Carina, to complement the techniques using sticky ends, etc., you can also use the "random" microseeding approach that I mentioned to Kavya, see below.
The great advantage in a project like yours, where you have a family of related constructs, is that you can use cross-seeding - that is, you can use crushed crystals of one construct to seed other target constructs. You can even mix several seed stocks together, although we always keep seed crystals grown in high-salt conditions separate from those grown in high-peg conditions. There are some very nice examples of cross-seeding and mixing seed stocks in this paper by Obmolova et al. Obmolova, G., Malia, T.J., Teplyakov, A., Sweet, R.W. and Gilliland, G.L., 2014. Protein crystallization with microseed matrix screening: application to human germline antibody Fabs. *Acta Crystallographica Section F: Structural Biology Communications*, *70*(8), pp.1107-1115. https://doi.org/10.1107/S2053230X14012552 More info https://www.douglas.co.uk/mms.htm Best wishes and good luck! Patrick _______________________ Hi Kavya 1. Make a seed stock from the globules or anything else that you think might be crystalline, and recreen. In other words, you should add your seed stock to *random screens* (not optimization experiments). There could be many conditions that are in the metastable zone of the phase diagram in your normal screens - this method can give you crystals in those conditions. If this works, you'll be in a better position anyway because you'll have more control - by diluting the seed stock, you can control the number of crystals per drop. References: D'Arcy, A., Villard, F. and Marsh, M., 2007. An automated microseed matrix-screening method for protein crystallization. *Acta Crystallographica Section D: Biological Crystallography*, *63*(4), pp.550-554. Shaw Stewart, P.D., Kolek, S.A., Briggs, R.A., Chayen, N.E. and Baldock, P.F., 2011. Random microseeding: a theoretical and practical exploration of seed stability and seeding techniques for successful protein crystallization. *Crystal Growth & Design*, *11*(8), pp.3432-3441. This is how we normally make the seed: https://www.douglas.co.uk/f_ftp1/rMMS_Procedure.pdf On Thu, Feb 8, 2024 at 11:26 AM careinaedgo...@yahoo.com < 000002531c126adf-dmarc-requ...@jiscmail.ac.uk> 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 > -- patr...@douglas.co.uk Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Patrick Shaw Stewart, Peter Baldock, Stefan Kolek http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 ######################################################################## 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/
