Seeing Herman's message reminds me that I forgot to send this reply to the BB as well.
Herman makes many of the same points, and a very good additional point that you want to examine whether the molecular replacement solution in the lower symmetry space group actually has the higher symmetry. One way to do this manually is to check whether the Fcalcs from the MR solution have higher symmetry, but I think the Zanuda server is doing that kind of thing automatically. Best wishes, Randy Read Begin forwarded message: > From: Randy Read <rj...@cam.ac.uk> > Date: 7 September 2012 09:10:19 GMT+01:00 > To: Qing Luan <patange...@gmail.com> > Subject: Re: [ccp4bb] poorly diffracting and twinned trigonal crystal > > Hi, > > There are a number of issues here. > > Refinement at 4.3A resolution is difficult at the best of times. I'm > assuming that, if you've chosen to use polyAla models for molecular > replacement, the sequence identity between the template and the target is > rather low, in which case you would expect very poor convergence for > refinement at this resolution. > > As an aside, there's very convincing evidence from a number of studies > (starting with a paper by Schwarzenbacher et al) that useful information is > lost by trimming back as far as a polyAla model. You keep a significantly > larger number of reasonably well-placed atoms by keeping all identical side > chains and keeping up to the gamma atom of non-conserved side chains (unless, > of course, the target amino acid is a glycine or an alanine). Various > programs, including the CCP4 chainsaw program and our sculptor program, can > carry out such model modification. > > It's not clear which twinning tests you are reporting. Twinning tests that > compare the intensities of potentially twin-related reflections to see if > they are more closely related than expected randomly (e.g. the H test or the > Britton plot) can't tell the difference between twinning and either higher > symmetry or pseudosymmetry. So you need to use a test based on intensity > distributions independent of twin laws (like the intensity moments or the L > test) to decide if your crystal is twinned, then if it is twinned you will > get a better idea of the twin fraction from tests comparing the twin-related > reflections. It's instructive to take a tetragonal lysozyme data set (true > space group P43212), merge it in P43, then run twinning tests to see what it > looks like when you assign too low symmetry. Based on the fact that you can > solve the structure in both P31 and P3121, either you have true P3121 > symmetry or P31 with pseudosymmetry close to P3121, so you have to be > cautious in your conclusions about twinning. Note that, as Garib has shown, > the R-factors are always lower when you assume the crystal is twinned! > > Best wishes, > > Randy Read > > On 7 Sep 2012, at 00:48, Qing Luan wrote: > >> I have a ~4.3 angstrom data set of a trigonal crystal of a seven subunit >> protein complex which I can scale in P3, P31, P32, P321, P3121 and P3221 >> with similar statistics: >> >> P3 >> Shell Lower Upper Average Average Norm. Linear Square >> limit Angstrom I error stat. Chi**2 R-fac R-fac >> 50.00 9.25 1296.8 89.2 23.5 1.233 0.064 0.077 >> 9.25 7.35 356.3 18.5 9.7 1.512 0.065 0.066 >> 7.35 6.42 97.1 8.2 7.5 1.584 0.143 0.140 >> 6.42 5.83 55.2 8.3 8.1 1.503 0.247 0.241 >> 5.83 5.42 51.4 9.4 9.3 1.438 0.297 0.284 >> 5.42 5.10 47.0 10.5 10.5 1.469 0.374 0.345 >> 5.10 4.84 48.3 11.8 11.9 1.421 0.398 0.383 >> 4.84 4.63 43.6 12.9 13.1 1.474 0.488 0.449 >> 4.63 4.45 40.3 14.1 14.2 1.530 0.546 0.477 >> 4.45 4.30 30.8 14.7 15.0 1.601 0.732 0.631 >> All reflections 203.8 19.6 12.3 1.477 0.125 0.085 >> >> >> P3121: >> >> Shell Lower Upper Average Average Norm. Linear Square >> limit Angstrom I error stat. Chi**2 R-fac R-fac >> 50.00 9.14 1242.9 51.8 18.3 1.200 0.057 0.068 >> 9.14 7.26 314.0 