Hi Kornelius,
On Mon, Oct 18, 2010 at 02:03:53PM +0200, Kornelius Zeth wrote:
> What I noticed was that the spot profiles in XDS looked not as the
> are supposed to, presumably because the mosaicity of the spots are
> 0.4-0.6 and spot integration may be hampered. Still, merging
> Rfactors are brilliant.
Be careful about the merging R-factors: they measure internal
consistency but not directly data quality! Yes, if everything is fine,
then the fact that symmetry-related reflections are very similar tells
you something about data quality. But if something goes wrong ...
Given that your data looks slightly twinned and your reflection
profiles are not ideal, this does look to me a bit like a problem with
the spots and potential overlap: either from a second lattice (split
crystal or non-merohedral twinning) or from the same lattice
(overlapped reflection).
As far as I understand XDS it will assign each pixel to the nearest
predicted hkl (experts: please correct me if I'm wrong). So if you
have overlapped reflections (because mosaicity, oscillation angle and
distance are not ideal) you will get systematically truncated
reflections. Since symmetry-related reflections will most likely be
truncated in a very simlar way you will get poor data but a low
Rmerge.
Similar for split crystals or non-merohedral twinning:
symmetry-related reflections could be affected in a very similar and
systematic way (giving low Rmerge) but the spots are just poor and
badly integrated.
I don't think this is a particular XDS problem - rather that other
programs might fail in processing those datasets at all and XDS is
just very good in getting as much out of it as possible.
The only thing that really helps here is to visually inspect
the images and the predictions: are all spots predicted or do you have
signs or split spots or multiple lattices? Don't forget to also look
at 30, 45, 60, 90 degree from image 1 ... Murphy's law dictates that
the first image always looks perfect ;-)
But maybe it is something different after all ... 3.8A SHARP phaser
going to 2.5A should give you something to work with: do you have NCS?
Do you see a significant difference in score for the two hands?
Cheers
Clemens
>
> Any hints + ideas are welcome!
>
> Best wishes
>
> Kornelius
>
> ----------------------------------------------
> Kornelius Zeth
> Max Planck Institute for Developmental Biology
> Dept. Protein Evolution
> Spemannstr. 35
> 72076 Tuebingen, Germany
> [log in to unmask]
> Tel -49 7071 601 323
> Fax -49 7071 601 349
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