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 >>>>> >>>> >>>> >>> >> >
