Hi Matt, You may be right, but how do you distuinguish you between a crystallographic 2-fold and perfect twinning? A solution that refines to an Rfree of 55.6% is not exactly a textbook example of a correct solution. Poor data, low-resolution and twinning/pseudosymmetry can cause all kinds of artefacts. I would really recommend to keep all options open until a solution is found that really refines.
Best regards, Herman -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Matthew Franklin Sent: Friday, September 07, 2012 3:57 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] poorly diffracting and twinned trigonal crystal Hi Qing - Just a couple of observations on your story, without a real solution: - Your space group is almost certainly P31 2 1. Your scaling statistics and Phaser results point to this - if your true space group was P31, then your statistics would be noticeably (even dramatically) worse in the higher symmetry space group. - Your twinning result does not mean that your crystal is twinned. In space group P31, you see a two-fold rotation axis along the ab unit cell edge; the twin fraction of 0.475 means that this is nearly perfect. Adding that two-fold to P31 gives you the P3121 space group. - I suspect that your molecular replacement solution is correct, although I've had Z scores that high before and not gotten a usable solution. I believe your data is simply too weak to permit any sort of refinement. I'm also a bit dubious about the 4.3 A limit; your useful data may be ending around 4.6 instead, despite the high I/sigma numbers. I'm guessing that you have very high redundancy in this dataset? Sadly, I have no advice for you other than to pursue larger crystals, with better X-rays. (I'm assuming the data you show us is from a synchrotron; if not, take these crystals there immediately!) How radiation sensitive are these crystals? Perhaps a microfocus synchrotron beamline would help; even if your crystals are large, you could shoot small parts of the crystal until they burned out, then move to a new region. You also have to consider whether a 3.5 A structure (probably the best you could hope for, barring a breakthrough in crystal quality) will answer the questions you have regarding this system, and be publishable. Best of luck, Matt On 9/6/12 7:48 PM, 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. > > -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
