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.
