Hi Venkat, 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?
Only experience can tell. You should try, if possible, to crystallize both constructs with and without the extra 20 amino acids. It is important to make sure that you try at least the option with 20 amino acids removed. They might interfere, for example, in DNA binding. 2. Its homologous protein shows good affinity for 31-mer. Shall I use same length of DNA for co- crystallization. Yes. But... you really want to have some reliable data for YOUR protein. Crystallization of protein-DNA complexes is a little more tricky because you have to decide not only how to crystallize it, but also which DNA sequence you want and how long the sequence should be. If you can do some binding experiments, that would really help. Determine which sequence binds best (if any, it is advantageous if you have sequence specificity) and what the optimal DNA length is. Alternatively, do some protection experiments: with which sequences can you see DNA digestion in the presence of your protein? What is the protein-protected sequence? 3. What is the length of DNA to be used? If you have experimental information, you try (example when 31 is best:) 30, 31, 32, 33, maybe 34. The additional DNA base pairs that will stick out of your protein may be important for crystallization and you will want to screen multiple lengths and vary the base pairs that stick out. Note that you can (in principle) have a non-blunt DNA sequences, i.e. one strand longer than the other. You should try that too. 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. My experience is that you will want HPLC grade. 3. Any other suggestions on Protein DNA co- crystallization. Characterize your protein and protein-DNA complex carefully before you try to crystallize. There are many variables and any information you can get from other experiments can help you in your choice how to go about crystallization of the complex. Note: if your protein is an enzyme, you may want to consider deactivating the enzyme. For example, many enzymes require a Mg co-factor. If you replace Mg with Ca, your complex may be more stable/robust and the crystallization may work better. Hope this helps. Mark -----Original Message----- From: venkatareddy dadireddy <venkatda...@gmail.com> To: CCP4BB <CCP4BB@JISCMAIL.AC.UK> Sent: Fri, Jan 3, 2014 4:26 am Subject: [ccp4bb] DNA Protein co- Crystallization 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
