Good evening, the large gap of 35% vs 45% indicates overfitting, e.g. too high a matrix weight in refmac. This would result in a flattening of the difference density so that you don't see additional features to improve your structure on. This is way it is better to first work on the model resulting from phaser based on the electron density map provided by phaser before doing any refinement.
The helix you show does not show any difference density map. If it were real, I would expect it to be green as much as blue. When you run refmac (or whichever refinement program you choose), look at the table at the bottom of the log file and ensure that the geometry is not too distorted (e.g. rms Angles approx 1.6, rms Bonds about 0.02). If they are higher, decrease the matrix weight (you could increase the number of cycles in turn). It cannot be stressed too much that you start with model building before you run any refinment, otherwise you may loose important information which is hard to recover in particular at medium to low resolution. Best, Tim Gruene On 12/13/2013 08:44 PM, 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. > -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
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