Following up on Prof. Rice's suggestions about trying different ends and reading the literature, you might have a look at
Crystallization of the yeast MATalpha2/MCM1/DNA ternary complex: general methods and principles for protein/DNA cocrystallization. J Mol Biol. 2000 Apr 7;297(4):947-59. PMID: 10736229 [PubMed - indexed for MEDLINE] Note, in particular, Table 1, which lists 82 oligos used for co-crystallization trials. Also see papers from Pabo's group, beginning with Systematic variation in DNA length yields highly ordered repressor-operator cocrystals. Science. 1985 Dec 20;230(4732):1383-5. PMID: 3906896 [PubMed - indexed for MEDLINE] Jack -- John J. Tanner Professor of Chemistry and Biochemistry University of Missouri-Columbia 125 Chemistry Building Columbia, MO 65211 Phone: 573-884-1280 Fax: 573-882-2754 Email: [EMAIL PROTECTED] http://www.chem.missouri.edu/TannerGroup/tanner.html > From: <[EMAIL PROTECTED]> > Reply-To: <[EMAIL PROTECTED]> > Date: Mon, 16 Jul 2007 11:26:42 -0500 > To: <CCP4BB@JISCMAIL.AC.UK> > Subject: Re: [ccp4bb] Protein-DNA complex for crystallization > > Dear Kumar, > One often has to try duplexes with many different > ends before getting decent crystals (e.g. 18 for > one project in my lab, even more for others). > Depending on your Kd, you might find that your > complex falls apart during gel filtration. > How long are your oligos? Gel purification > sometimes helps us and sometimes doesn't, but > longer ones (> 20-30nt) are more likely to benefit from it. > Did you check the concentrations yourself before > annealing? (my students get better results when they do). > Can your oligos hairpin? If you have a dimer > that binds a symmetric DNA site, having > individual monomers bound to hairpinned oligos > would certainly make a mess of your xtals. > If you think you have an excess of one single > strand, you could try annealing small amounts at > several different ratios, and check how much is > really duplex on a native agarose or acrylamide gel. > Have you checked your prep for nuclease > contamination? Just incubate some with a > supercoiled plasmid and ~10mM Mg++ for a couple > hours and see if the plasmid stays > supercoiled. This is a beautifully sensitive > assay because cleaving only 1 bond out of > thousands will change the plasmid's mobility - > but bear in mind it will only reveal endonucleases, not exonucleases. > Finally, its always a good idea to pull up a pile > of old papers and skim their methods sections for inspiration. > Good luck! > Phoebe Rice > > At 11:01 AM 7/16/2007, you wrote: >> Hi, >> I am trying to crystallize a protein-DNA >> complex. I purify the protein finally >> using gel filtration. I purchase >> single stranded complementary oligos (desalting from idtdna.com), mix them up >> and make DNA duplex by >> heating to 95 degree C and cooling to room >> temperature. I mix protein and DNA, >> concentrate and use it >> for crystallization. >> I am geting small crystals consistently under a specific condition. These >> crystals take up IZIT dye but are >> not well shaped. I am not able to improve the size and shape of the crystals >> substantially even after >> screening with additives (Hampton research). >> I suspect that purity of the duplex DNA (presence of unpaired oligos) is >> limiting the chances of obtaining >> better crystals. >> >> How can I purify the duplex DNA further? >> >> Are there better ways of making protein-DNA complex for crystallization? >> >> If I make the protein DNA complex and then do the gelfiltration, will the >> complex purified so be a better >> choice for crystallization? >> >> Thank you >> Kumar >> >> Dept. of Biochemistry, Cellular and Molecular Biology, >> Walters Life Science, # 406, >> University of Tennessee, TN, Knoxville, USA > > ------------------------------------------------------------------------------ > --------------------------------------------- > Phoebe A. Rice > Assoc. Prof., Dept. of Biochemistry & Molecular Biology > The University of Chicago > phone 773 834 1723 > fax 773 702 0439 > http://bmb.bsd.uchicago.edu/index.html > http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
