Re: [ccp4bb] tips on crystallizing a Protein-DNA complex

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: tips on crystallizing a Protein-DNA complex
From: "clare stevenson (JIC)" clare {- dot -} stevenson {- at -} BBSRC {- dot -} AC {- dot -} UK
Date: 2009-07-16

Next message:
Subject: Re: tips on crystallizing a Protein-DNA complex
From: Phoebe Rice price {- at -} UCHICAGO {- dot -} EDU
Date: 2009-07-16

Subject: Re: tips on crystallizing a Protein-DNA complex
From: Kushol Gupta kushol {- dot -} gupta {- at -} GMAIL {- dot -} COM
Date: 2009-07-16

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

cheers,

Kushol

Kushol Gupta, Ph.D.

Mathilde Krim Fellow in Basic Biomedical Research

Van Duyne Laboratory - Univ. of Pennsylvania School of Medicine

kgupta@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

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: tips on crystallizing a Protein-DNA complex
From: "clare stevenson (JIC)" clare {- dot -} stevenson {- at -} BBSRC {- dot -} AC {- dot -} UK
Date: 2009-07-16

Next message:
Subject: Re: tips on crystallizing a Protein-DNA complex
From: Phoebe Rice price {- at -} UCHICAGO {- dot -} EDU
Date: 2009-07-16