First - I dont think you have a 3rd molecule where you have put it - or at least not one with full occupancy. Those maps are a clear indication that something is wrong. What is the Matthews coefficient for the numbers in the asymmetric unit?
Presumably your processing gave you a lattice which fitted the diffraction spots? ie you didnt miss a set of observations? You should see that at the data processing stage, and the different integration programs also try to report it. If there is non-crystallographic translation that can confuse things a bit; some classes of reflections might be systematically weak, but you can find if there is such a phenomena by doing a patterson. Or run ctruncate after merging the data - it checks this, and so does Xtriage. All these options will also check for twinning. If there is NCT then that could explain the high Rfactor. Are the spots nicely shaped? There are some cases of sheared crystals, which usually show up in distorted diffraction spots. If this is so and you have integrated the data according to an orthogonal lattice, there is nothing to stop you merging those observations in a low symmetry. Pointless gives you good statistics on the scoring for different symmetry operators. You can either run MR again in that symmetry - check all SGS consistent with the pointgroup, or try to work out how to position your P22121 solution in the new SG. There may well be 2n+1 copies of your molecule when you double the size of the asymmetric unit - all hard to check without more information. Good luck Eleanor On 22 March 2013 17:54, Andrey Nascimento <andreynascime...@gmail.com>wrote: > Dear all, > > I have tried the procedure recommended by Zbyszek, expanding data from a > higher symmetry and keeping the R-free set. But the map for third molecule > (new molecule placed) are still very bad, even when a tried to reprocess > data in P1 or P2 (P 1 21 1). The previous placed molecule (present in P2 21 > 21 ASU) and its symmetry related on P21 shows a very good map, but the > third molecule are almost completely wrong (~50 residues in 470 are placed > in quite good map) and map does not have connectivity to build a new > molecule (even in lower sigmas, 0.8-1.0). I have tried automatic model > building (AutoBuild and ARP/wARP) but they cannot build anything that make > some sense or build a random chains without any sense. > > > I do not have an extensive knowledge of crystallography, but I have been > thinking about some questions: > > > If the third molecule (the bad one) is lying on the 2-fold symmetry axis > on P 2 21 21, and since it does not have an intrinsic 2-fold symmetry axis > (like protein molecule), how can I merge the structure factors (or > intensities) related by symmetry and expand to lower symmetry afterwards? > In this case the molecule lying on the 2-fold symmetry axis will have the > structure factors wrongly merged, since the molecule is not symmetric, is > it ok? > > > If the third molecule is lying on the 2-fold symmetry axis on P 2 21 21, > and only another two molecules can be related by the crystallographic > symmetry, is it a case of pseudo-symmetry? But in this case, the third > molecule is disordered in the crystal packing (as Zbyszek said), and > probably have a long range disorder, because I cannot get a good maps for > this third molecule even in P1. (pseudo-symmetry + order/disorder????). > > > And a more philosophical question… what is the problem in process data in > a lower symmetry? Are there mathematical/statistical problems related that > can lead to “false-good” data? > > > I put a new .pdf file (ccp4bb_maps_P21.pdf) with map figures in this link: > https://dl.dropbox.com/u/16221126/ccp4bb_maps_P21.pdf > > > I am sorry for so many questions and thanks in advance. > > > Cheers, > > Andrey > > 2013/3/20 Jrh <jrhelliw...@gmail.com> > >> Dear Zbyszek, >> I am concerned that the unmerged data would be bypassed and not preserved >> in your recommendation. I also find it counter intuitive that the merged >> data would then be unmerged into a lower symmetry and be better than the >> unmerged data; there is I imagine some useful reference or two you can >> direct me to that may well correct my lack of understanding. Thirdly I >> think this a very likely useful case to preserve the raw diffraction images. >> All best wishes, >> John >> >> Prof John R Helliwell DSc >> >> >> >> On 19 Mar 2013, at 14:37, Zbyszek Otwinowski <zbys...