I guess my hunch would be that there is some sot of order-disorder
problem; either twinning or crystal dislocation.
Some ideas -
reindex the PG222 data set h/2,k,l so that a~=b and test that data for
twinning - you can just run truncate on the output Is and see the
moments and cumulative intensity plots.
Try solving the structure with that data set to find 2 molecules.
The new SG is likely to be P2 212 21 (or possibly P 21 21 21. Look at
the new set of h' 00 to see if there are any absences)
It might suggest something..
Eleanor
James Irving wrote:
> Dear cpp4bb,
>
> I'm wrestling with a crystal form that is causing us a great deal of
> trouble, and I was hoping for some general suggestions that might get us
> working in the right direction. The protein in question is 40kDa. Details
> are provided below. The summary is that at the model building/refinement
> stage this data is behaving as though it is twinned, or merged in the wrong
> space group, or there is some other fundamental anomaly that needs to be
> accounted for, but the tests we've performed indicate that it is solved, not
> twinned, in the correct space group.
>
> Here are some details:
>
> 1. It integrates and merges well in P2(1)2(1)2(1) without any trouble (see
> table pasted at bottom). We've collected two datasets, around 2.5-2.6A
> each, one is of a selenomethionine derivative. To get as strong data as
> possible for MIRAS we collected ~400 deg of the derivative, and around 205
> deg of the native. Scaling and merging in XDS (although we've used
> mosflm/scala as well) gives rather good stats at the low resolution (rmerge
> 1-2% <3.7A) and deteriorate quite markedly in the high resolution bin (~50%
> at 2.55-2.7A), with I/sigI of 60 for the former and 3.0 for the latter.
> Systematic absences very strongly support a P212121 space group.
>
> 2. There is no visible sign that this is part of a superlattice, or
> comprises a superlattice. Virtually all spots are accounted for during
> integration. By eye there are no systematically weak and strong
> reflections. Playing with the minimum I/sigI at the indexing stage doesn't
> do anything, nor does deleting strong reflections and indexing only with
> weak ones, nor does indexing using only high, or only low, resolution spots.
>
> 3. Unit cell: a=42.6 b= 85.3 c=108.5 90 90 90. a is almost exactly 2*b.
>
>
> 4. Wilson plot looks normal. There is no detected pseudotranslation.
> Cumulative
> intensity distribution in truncate appears *very slightly* sigmoidal. Is it
> twinned? More on that in a moment...
>
> 5. There is a reasonably close homolog of this protein that has been
> crystallised (~50% identity) - we were expecting an easy MR solution.
> Phaser gives Z-score in the rotation function of ~17, and ~11 in the
> translation function for a single molecule in the AU, as expected. 2FoFc &
> FoFc maps look absolutely rubbish, very much worse than would be expected
> for this protein at this resolution with this solution. Correction for
> anisotropy doesn't improve maps much here or at any other stage in the
> building/refinement.
>
> 6. Scaling and merging in P21 on the off-chance of perfect twinning or
> pseudosymmetry gives exactly two solutions with very good Z-scores. Maps
> still look rubbish. Phenix.xtriage, as would be expected, suggests a
> twinning operator with alpha ~0.5 that is identical to a crystallographic
> operator in P212121. Rigid body refinement using phenix.refine and this
> twinning operator gives Rfactor/Rfree that are low but again maps are
> uninterpretable. Conclusion: this isn't perfectly pseudomeroherally twinned
> in P21.
>
> 7. Went back to basics in P1. Same deal as P21.
>
> 8. All other enantiomorphs in monoclinic and orthorhombic give significantly
> lower, and poorly distinguished translation Z-scores in MR.
>
> 9. The selenomethionine dataset was solved using MIRAS in SHARP/autoSHARP.
> The experimentally phased electron density yields contiguous tracts of
> density in the right place, unbiased density indicates a good solution.
> Model building was conducted in P212121, initially into the experimental
> maps and later with refinement in refmac using HL-coefficient-based
> restraints. In some regions, sequence can easily be deduced from clean
> electron density (for the resolution). In other regions, side chains are
> missing, and in others, density is completely inconsistent with the
> connectivity of the chains and highly conserved structural elements. As
> occurs sometimes with twinned data, many loops cannot be modelled at all,
> and the Rfree does not drop below 0.41 with an Rfactor of 0.34. The result
> is a model that is about 60-70% complete. Refinement was performed with and
> without B-factor refinement.
>
> 10. Using density modification in SOLOMON, a structure-based solvent mask in
> DM or statistical modification in PIRATE fails to elucidate these
> additional, significant missing regions (which includes three helices, 1.5
> beta sheets and several loops). Tellingly, comparing final models to the
> original experimentally phased maps shows truncation of the model at the
> same places as "truncation" occurs in the electron density. During the
> building process as much care was taken as possible that the structure was
> not being built into a "local minimum".
>
> 11. phenix.autobuild is able to build a polyalanine model that covers about
> 25% of the molecule.
>
> 12. The native and derivative datasets scale extremely well together: they
> are strongly consistent. This is often not the case with twinned crystals.
>
> Any suggestions would be greatly appreciated!
>
> Thanks,
> James
>
> OUTPUT FROM CORRECT IN XDS:
>
> SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION
> RESOLUTION NUMBER OF REFLECTIONS COMPLETENESS R-FACTOR R-FACTOR
> COMPARED I/SIGMA R-meas Rmrgd-F Anomal SigAno Nano
> LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed
> expected Corr
>
> 7.02 4906 697 746 93.4% 1.9% 2.2%
> 4897 77.46 2.0% 1.1% 11% 0.849 447
> 5.04 9074 1152 1152 100.0% 2.1% 2.5%
> 9074 66.65 2.3% 1.4% 1% 0.797 890
> 4.13 11742 1445 1446 99.9% 2.3% 2.6%
> 11742 65.87 2.5% 1.5% -7% 0.755 1178
> 3.59 13929 1685 1685 100.0% 3.4% 3.6%
> 13929 45.70 3.6% 2.8% 2% 0.763 1428
> 3.21 15675 1884 1884 100.0% 6.5% 6.5%
> 15675 28.42 6.9% 5.7% -1% 0.811 1618
> 2.94 17324 2066 2066 100.0% 14.1% 14.2%
> 17324 14.14 15.0% 14.5% 0% 0.787 1804
> 2.72 18743 2232 2232 100.0% 27.5% 27.7%
> 18743 7.61 29.3% 29.9% 0% 0.749 1965
> 2.55 20107 2388 2388 100.0% 46.1% 45.4%
> 20107 4.85 49.1% 51.7% -2% 0.717 2123
> 2.40 20056 2466 2545 96.9% 73.6% 72.2%
> 20023 3.04 78.5% 81.1% 1% 0.707 2144
> total 131556 16015 16144 99.2% 5.4% 5.6%
> 131514 26.34 5.7% 10.6% 0% 0.758 13597
>
> Attached figures:
> Data scaled in P2, self-rotation function in MOLREP
> Data scaled in P222, self-rotation function in MOLREP
> Cumulative intensity distribution in TRUNCATE
>
>
>
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