Hello Careina,
In my hands, DNA protein complex crystals may be frustrating, because
often we get good looking crystals which don't diffract at all and are
actually not easy to improve.
I remember obtaining a lot of crystal looking a bit like "STOP" road
sign (octogonal shape for one axis) which never diffracts. (Often
containing only DNA not well organized)
So long story short, In my hand (transciption factor bound with
homeodomain for example). I had good results with DNA sequence which
results in hoverhangs. The idea was to bet on a "infinit" DNA helix
which should help the packing.
I strongly encouraged you to rely on any other information you can have
to be sure of what is the best minimal sequence (like band shift
assay). Also if you can purify the entire complex before crystallization
assay (I don't know your protocol, but ideally, I would prepare the
complex prot-DNA and put it on size exclusion).
The point is, you don't know /a priori /what kind of crystal packing you
will have. It may be only due to protein protein contact and not related
to the DNA directly.
Also, I often get good results with crystal growing condition containing
MPD or PEG (makes me using PEG screen Familly as first approach).
I invite you to read the Timothy Richmond teams Papers on nucleosome
they spend some times improving the resolution on very large complex.
(Luger etal 1997).
There is many parameters, DNA sequence also change a bit the DNA
geometry (look for A-tract), You may want to introduce such sequence to
maybe improve the "rigidity".
Also if your DNA fragment are small, be careful with the temperature.
The annealing and the DNA duplex formation is critical and you should be
careful on your procedure.
I remember that small cation like Li, may help too.
HTH
Nicolas
On 08/02/2024 12:25, [email protected] wrote:
Hello all.
I am struggling to get defracting crystals with a protein DNA complex.
The crystals are plentiful but they do not diffract. I am going back
to the grind stone and relookong at my DNA sequence.
Is there any wisdom you could give me with regards to what works best
with DNA in crystals?
From my reading it seems if the length is a multiple of 7 (for B DNA)
and blunt ended, it will stretch over the length of the crystal and
improve crystalisability. But if you want crystals that diffract
better, you will need to play with length and even making it only one
base longer or shorter can make a difference, even changing the
morphology of the crystal? Longer is better than shorter, and
overhangs are good for improving diffraction? Presumably because they
stabilize contacts? It is expensive to synthesize a while bunch of
sequences so I need to be strategic in my choice. Would appreciate any
advice.
Thank you
Careina.
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--
Nicolas Foos PhD - ARISE fellow
https://orcid.org/0000-0003-2331-8399
EMBL Grenoble, McCarthy Team
71 av. des Martyrs,
38000 Grenoble FRANCE
+33 4 57 42 84 67
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