Hello,
If you want to promote "infinite" helix, you should go for overhangs
(sticky end) with compatible sequence. I try to explain better what I
have in mind (and I actually did).
example :
5'ATCCCTAAATCGGCGTGTGCT---3'
3'---GGATTTAGCCGCACACGATAG5'
Hoping that this results in something like :
5' ATCCCTAAATCGGCGTGTGCTATCCCTAAATCGGCGTGTGCTATCCCTAAATCGGCGTGTGCT---3'
3' ---GGATTTAGCCGCACACGATAGGGATTTAGCCGCACACGATAGGGATTTAGCCGCACACGATAG5'
With the blunt end, you may have the helix, or not, in my opinion your
are not really promoting the "infinite helix".
Nicolas
On 09/02/2024 10:59, careinaedgo...@yahoo.com wrote:
Thank you for this insight, Nicolas. It is very helpful.
Yes I have also had a soccer ball shaped crystal that does not
diffract as well as, and more recently, many plate like crystals but
they do not diffract either.
I do know I have both protein and DNA in my crystals but I do not
know, as you say, exactly what is forming the crystal contacts.
Just to be clear, do you say overhangs are helpful? Surely overhangs
won't promote an infinite helix? If one wants an infinite helix, would
the DNA not have to be blunt ended?
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On Thu, Feb 8, 2024 at 4:59 PM, Nicolas Foos
<nicf...@embl.fr> wrote:
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, careinaedgo...@yahoo.com
<mailto:careinaedgo...@yahoo.com> 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
<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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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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