Dear All,

 

First I would like to thanks everyone who took the time to respond.  It never fails to amaze me what an awesome forum the ccp4bb board is and how willing people are to share their expertise!

 

Below is a summary of the replies I received

 

1.       Do people routinely try different lengths of DNA?

 

Yes, this seems to be the biggest variable

 

2.       Do you start with blunt or sticky ends?

 

The general consensus seems ideally to try both.  Sticky ends seems the best option to try first though with complementary ends 

 

3.       Would purification of the resultant complex by gel filtration be a good idea as the interaction is so tight?

 

Most replies seem to suggest just mixing protein and DNA together is just as likely to get good results

 

4.      Which screens would you try first?

In no particular order

Natrix, Nucleix, PEGS, Index, JCSG core, MPD screen, Peg ion screen, home made PEG salt screen (=/- Mg++)

 

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?

 

A wide range of suppliers were suggested.  Didn’t seem to be a great preference for one over another

Some people purify their own DNA, others order HPLC purified and some use un-purified for screening. 

 

6.      We expect 1:1 binding. What ratios of protein to DNA are generally used (bearing in mind the inaccuracies of protein estimation)?

 

1/1.2 seems to be the best option.  People incubate together and then dialyse prior to crystallisation

 

7.      Any other useful tips?

 

Protein DNA crystals can be very fragile so good idea to screen with cryoprotectant in the screen to save manipulations

Test crystals with a gel to see if DNA is present

Use of SAXS to get molecular envelop of complex

 

 

Several papers were suggested

 

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.

 

Tan et al, Crystallization of the Yeast MATa2/MCM1/DNA Ternary Complex: General Methods and Principles for Protein/DNA Cocrystallization J. Mol. Biol. (2000) 297, 947±959

 

Cannone et al, Crystallization of bFGF-DNA aptamer complexes using a

Sparse Matrix designed for protein–nucleic acid complexes Journal of Crystal Growth, 2001 232 (2001) 409–417

 

Book chapter 8 from Crystallization of nucleic acids and proteins: a practical approach.

Edited by A.Ducruix and R. Giege (Oxford University Press)

 

 

Thanks again for all your replies

 

Best wishes

 

Clare

 

 

Dr. Clare E. M. Stevenson

John Innes Centre,

Department Biological Chemistry

Colney Lane

Norwich

Norfolk

NR4 7UH