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The diffraction patterns clearly show an overlap of two or more lattices, which either means you didn’t have a single crystal at the start or it was damaged during the flash-cooling process. PEG 400 is generally a good cryoprotectant at ≥20%, but I would recommend using at least 25% to be certain no ice formation damages the crystal lattice. The high mosaicity makes interpretation of the diffraction pattern open to debate. Some of the reflections look like they could originate from protein, since they indicate long distance repeats. Some double-spots suggest a cracked crystal with high mosaicity. It’s a difficult call for me, but I think the layer lines look roughly like an hour glass shape, which is characteristically seen in DNA patterns and arises from the double-helical structure. Again, the layer lines suggest at least 2 or 3 lattices. To pin down whether you’re really looking at double-helical DNA, you need to measure the real space distance suggested from layer lines (measure the equiv. resolution from the reciprocal space origin). I don’t have time to look it up now, but there should be tell-tale layer line repeats (if I recall correctly) at about 10 A and 34 A. You can look for some old papers by Francis Crick on helical theory (sorry I don’t have the reference at my fingertips). In summary, I would cautiously surmise that you have a protein (small round spot reflections), bound by DNA that is much more mobile (highly mosaic layer lines), and that both the protein and DNA exist in multiple (probably three) lattices, but, again, the distance repeats of the layer lines have to be measured to see if they are consistent with double-helical DNA. Try to get crystals that diffract better for more clarity. Flash cool in 25% PEG 400 (or try combination of PEG 400 and PEG 200), or vary the MPD concentration. Good luck. I hope this helps. -Daniel ____________________ [image: RnNZfQn2o2xpggJQqefCOervMbPIci5mujDPJnvl43kv6Rtxjyh5gHN_JKVzeU-aaGz3pePFgxfoAAtZJZNx8mveVTc-11j98EfuAJVcumUenA=s0-d-e1-ft.gif]Daniel M. Himmel, Ph. D. URL: http://www.DanielMHimmel.com <http://www.danielmhimmel.com/> On Mon, Mar 12, 2018 at 11:24 AM, Joseph Ho <[log in to unmask]> wrote: > Dear Xiao and Hans: > > Thanks for your reply. We tried to index it but failed. > > Joseph > On Mon, Mar 12, 2018 at 11:15 PM, Xiao Lei <[log in to unmask]> wrote: > > did you try to index it? the cell dimensions may give you hint. > > > > On Mon, Mar 12, 2018 at 4:40 AM, Joseph Ho <[log in to unmask]> > wrote: > >> > >> Dear all: > >> > >> > >> I would like to seek your wisdom on our latest diffraction pattern. We > >> have been working on protein/DNA complex. The protein and DNA have > >> similar MW. By binding assay, we know the minimal length of DNA. (The > >> Kd is 0.1-1 microMolar and we can see the complex formation in size > >> exclusion chromatography up to 200mM NaCl but also some unbound form) > >> After trying different length of DNA, we recently obtained many > >> crystal hits (the percipient is either PEG400 or MPD). The final ratio > >> (prior to protein crystallization) between protein and DNA is 1:1.6 > >> considering some loss of protein during concentration. The crystal is > >> birefringent. Since high conc. of PEG400 (MPD), the crystals were > >> directly frozen in liquid N2. However, crystals only diffract to 8-10 > >> angstrom (anisotropic) and also weird striking line are present > >> (please see attachment). Do you think if it is DNA alone crystal or > >> protein/DNA complex crystal? > >> How should I improve the diffraction quality? > >> > >> > >> > >> PS. We have done some tests. For example, set up the same conditions > >> with DNA alone. I also tried to dissolve crystals in Bradford assay > >> solution and I believe I saw some blueish color. But none of these > >> tests are conclusive. > >> > >> Thanks for your suggestion. > >> > >> Joseph > > > > >