Hi Phil, Thank you, I appreciate the input. We’ll try a few more things. However, it seems that also in P1 we don’t really see a break in the DNA density (it slowly spirals in the crystal packing, as the protein bends it in between two protein monomers). My thinking is that this can be explained by the “sliding” nature of the bining, since there is no sequence specificity here (it’s a poly-A). We of course know that the DNA is 60 bases long, but my guess is that the breaks between strands occur in different sites in the crystal and therefore gets averaged out in the observed density. Just don’t really know how to properly handle it in the modelling.
In principle the P212121 model captures the structure in its completeness, since the next stretch of DNA and 3 proteins can be reproduced by the ASU in that space group (even though I’m fully aware that in theory this ASU should be bigger to accommodate the 60 bases long DNA). I’ll think about it some more.. Best wishes, Ronnie ---------- Ronnie Berntsson, PhD Chair of the Young Academy of Sweden Department of Medical Biochemistry and Biophysics, Umeå University 90187 Umeå, Sweden e-mail: ronnie.bernts...@umu.se<mailto:ronnie.bernts...@umu.se> phone: +46 90 7865235 web: https://www.biostruct.umu.se/principal-investigators/ronnie-berntsson/ From: Phil Jeffrey <pjeff...@princeton.edu> Date: Monday, 31 July 2023 at 17:10 To: Ronnie Berntsson <ronnie.bernts...@umu.se> Subject: Re: [ccp4bb] Connecting DNA between asymmetric units? Hi Ronnie Well, technically, your asymmetric unit is only an approximation, since your true asymmetric unit would hold one entire DNA duplex. One could argue that you've misindexed, you've got pseudo-centering, and you should test a 4x/16x/64x larger cell to see if you can see the ends of the DNA. You should go back to the original diffraction images and see if there are any non-integrated spots between the indexed spots with your current/smaller cell. If there are, this might be a priori experimental evidence that your true cell is larger than your indexed cell. These spots might be weak (because your smaller/current unit cell is an ok approximation) but if they are there then you should treat the cell differently. If they're not there, then your DNA is disordered in the formal sense, and you should probably come up with a model that encapsulates that disorder with multiple DNA sequences overlapping in space, which then shows up in the PDB file. If your 60 bp DNA folds into 4 (?) asymmetric units, even without bending, I've got to thing that there is evidence of multi-conformation visible somewhere, which you should then model. You'll still need some explanatory text in the header - there's no single model in the smaller unit cell that can convey what you think is going on in this case. Cheers Phil Princeton On 7/31/23 10:12 AM, Ronnie Berntsson wrote: > Hi all, > > We have recently solved a structure of a DNA binding protein in complex > with DNA to ca 2.5 Å. The space group is P212121 and contains 3 protein > molecules in the asymmetric unit (current Rwork/Rfree = 20.4/24.1). The > protein was crystallized together with a 60 bases long poly-A oligo > (ssDNA). Each protein molecule binds to a stretch of this DNA. However, > only 15 bp of the DNA is part of the asymmetric unit, with each protein > molecule binding 5 bp. The DNA then goes on to the next ASU (with its 3 > proteins) and so forth. There is no sequence specificity to the DNA, so > it can “slide”. Redoing the structure in P1 does not really help, as due > to its “sliding” nature the density is still continuous in between ASUs. > > Currently, the structure model thus consists of 3 protein molecules and > one 15 bases long DNA. However, the density for the DNA is as I wrote > continuous into the neighboring ASUs. Any advice for how to deal with > this in the model would be most welcome! We can of course explain it in > the manuscript that we’ll submit, but would feel good if one can > represent it in a better way also in the pdb. 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