Dear Michael, throwing a few other suggestions into the mix (on top of the good advice you got already from Graeme and Phil):
* The low resolution I/sigI is quite large and although we have seen such values for extremely good crystals collected very carefully on very good instruments, it is rather uncommon for "standard" (whatever that means) protein crystals that diffract to lowish resolution. So Phil's suggestion about problems in your error model is quite likely - and since refinement programs usually take sigmas into account, this might explain why there is no effect when using higher-resolution data. * You have quite high multiplicity so that you can easily check for possible effects of radiation damage, which could also be at play here: is there an increase in cell dimensions, a "smiley" like shape to Rmerge-vs-Image number plots, visible radiation damage in F(early)-F(late) maps ... ? * Any ice-rings present? The 3.5A range is known for those ;-) * Did you mask your beamstop shadow best/correctly? This shouldn't impact the high-resolution limit, but sometimes things can still go wrong there. * Completeness as calculated in that table is only ever completeness of Miller indices and not necessarily observations (i.e. significant data) [1]. * If there is a very large empty detector area (because the crystal-detector distance was over-optimistic) and integration/scaling is not restricted to something more sensible, things can sometimes go wrong in those steps: programs are very good in dealing with noise, but if nearly all incoming data is pure noise even they can sometimes go off into a wrong minimum (during scale or error model parameter refinement). * Have a close look at the detailed processing output from the program(s)/pipeline(s) you used for processing. Especially if you picked up your data directly from the automatic processing options available at a synchrotron, you have to be aware that sometimes sensible restrictions imposed by hardware, data policy or time requirements mean that some programs/pipelines can not necessarily be run in recommended default mode (assuming that the defaults are chosen by the developers for very good reasons). If you processed your data yourself: all programs and pipelines should give you plenty of clear warning messages early on that can highlight any possible problem mentioned above. There are some more potential issues one could think of I guess ... see also [2] and [3]. Cheers Clemens [1] http://staraniso.globalphasing.org/ [2] http://www.globalphasing.com/autoproc/manual/autoPROC7.html [3] http://www.globalphasing.com/autoproc/ On Thu, May 18, 2017 at 11:47:30AM +0000, Michael Jarva wrote: > Dear all, > > I have a dataset that have two very interesting properties: a) It's in I432, > and b) has a whooping 75% solvent content. > You might think that the solvent content obviously is a big red flag, and so > did I, but I have phased this successfully with just one monomer, and the > packing result does makes a lot of sense. The resulting maps contain no extra > umodelled blobs, and trying to phase it with an additional molecules does not > give a good solution. > > The problem I have is that the diffraction intensity/Rmerge plummets/explodes > around the 3.5Å mark (I assume because of the high solvent content) to such > an extent that even though I have little radiation damage, 100% completeness > in high resolution shells, and very high redundancy, any attempt to merge the > dataset at a higher resolution has so far given no improvement to the maps. > > I'm hoping that there might be a few tricks out there I can apply to the spot > finding/integration/scaling steps have it merge in a even slightly higher > resolution than I currently have been able to do. > Although I have a feeling that the only thing I can do is to grow another, > much bigger, crystal… > > many thanks for any feedback > /michael > > See below for sample outputs from aimless: > > Overall InnerShell OuterShell > Low resolution limit 43.50 43.50 3.32 > High resolution limit 3.10 8.78 3.10 > > Rmerge (within I+/I-) 0.079 0.010 21.891 > Rmerge (all I+ and I-) 0.081 0.011 22.502 > Rmeas (within I+/I-) 0.084 0.011 23.102 > Rmeas (all I+ & I-) 0.084 0.011 23.169 > Rpim (within I+/I-) 0.027 0.004 7.335 > Rpim (all I+ & I-) 0.020 0.003 5.450 > Rmerge in top intensity bin 0.010 - - > Total number of observations 34917 1495 6448 > Total number unique 2057 112 362 > Mean((I)/sd(I)) 18.3 130.9 0.1 > Mn(I) half-set correlation CC(1/2) 1.000 1.000 0.533 > Completeness 99.9 97.4 100.0 > Multiplicity 17.0 13.3 17.8 > > Overall InnerShell OuterShell > Low resolution limit 43.50 43.50 3.84 > High resolution limit 3.50 8.58 3.50 > > Rmerge (within I+/I-) 0.052 0.011 2.422 > Rmerge (all I+ and I-) 0.056 0.012 2.659 > Rmeas (within I+/I-) 0.055 0.011 2.553 > Rmeas (all I+ & I-) 0.058 0.013 2.738 > Rpim (within I+/I-) 0.017 0.004 0.804 > Rpim (all I+ & I-) 0.014 0.003 0.644 > Rmerge in top intensity bin 0.010 - - > Total number of observations 24596 1690 6071 > Total number unique 1462 120 343 > Mean((I)/sd(I)) 25.8 132.0 1.0 > Mn(I) half-set correlation CC(1/2) 1.000 1.000 0.771 > Completeness 99.8 97.6 100.0 > Multiplicity 16.8 14.1 17.7 > -- *-------------------------------------------------------------- * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * Global Phasing Ltd., Sheraton House, Castle Park * Cambridge CB3 0AX, UK www.globalphasing.com *--------------------------------------------------------------
