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?
Sent from Yahoo Mail on Android 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 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. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 -- 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 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
