See previous answer re spacegrroup - if phaser has output coordinates with SG P6522, but you are refining against the originally processed data that may well have SG P6122 in the header.
Refmac DOES NOT check this ( Why not Garib!!!!! ) Eleanor On 16 December 2013 09:47, <herman.schreu...@sanofi.com> wrote: > Dear Bonsor, > > I fully second James suggestions but have a few additional comments: > If you get a solution in P6522 with one molecule, you should get the same > solution in P65 with 2 molecules. One of the "crystallographic" symmetry > operators would then be "non-crystallographic". > The current version of Refmac will test all possible twinning operations, so > there is no need to do it yourself (provided of course that you get a > molecular replacement solution). > I would also try your rebuilt model with extended helix as a model for MR. > I suspect that the dimer which has formed is asymmetric and that it may be > randomly packed in your crystal. If the helix is a small compared to the > complete protein, it may not show up in twinning tests. > > Good luck! > Herman > > > > -----Ursprüngliche Nachricht----- > Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von James > Holton > Gesendet: Sonntag, 15. Dezember 2013 23:29 > An: CCP4BB@JISCMAIL.AC.UK > Betreff: Re: [ccp4bb] Wrong Space Group? > > Its possible you are in a lower space group, perhaps with some twinning, but > your search model is different enough to only find a "solution" when things > are over-merged. > > Try refining your P6522 model against data merged in P65. If the other copy > (symmetry mate in P6522) does not show up, you may be in trouble (wrong MR > solution). I'd also try refinement/building in the other triogonal/hexagonal > space groups, but again, start with the PDB file that you got for P6522. > Just change the space group in the header, and switch out the MTZ file. You > will need to merge your data in each space group and also check the a-b > "flip" re-indexing for most of them. Have a look at the CCP4 "reindexing" > list for the h,k,l operators to try: > http://www.ccp4.ac.uk/html/reindexing.html > note how similar they are to the "twinning" operators: > http://www.ccp4.ac.uk/html/twinning.html > If I have counted right, that means you have 36 jobs to run. > > I'd also recommend turning the "TWIN" option in refmac off and on for each > of these cases. This will always give you a lower R factor, because of the > dynamic range compression you get with twinning, but if one particular > combination of twinning with a particular space group and axis reindexing is > markedly better than all the others, then you have just found your right > space group. So, now we are up to 72 jobs, but hardly a lot of work compared > to growing the crystals in the first place. > > You might also want to try being "clever" and generating the symmetry mates > of your P6522 model and refine these partners as separate molecules as you > reduce the symmetry of the data. It's tricky, but think of it as an > exercise. Which real-space operator becomes what reciprocal-space operator? > You can check your answer by loading it up in coot and seeing if symmetry > mates clash with the input coordinates. > > Yes, its a lot of work to try all these combinations, but that's the annoying > thing about twinning, it opens up a lot of ambiguities. > > Good luck! > > -James Holton > MAD Scientist > > On 12/14/2013 6:44 AM, D Bonsor wrote: >> Dear all, >> >> I have collected ~160 degrees of data on a new crystal form of a protein >> which has already been solved. Data was processed with XDS and reindex, >> scaled and truncated with Aimless. Both XDS and Pointless suggested a Laue >> group of P6/mmm with a possible space group of P6122 or P6522. Stats showed >> an overall Rmerge of 0.131 but an Rpim of 0.041 (multiplicity/redundancy of >> 19.1), a completeness of 99.1% and resolution of 2.8Ang. >> >> With cell dimensions of 63.1 63.1 243, only one protein chain can be found >> in the asymmetric unit (two copies would leave a solvent content of 8%). I >> ran phaser with all alternative space groups and a single solution in P6522 >> with a TFZ of 10.0. >> >> I then performed 20 Refmac cycles ending up with an R/Rfree of 35.5/45.5. I >> open the structure and map in Coot and could see that there was a large >> conformational change of helix-turn-helix actually becoming just a long >> helix (https://www.dropbox.com/s/4s6g8apatsi5xcg/Before_Building.png) and >> then dimerizing through the long helix with one of the symmetry mates. >> >> This section was rebuilt >> (https://www.dropbox.com/s/5j7tv0i5yq3mxxx/After_Building.png) and ran >> through Refmac again resulting in an R/Rfree of 35.5/44.3. Looking through >> the rest of the structure I see nothing else really to be modeled. Nothing >> that could bring the Rfactors down to a reasonable range. >> >> I have therefore tried several things. I ran the structure through Zanuda >> server to look at other space group possibilities. The server suggested I >> was in the correct space group. However I did reprocess the data to P6, P3, >> P312, P321, C2221, P2 and C2, and reran phaser in "search all alternative >> space groups" using the original search model but found no solutions. I did >> reprocess the data in P1, though I did not collect enough data. >> >> Twinning tests show no twinning. Although that does not mean there is no >> twinning, I can see that P6522 has no twin laws. Does that mean no twinning >> can occur in P6522 or that it can occur but there is no law to be able to >> separate the amplitudes? >> >> I also collected data on a single point mutation of the protein. Although >> this diffracted to a slightly weaker resolution (3.2Ang), I also observe the >> same problem of good maps in P6522 but no solution in the groups described >> above, a clear indication that this helix has elongated but terrible >> Rfactors. >> >> Based upon that the maps look good in P6522 do you believe that I have >> solved the structure in the correct space group but my data collection is at >> fault or in fact that I have some form of pseudosymmetry or something else >> going on and that the space group has lower symmetry but not in the space >> groups I have checked. Or is it something else. >> >> Any suggestions, criticisms or you need further information please contact >> me and enjoy your weekend.