11.2 6.5 1.454 0.070 0.069 >> 7.26 6.35 86.9 5.3 5.0 1.499 0.158 0.152 >> 6.35 5.77 51.9 5.5 5.3 1.248 0.264 0.252 >> 5.77 5.35 46.9 6.1 6.0 1.213 0.330 0.305 >> 5.35 5.04 44.3 6.9 6.7 1.137 0.393 0.363 >> 5.04 4.79 43.4 7.7 7.4 1.109 0.434 0.407 >> 4.79 4.58 39.2 8.5 8.1 1.128 0.533 0.478 >> 4.58 4.40 34.2 9.1 8.6 1.115 0.634 0.549 >> 4.40 4.25 24.9 9.9 9.3 1.064 0.872 0.766 >> All reflections 199.0 12.4 8.1 1.216 0.127 0.080 >> >> Unit cell parameters: 129.653 129.653 358.280 90.000 90.000 >> 120.000 >> >> The systematic absences are consistent with either P31, P32, P3121, or >> P3221. Analyzing the cell contents in P3121 suggests either 1 (Matthews >> coefficient of 3.86, 68.2% solvent) or 2 mol/ASU (Matthews coefficient of >> 1.93, 36.38% solvent) >> >> >> I built a molecular replacement model (a polyala model containing about 2/3 >> of the protein complex) and ran phaser in multiple space groups with one >> (for P3121 or P3221) or two (P31, P32) copies of the model. Runs in P32 or >> P3221 gave no solutions or solutions with TFZ around 4-5. When run in P31 or >> P3121, phaser output solutions with TFZ> 11.0 and what appeared to be good >> packing. >> >> Rigid body refinement on the P3121 solution failed to improve the Rfactor >> (it hovered around 55.3%). Adding the missing subunits (as polyala chains) >> based on the phaser solution and refining with rigid body refinement >> resulted in a model with an Rfree to 48.5. Refining with torsion angle >> dynamics and restrained group B-factor refinement made the Rfree worse – it >> jumped up to about 55.6%. The Rwork values were similar to the Rfree values >> for each attempt. I also tried DEN refinement with similar results. >> >> Rigid body refinement of the P31 phaser solution gave an Rfree of about >> 54.4%. Adding the missing subunits and running rigid body refinement again >> improved the Rfree to 53.0. Refining with torsion angle dynamics and >> restrained group B-factor refinement again made the Rfree worse (increased >> to 54.5%). >> >> I analyzed the reflection file processed in P31 using detect_twinning.inp in >> cns. The data did not appear to be perfectly merohedrally twinned, but in >> the test for partial merohedral twinning, the twin fraction calculated for >> “2 along a,b” was 0.475. I repeated rigid body refinement, then torsion >> angle dynamics with restrained group b-factor using the calculated twinning >> parameters. This brought the free R down to 46.3%, but caused significant >> divergence between Rwork and Rfree (Rwork =21.4%(!)). The Rfree is fairly >> constant across resolution shells, but Rwork drops dramatically with low >> resolution reflections (In the 50 – 9.14 ang shell, Rwork = 12.3%!). >> >> I’m guessing that because the twinning fraction is near 0.5, detwinning is >> not working. Does anyone have any suggestions about how to successfully >> refine this structure (assuming it is possible)? Should we average the twin >> related reflections to generate perfectly twinned data, and if so, how do we >> do that? Is the twinning likely responsible for our difficulty refining the >> structure or could there be a problem with the space group assignment? Why >> does including the partial twinning in our refinement cause Rfree and Rwork >> to diverge so dramatically? Given the trouble I’ve had so far and the poor >> quality of the data, I’m about ready to give up on this structure, but if >> anyone has any ideas please let me know. > > ------ > Randy J. Read > Department of Haematology, University of Cambridge > Cambridge Institute for Medical Research Tel: + 44 1223 336500 > Wellcome Trust/MRC Building Fax: + 44 1223 336827 > Hills Road E-mail: rj...@cam.ac.uk > Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk > ------ Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills Road E-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
