Hi Jhon, Here are a few hints for protein-DNA crystallization: 1.) Starting point for protein:DNA ratio is 1:1.2 for a tight binder. In your case it would be protein complex to DNA 1:1.2. I like to run size-exclusion chromatography (if applicable) to determine an optimal ratio with a slight excess of DNA. In a recent case, we reduced it to 1:1.05. However, it can be beneficiary to increase the DNA amount in order to keep the protein happy (increased solubility). Moreover, have a look at the biochemistry of your complex: What is the Kd or does it bind more than one DNA molecule or what kind of DNA (length/sequence) does it bind?
2.) Starting protein concentration for crystallization screening is 5-10 mg/ml. This is a rough estimate and it depends on solubility, availability, size and behavior in crystallization screening. 3.) I like to start with a broad crystallization screen like the Hampton Index. This depends on your personal preference. Most protein-DNA complexes were crystallized in PEG at a slightly acidic pH (around 6) using vapor-diffusion. 4.) From my experience, you can get protein-DNA crystals in hanging-drop or sitting-drop vapor-diffusion, microbatch under oil or counter-diffusion experiments. Did I forget something? This best is to decide which technique is the most practical for you, e.g. Do you have a crystallization robot available... 6.) Keep in mind that the choice of DNA - in particular the length - is critical for most protein-DNA crystallization trials. 5.) There are many good crystallization notes published and there is the classic book of Ducruix, A. & Giegé, R. (ed.), Crystallization of Nucleic Acids & Proteins, 1999 Good luck! christian Jhon Thomas wrote: > Hello all > > I am novice to this field and trying to crystallize a protein-protein > complex verses DNA i.e a ternary complex of protein complex and DNA, > which stochiometry is 2:!. > I am wondering > 1-what should be the DNA and protein ratio for the ternary complex like > this typically for binary complex it is taken as 1.2 :1. > > 2- what should be the minimum concentation of DNA in the > crystallization drop while protein-DNA complex (binary complex of > protein-DNA) crystallization. similary what care should be taken for the > ternary complex crystallisation. > > 3- What are the screen would be better to start the screening of > crystallisation of the complex. > > 4- Whether crystallisation under oil could be done for the complexes or > hanging drop would be better than the crystallisation under oil in this > concern. > > I would really apprecitae the suggestions. > > Thanks in advance > thomas -- _______________________________________________________________________ Dr. Christian Biertümpfel Laboratory of Molecular Biology NIDDK/National Institutes of Health phone: +1 301 402 4647 9000 Rockville Pike, Bldg. 5, Rm. B1-03 fax: +1 301 496 0201 Bethesda, MD 20892-0580 USA email: biertumpf...@niddk.nih.gov _______________________________________________________________________
