Acoot, Remember that when you crystallize something, you want to build a lattice of *the same thing*. So it is better to make a homogeneous complex (if it is not mandatory). This means that the stoichiometry of the protein and DNA has to be a constant (we used 1:1.05 with excess DNA because the protein alone would not crystallize at all) and also that the complex shape has to be as stable as possible. If you can get a DNA sequence with specificity, that would help because you are more sure that the complex is homogeneous.
These experiments are always expensive because the DNA cost is high. You are facing trials with multiple DNA sequences and I would guess that you are lucky if "multiple" equals three in the case the binding is well behaved (sequence specific), you have a good grasp on the sequence length (say +/- 1) and then you still have to decide what the best "end sequence" is. Frequently these ends make crystal contacts with something else (it is fashionable to think that you can build a pseudo-continuous DNA helix throughout the crystal by lining up subsequent molecules) but in the end it is not known what these contacts are until you solve the structure. Trial and error, as usual. Mark -----Original Message----- From: Acoot Brett <[email protected]> To: CCP4BB <[email protected]> Sent: Fri, Jan 3, 2014 9:23 pm Subject: Re: [ccp4bb] DNA Protein co- Crystallization Dear All, For the question, I think for a protein-DNA interaction, the protein may interact with any sequences of DNA, which will give a lot of combination of protein-DNA sequence for crystallization screening. Or do anyone regard to just try the crystallization of the protein-one specific sequence DNA fragment for the trial (for example the DNA sequence with the highest binding affinity)? In another word, does the easiness of the crystallization has relation with how strong the protein interacts with the DNA sequence? Acoot On Saturday, 4 January 2014 11:02 AM, rajakumara eerappa <[email protected]> wrote: My suggestions are 1 try complementary and non-complementary overhangs which can form Watson-crick and/ or Hoogstein base pairing. 2. If it binds self-complementary duplexes then try them also. 3. Peg conditions with slight acidic pH are more suitable. 4. Divalent cation salts (Ca, Mg, Mn) in crystallization. Wish you good luck Raj On Friday, January 3, 2014, venkatareddy dadireddy wrote: 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
