Dear Venkat, We recently solved a protein DNA complex structure and I had all the same queries as you have at the outset. We decided to use SPR as a techniques to study the interaction of the protein with the DNA and to use this to guide the choice of DNA for co-crystallisation. The work has recently been published (Stevenson C. E., Assaad A., Chandra G., Le T. B., Greive S. J., Bibb M. J., Lawson D. M. (2013) Investigation of DNA sequence recognition by a streptomycete MarR family transcriptional regulator through surface plasmon resonance and X-ray crystallography. Nucleic Acids Research 41 7009-7022).
As with all crystallisation attempts there are many variables that can be screened but below I have tried to give you some suggestions: 1. The additional vector amino acids may have an effect on the crystallisation. Have you tested whether this protein construct binds to the DNA (I would use SPR but can be done using EMSA as well). In an ideal world I would try with and without the additional amino acids 2. The length of DNA used in the crystallisations is probably the most important variable. I would test to see whether this length shows good binding. I would test a range of different lengths of DNA. Again I did this using SPR and used the shortest oligo that gave good binding affinity. You may want to screen several DNA lengths with blunt and sticky ends 3. See 2. 4. Normal desalted oligos were ok for us 5. I used a slight excess of DNA in the crystallisations Hope that helps. Good luck Clare Dr Clare E.M. Stevenson John Innes Centre Norwich Research Park Colney Lane Norwich NR4 7UH Tel 01603 450734 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of venkatareddy dadireddy Sent: 03 January 2014 11:16 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] DNA Protein co- Crystallization Hi, I'm working on DNA binding protein, looking to co-crystallize protein- DNA complex and have no previous experience. Your suggestion would be very precious on the following queries. 1. My protein is 646 amino acid long and it exists as homodimer. It is also having around 20 amino acid extra sequence from vector. Will vector sequence affect crystallization? 2. Its homologous protein shows good affinity for 31-mer. Shall I use same length of DNA for co- crystallization. 3. What is the length of DNA to be used? 4. What is purity of oligos to be used? Is it HPLC pure or normal desalted ones. I have read on CCP4 mails for screening purpose normal oligos are fine. Please comment on that. 3. Any other suggestions on Protein DNA co- crystallization. Thanks venkat
