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Andreas, Is Phaser putting your dimer search model directly on the crystallographic 2-fold? This will generate a very large number of clashes between monomer 1 and monomer 2 during the packing routine. The simplest way around this is to re-run MR_PAK after trimming your search model to 1 monomer. If the dimer search model is not placed directly on the crystallographic 2-fold, does your data demonstrate a large patterson peak (other than the C-centering)? Eric On Thu, 16 Nov 2006 [EMAIL PROTECTED] wrote: > *** For details on how to be removed from this list visit the *** > *** CCP4 home page http://www.ccp4.ac.uk *** > > > Dear molecular replacement cracks out there, > > > I'm working on a non-trivial molecular replacement problem (bad model, > poor data, many molecules in au) and am hitting the wall. This might be > because the problem cannot be solved or because I'm blind to the solution. > > Run on a list of normal mode-perturbed models, Molrep didn't find obvious > solutions but suggested a dimer I should use as model instead of the > monomer. This makes good sense, as my protein is like dimeric (though I > can't see NCS in the data). What's the basis of this suggestion, Alexei? > > Using the dimer didn't help Molrep find the solution, but Phaser suggested > a rotation function (Z-score = 3) whose 26 associated translation > functions have Z-scores between 7 and 9. All solutions are rejected > because of clashes (~100). > > Forcing the transformations I can write out 26 potential solutions and > look at them in PyMOL. They are lined up along the a axis of the unit > cell (c222_1). The a axis is also coincident with the two-fold that > relates the two monomers of the dimer (thus the nearly complete overlap > indicated by 100 clashes). One monomer is symmetry-related to the other. > > Why does Phaser put the dimer on a symmetry axis? Would this not give > wrong solutions in any case where you search with a dimer? Is it possible > (and sensible) to define the a axis in C222_1 as a dyad axis for molecular > replacement? > > At first I thought my problems were due to misindexing, but running phaser > or Molrep with data reindexed in all other possible spacegroups (p1, p2, > p2_1, c2, c222, see statistics at the end of the email) didn't give > solutions (nor high Z-scores in Phaser). For example, in P1, Molrep > places 4 dimers but the contrast values decrease with every placed dimer > (from 5 to 2). Does that tell me that I'm completely off? > > Next I was thinking twinning but there is no indication for that (Yeates, > sfcheck). > > Currently I'm running Molrep with the dyad option and half of the > suggested dimer (i.e. the initial monomeric model). In C222 and C222_1 > Molrep comes up with a solution containing four models (as expected from > Matthews coefficient) and good contrast values (11 and 120 for C222; 92 > and 85 for C222_1). Looking at the solutions, I don't see dimers. The > other space groups are work in progress... > > Furthermore, I plan to do the dyad search with a fixed model running > Molrep 26 times with all solutions from Phaser. > > > Am I chasing waterfalls, or does it sound like I'm up to something? Any > comments or suggestions are highly appreciated. And, if you got that far, > thanks for reading through all this ;-) > > > > Andreas > > > > And yes, I have whipped myself and a student for the last six months > trying to get better crystals, derivatives, data, etc. > > > > > Indexing in different space groups - output from XDS for signal/noise >= > 0, all data: > > RESOLUTION NUMBER OF REFLECTIONS COMPLETENESS R-FACTOR > LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed > > P1 a total 105246 62621 108903 57.5% 8.2% > P1 b total 105224 62606 108903 57.5% 8.2% > P2 total 106172 50601 56092 90.2% 9.0% > P2_1 total 106164 50594 56081 90.2% 9.0% > C2 a total 108603 38721 55096 70.3% 10.4% > C2 b total 107130 48446 55231 87.7% 9.2% > C222_1 total 110472 27435 28860 95.1% 11.1% > C222 total 110479 27441 28871 95.0% 11.1% > > R-FACTOR COMPARED I/SIGMA R-meas Rmrgd-F S_norm/ > expected S_ano > > P1 a 8.3% 85250 7.52 11.7% 26.4% 0.00 > P1 b 8.4% 85236 7.51 11.7% 26.4% 0.00 > P2 9.1% 102652 8.35 12.3% 26.6% 1.12 > P2_1 9.1% 102650 8.35 12.3% 26.6% 1.12 > C2 a 10.6% 102377 8.75 12.5% 23.5% 1.06 > C2 b 9.3% 101481 8.34 12.2% 25.4% 1.08 > C222_1 11.4% 110384 10.50 12.9% 19.9% 1.02 > C222 11.4% 110386 10.50 12.9% 19.9% 1.02 > > > >