@work.swmed.edu> >> wrote: >> >> > It is a clear-cut case of crystal packing disorder. The tell-tale sign >> is >> > that data can be merged in the higher-symmetry lattice, while the number >> > of molecules in the asymmetric unit (3 in P21) is not divisible by the >> > higher symmetry factor (2, by going from P21 to P21212). >> > From my experience, this is more likely a case of order-disorder than >> > merohedral twinning. The difference between these two is that structure >> > factors are added for the alternative conformations in the case of >> > order-disorder, while intensities (structure factors squared) are added >> in >> > the case of merohedral twinning. >> > >> > Now an important comment on how to proceed in the cases where data can >> be >> > merged in a higher symmetry, but the structure needs to be solved in a >> > lower symmetry due to a disorder. >> > >> > !Such data needs to be merged in the higher symmetry,assigned R-free >> flag, >> > and THEN expanded to the lower symmetry. Reprocessing the data in a >> lower >> > symmetry is an absolutely wrong procedure and it will artificially >> reduce >> > R-free, as the new R-free flags will not follow data symmetry! >> > >> > Moreover, while this one is likely to be a case of order-disorder, and >> > these are infrequent, reprocessing the data in a lower symmetry seems to >> > be frequently abused, essentially in order to reduce R-free. Generally, >> > when data CAN be merged in a higher symmetry, the only proper procedure >> in >> > going to a lower-symmetry structure is by expanding these >> higher-symmetry >> > data to a lower symmetry, and not by rescaling and merging the data in a >> > lower symmetry. >> > >> > Zbyszek Otwinowski >> > >> >> Dear all, >> >> We have solved the problem. Data processing in P1 looks better (six >> >> molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three >> molecules >> >> in >> >> ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round >> >> of refinement (without put waters, ligands, etc.). >> >> >> >> Indeed, there were one more molecule in ASU, but the over-merged data >> in >> >> an orthorhombic lattice hid the correct solution. >> >> >> >> Thank you very much for all your suggestions, they were very important >> to >> >> solve this problem. >> >> >> >> Cheers, >> >> >> >> Andrey >> >> >> >> 2013/3/15 Andrey Nascimento <andreynascime...@gmail.com> >> >> >> >>> *Dear all,* >> >>> >> >>> *I have collected a good quality dataset of a protein with 64% of >> >>> solvent >> >>> in P 2 21 21 space group at 1.7A resolution with good statistical >> >>> parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; >> >>> Complet.=93% >> >>> Redun.=2.4, the overall values are better than last shell). The >> >>> structure >> >>> solution with molecular replacement goes well, the map quality at the >> >>> protein chain is very good, but in the final of refinement, after >> >>> addition >> >>> of a lot of waters and other solvent molecules, TLS refinement, etc. >> ... >> >>> the Rfree is a quite high yet, considering this resolution >> >>> (1.77A).(Rfree= >> >>> 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a >> lower >> >>> symmetry space group (P21), but I got the same problem, and I tried >> all >> >>> possible space groups for P222, but with other screw axis I can not >> even >> >>> solve the structure.* >> >>> >> >>> *A strange thing in the structure are the large solvent channels with >> a >> >>> lot of electron density positive peaks!? I usually did not see too >> many >> >>> peaks in the solvent channel like this. This peaks are the only reason >> >>> for >> >>> these high R's in refinement that I can find. But, why are there too >> >>> many >> >>> peaks in the solvent channel???* >> >>> >> >>> *I put a .pdf file (ccp4bb_maps.pdf) with some more information and >> map >> >>> figures in this link: >> https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf* >> >>> >> >>> * >> >>> * >> >>> >> >>> *Do someone have an explanation or solution for this?* >> >>> >> >>> * * >> >>> >> >>> *Cheers,* >> >>> >> >>> *Andrey* >> >>> >> >> >> > >> > >> > Zbyszek Otwinowski >> > UT Southwestern Medical Center at Dallas >> > 5323 Harry Hines Blvd. >> > Dallas, TX 75390-8816 >> > Tel. 214-645-6385 >> > Fax. 214-645-6353 >> > >
