On Fri, 13 Dec 2013 19:44:44 +0000, D Bonsor <dbon...@ihv.umaryland.edu> wrote:
Dear D, I agree with Tim and Jürgen that a) the map after Phaser, and before refinement is the most unbiased and should be used for sequence assignment. b) there may be a sequence register shift error that is responsible for the high R-values, and is masked by overfitting So I would try a) To stay on the safe side, you could chop the Phaser model into secondary structure elements and do rigid-body refinement. This yields maps that are largely unbiased. b) sharpening (very easy in coot) and inspection of these unbiased maps, to confirm the sequence register c) submit the Phaser model to Arp/wArp re-building, and also try the buccaneer/refmac iterative re-building, and maybe phenix.autobuild But the problem may also be your data. a) Maybe every second reflection is not integrated because it is weak? That is easy to check with XDSGUI using Tools/"show frame with predicted spots" b) other pathologies like spot overlap or experimental instability (what is the value of ISa in CORRECT.LP ?) - you could post FRAME.cbf and IDXREF.LP, INTEGRATE.LP and CORRECT.LP and pointless/xtriage statistics If the true space group is P6x22, then the data cannot be twinned. But if the true space group has lower symmetry the data may appear to be P6x22 . HTH, Kay P.S. XDSGUI latest version can be obtained from http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/XDSGUI >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.
