Dear Herman, I would like to mention one more information, maybe I have forgotten. When a process the data in P21212 and run the sfcheck it do not appear to have twinning (even when a ran phenix Xtriage with older process data in P21212). I will send direct to your e-mail the .pdf file with sfcheck analysis to both space groups. Thank you very much. Andrey
2013/3/28 Andrey Nascimento <[email protected]> > >> Dear Appu, >> I am sorry, I do not have the script file. I ran it basically from >> default parameters in CCP4 GUI. I just ran the sfcheck for 'check >> experimental data only' and I got the twin law and twin fraction from the >> output (post script file). So, I put these information on the detwin and >> ran it. I will send a .pdf file with the a print screen of ccp4 GUI. >> I am not a experienced crystallographer, but I hope it helps you. >> Good luck, >> Andrey >> >> >> 2013/3/28 Appu kumar <[email protected]> >> >>> >>> Respected sir, >>> I have same problem what you have. I am running >>> the detwin on mtz file but it getting failed. Could you please tell me how >>> you did this. If possible send me your script file. It will be a great help >>> for me. >>> Thank you in advance. >>> Appu >>> >>> On 28 March 2013 05:10, Andrey Nascimento <[email protected]>wrote: >>> >>>> Dear all, >>>> >>>> >>>> As I said in the latest topic, I could not model the third molecule. >>>> But when I superpose the two trimmers found in P1 MR solution (link below), >>>> I get the first two molecules perfect aligned and the third molecule >>>> inverted! (It is also possible to see the 2-fold axis and the third >>>> molecule lying on it!) >>>> >>>> >>>> >>>> I tried to run a MR with a model with two alternative positions and >>>> adjusted occupancy for the third molecule, but the Rfactor/free get higher >>>> (> 40%) and the map becomes worse – even the good ones (molecules 1 and 2) >>>> and for third molecule it remains bad (or worse). >>>> >>>> >>>> >>>> A procedure that “solved” the problem (decreased the Rfactor/free and >>>> gave good maps for third molecule) was the following: I integrated and >>>> scaled the data in P21, then I ran the sfcheck and it showed a twinned data >>>> (probably because of the (pseudo) higher symmetry present – P21212). So, I >>>> detwinned the data (with detwinn) and run a MR with detwinned data that >>>> gave a very good solution with tree molecules in ASU (it have never >>>> happened before!). After the MR I refined this MR solution against the >>>> original P21 data (without detwinn procedure) with amplitude based twin >>>> refinement in Refmac5 and, finally, it gave a good statistics (R factor / >>>> free about 0.19 / 0.22; FOM ~0.8) in the first round of refinement. I think >>>> that procedure probably discard reflections related to other positions >>>> making increasing the signal of the most frequent position. >>>> >>>> >>>> >>>> Link (.pdf): https://dl.dropbox.com/u/16221126/superposition.pdf >>>> >>>> >>>> >>>> Is there some problem in procedure described? If so, does anybody have >>>> a suggestion how can I model these disorder? Moreover, it seems to be a >>>> long range disorder (multiples positions along the all lattice), since even >>>> in P1 the maps for this third molecule are very bad. >>>> >>>> >>>> >>>> Thank you for all the suggestions. >>>> >>>> >>>> >>>> Cheers, >>>> >>>> Andrey >>>> >>>> 2013/3/25 Eleanor Dodson <[email protected]> >>>> >>>>> 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 >>>>> <[email protected]>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 <[email protected]> >>>>>> >>>>>>> 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 <[email protected]> >>>>>>> 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 <[email protected]> >>>>>>> >> >>>>>>> >>> *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 >>>>>>> >>>>>> >>>>>> >>>>> >>>> >>> >> >
