trials
If precipitation is occurring, consider running DLS to help determine at what concentrations precipitation is occurring
References:
http://mcl1.ncifcrf.gov/nihxray/Tips-and-Tricks_Crystallization_Protein-DNA_updated.pdf
http://bstr521.biostr.washington.edu/PDF/2011%20Protein-DNA%20complexesV06.pdf
I hope that helps in getting you started, take care!
Sean Seaver
P212121
http://store.p212121.com/
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