On Sun, 19 Jul 2015 17:15:56 +0100, Christopher Barnes <[log in to unmask]> wrote:
>Harry- Yes we have used pointless when merging multiple sets, and we always come back with p212121.
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>Tim - Yes i am confident that the values are not a by-product of generally little data per wedge, as some wedges containing more data (because of thicker regions of the crystal) produce the I/sigI or Isa values that I mentioned in my previous message.
I think Tim's hypothesis is a good one, because for few-degree wedges the ISa (and CC1/2, and Rmeas) values are based on few reflections only (those that have sum-related reflections in the same dataset) and are thus not very predictive.
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>Kay - As for the indexing ambiguity, two of the axes are pretty close (within 5 Angstroms of each other), and so we do sometimes see programs index in higher symmetry (P4) spacegroup. However, I have collected multiple low dose datasets from single crystals to ~4.4 Angstrom (at a BM-line, so no translation), where you can clearly see that P212121 is most likely the right spacegroup (ISa for P212121 > 30 whereas ISa for P4 related spacegroups is never greater than 2, also determined using pointless as well). In addition, these crystals are composed of a multi-protein complex, where 75% of the components are structurally known; therefore, molecular replacement in p212121 gives a clear solution with no symm-related clashes, and initial rigid/bgroup refinement gives Rwork/Rfree values of 0.29/0.34. Maps also show a good fit for the known parts of the model, with clear electron density in the difference map for the novel components.
I was only asking this because you had not given the space group in you first posting, and inconsistent indexing would have been the prime suspect.
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>As for the radiation damage, I wonder why this would cause such a problem, since we are only losing the high res reflections? So while we have datasets to 4.5 Angstrom, at that resolution there is no way to trace the novel components (se-mets are sparse, and generally reside in predicted disordered loop regions which would not be much help for tracing). So translating after a few degrees is the only way to maintain the high intensity reflections we need at the higher resolution (~3.5 Angstrom, which already is pretty weak).
Sorry, that's a weird argumentation; you cannot argue with Nature in this way, i.e. effectively saying "I need to expose so strongly because I want to see this effect". It works the other way round: do the experiment properly, i.e. don't over-expose, merge non-overexposed data sets, and see what you get. If you don't see enough, use additional crystals and average.
If you "only lose the high res reflections" you have already distorted the order and contents of your crystal, and you cannot expect to recover information from those data. Loss of high resolution tells you that your crystal is severely damaged, and even though you don't see it visually at low resolution, the low resolution intensities are just bad.
However, reflections less than 4.2 stay consistent (at least visually), so why would radiation damage between the last frame from one wedge and first frame of the next wedge cause such a dramatic drop even for the low res data, and is there any way I can correct for this?
I'm afraid that no amount of data massaging will help. My advice would be: repeat your experiment at a beamline with Pilatus detector (if you have not been using one for this experiment already), do fine-slicing, and expose e.g. 10 times less per degree but collect twice as many degrees per wedge. You could overlap the wedges in phi, since you are anyway processing them individually.
With your current data, you could try the following: discard the second half of each wedge, and merge the first halves only.
HTH,
Kay
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>Thanks,
>Christopher
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