Hello Careina, In addition to all the good suggestions you already got, I can point to these very good references:
- Pryor EE Jr, Wozniak DJ & Hollis T (2012) Crystallization of Pseudomonas aeruginosa AmrZ protein: development of a comprehensive method for obtaining and optimization of protein-DNA crystals. Acta Crystallogr Sect F Struct Biol Cryst Commun 68: 985–993 https://doi.org/10.1107/S1744309112025316 - Hollis T (2007) Crystallization of protein-DNA complexes. Methods Mol Biol 363: 225–237 https://doi.org/10.1007/978-1-59745-209-0_11 - And these lecture notes also have very useful info: https://gradebuddy.com/doc/2627393/protein-dna-complexes/ (Now I can’t find the link on the author’s website, where I originally found it…) The 2012 paper presents a PEG-salt screen of 48 conditions rationally designed for protein-DNA complexes. It is commercially available (I can’t find the company selling it now), but also not difficult to prepare from scratch. Might be worth a try if the conditions in this screen differ significantly from your current best conditions. The 2007 review has very good advice on all the critical points specific to protein-DNA complexes, and suggestions for DNA construct design. If you are on a budget, and in any case if you want overhangs, you should buy single-stranded oligonucleotides and anneal them yourself. One way to minimize the number of oligos you need to buy is to design one top strand with the sequence you want, and three different bottom strands: on that will make blunt ends, one that will make 5’-overhangs and one that will make 3’-overhangs. Ideally you want to test the effect of overhangs, so you don’t want to change the base composition. The easy way is to remove one letter from the 3’-end and put it back at the other end (for the 5’-overhang) and vice versa (for the 3’-overhang). I don’t remember where I read this (likely in one of these references), but it seems that sticky ends with a 1-bp overhang and A/T pairing are better at promoting crystallization than sticky ends with longer overhangs and ending on a G/C pairing (somewhat counter intuitively). Another thing to optimize is the length of the DNA. You might be constrained by other factors, mainly the length of the motif the protein binds to. But also consider that, when looking at all crystal structures of protein-DNA complexes in the PDB, it is apparent that certain DNA lengths are much more common, with 12 and 16 bp largely over-represented, presumably because different lengths crystallize less easily (or because people solving such structures don’t deposit them…). See the histogram of number of PDB entries by DNA length here: https://guillawme.github.io/insights-from-the-pdb/dna-length-in-protein-dna-complexes.html#0-150_bp_range This bias in DNA length is even more pronounced in crystals of free DNA, with 6, 8, 10 and 12 bp vastly over-represented: https://guillawme.github.io/insights-from-the-pdb/free-dna.html#DNA_length_in_crystal_structures_of_free_DNA So, for free DNA, an even number of base pairs and somewhere around an integer number of DNA turns (~10 bp) seem most favored for crystallization. Once you add a protein, you have to take into account the length of the binding motif, and if you decide to design an oligo with two binding motifs then you also need to think about the spacing between the two (if it’s not constrained by the binding mode of the protein) because this spacing will also of course affect the relative rotation between the two bound proteins around the DNA axis, which might affect packing. (I last updated these histograms in December, so this is relatively fresh data.) I hope this helps, Guillaume On 9 Feb 2024, at 13:09, Patrick Shaw Stewart <[email protected]<mailto:[email protected]>> wrote: 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 [email protected] <[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. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 -- [email protected] Douglas Instruments Ltd. 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