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
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_______________________________________________________________________
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: [log in to unmask]
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