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.