[ccp4bb] Summary - [ccp4bb] tips on crystallizing a Protein-DNA complex
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
[ccp4bb] tips on crystallizing a Protein-DNA complex
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
Re: [ccp4bb] tips on crystallizing a Protein-DNA complex
Hi Clare, Two papers that might be worth checking out that address some of your questions - 1. 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 2. Cannone et al, Crystallization of bFGF-DNA aptamer complexes using a Sparse Matrix designed for proteinnucleic acid complexes Journal of Crystal Growth, 2001 232 (2001) 409417 cheers, Kushol Kushol Gupta, Ph.D. Mathilde Krim Fellow in Basic Biomedical Research Van Duyne Laboratory - Univ. of Pennsylvania School of Medicine BLOCKED::mailto:kgu...@stwing.upenn.edu kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 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
Re: [ccp4bb] tips on crystallizing a Protein-DNA complex
1. Do people routinely try different lengths of DNA? Yes, its usually the most important variable because the ends like to stack against each other to form a pseudo-continuous helix. 2. Do you start with blunt or sticky ends? Both. Plan on a dozen or so different duplexes to start (you can usually mix-n-match top bottom strands to get more variety at the ends). 3. Would purification of the resultant complex by gel filtration be a good idea as the interaction is so tight? Probably wouldn't hurt, but probably not necessary. You can screen a lot more DNAs if you start out with mix-n-pray. 4. Which screens would you try first? PEG/ion AND hand-made PEG vs. salt, +/- Mg++, with concentrations that suit our complexes' personalities. 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? We've had good luck with IDT. For screening, even for lengths in the low 30s, we just use the unpurified stuff. Its much faster and cheaper that way! Then try purifying the oligos that give you hits. Sometimes it matters, sometimes it doesn't. 6. We expect 1:1 binding. What ratios of DNA to protein are generally used (bearing in mind the inaccuracies of protein estimation)? Usually a bit of extra DNA. 7. Any other useful tips? Protein-DNA cocrystals are often (at least in my lab) fragile and a pain to freeze. Do at least some of your screens with enough glycerol around to act as a cryoprotectant, so that you won't have to do extra crack-inducing manipulations later. 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 Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] tips on crystallizing a Protein-DNA complex
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
