Dear Quing, My first suggesting is a no-brainer: try to get better data. At 4.3Å model building and refinement will remain painful, whatever you try. Getting better data includes improving your crystals (ligands, additives etc.) and the cryoconditions. You could also try to collect data on say 100 crystals to see if there is one crystal that diffracts better, or is less twinned. However, from your question this is probably not an option.
Concerning your current data set, I would also try molecular replacement with the complete model (no poly-ala), at least with the complete core. E.g. if your protein has an Ile at a certain position and your model has a Leu, this Leu is closer to your protein than the Ala you are using now. I would run molecular replacement in the lower symmetry P3x space groups since in this case no asumptions are being made whether the 2-folds are crystallographic, non-crystallographic, or generated by twinning. Then I would analyze the packing to look if 2-folds are present and whether they could be crystallographic, or must be non-crystallographic and whether the packing makes sense. If there are no 2-folds present, the twofold symmetry of your data must be caused by twinning. If there are 2-folds present, there still could be twinning and I would try to generate the twin-related molecule as well and examine them together on the graphics to see what the implications are. At low resolution, you get severe model bias giving a large split between R and Rfree and having twinned data or data with pseudo-crystallographic symmetry will not improve the situation either. Probably Randy or Garib could tell you more precisely what that means for your R/Rfree's. My 2 cents, Herman -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Qing Luan Sent: Friday, September 07, 2012 1:49 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] poorly diffracting and twinned trigonal crystal 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.
