Dear Ho,
On Fri, Feb 13, 2009 at 04:45:29PM -0800, Ho-Leung Ng wrote:
> Can you elaborate on the effects of improper inclusion of low
> resolution (bogus?) reflections? Other than rejecting spots from
> obvious artifacts, it bothers me to discard data. But I can also see
> how a few inaccurate, very high intensity spots can throw off scaling.
I completely agree: it also "bothers me to discard data". However, the
crucial word here is 'data' - which is different from Miller indices
HKL.
So I am mainly concerned with two types of reflections (HKL) that
aren't really 'data':
1) overloads
These are obviously not included into your final reflection file
(unless you explicitely tell the integration software to do that
- in which case you know exactly what you are doing anyway). So
there is no problem ... or is there?
Overloaded reflections are only very few at low resolution - and
the most important reflections are obviously the ones at 1.94A
resolution so that one can have a 'better-than-2A' structure in
the end ... ;-) ... So still no problem, right?
And who cares if the completelness of the data isn't 100% but
rather 99.4%? Exactly ... so where is the problem?
But: these few missing reflections are systematically the
strongest ones at low(ish) resolution, and any systematically
missing data is not a good thing to have.
Solution: always collect a low-intensity pass to measure those
strong reflections if there is a substantial amount of overloads.
2) beamstop
The integration software will predict all reflections based on
your parameters (apart from the 000 reflection): it doesn't care
if such a reflection would be behind the beamstop shadow or
not. However, a reflection behind the beamstop will obviously not
actually be there - and the integrated intensity (probably a very
low value) will be wrong.
One example of such effects in the context of experimental
phasing is bogus anomalous differences. Imagine that your
beamstop is not exactly centred around the direct beam. You will
have it extending a little bit more to one side (giving you
maybe 20A low resolution) than to the other side (maybe 30A
resolution). In one orientation of the crystal you might be able
to collect a 25A (h,k,l) reflection very well (because it is on
the side where the beamstop only starts at 30A) - but the
(-h,-k,-l) relfection is collected in an orientation where it is
on the 20A-side of the beamstop, i.e. it is predicted within the
beamstop shadow.
Effect: you have a valid I+ measurement but a more-or-less zero
I- measurement, giving you a huge anomalous difference that
shouldn't really be there.
Now if you measured your data in different orientations (kappa
goniostat) with high enough multiplicity, this one bogus
measurement will probably be thrown out during
scaling/merging. You can e.g. check the so-called ROGUES file
produced by SCALA. But if you have the usual multiplicity of only
3-4 the scaling/merging process might not detect this as an
outlier correctly and it ends up in your data. Sure, programs
like autoSHARP will check for these outliers and try to reject
them - but this is only a hack/fix for the fundamental problem:
telling the integration program what the good area of the
detector is.
Solution: mask your beamstop. All integration programs have tools
for doing that (some are better than others). I haven't seen
any program being able to do it automatically in a reliable way
(if reliable would mean: correctly in at least 50% of cases) -
but I'm no expert in all of them by a long shot. it usually takes
me only about a minute or two for masking the beamstop by hand. A
small investment for a big return (good data) ;-)
There are other possibly problematic reflections at ice-rings etc:
these can also have effects seen in the maps. But the above effects
have one thing in common: they happen mainly at low-resolution. And
our models can be seen to consist of basically two real-space
components (atoms in form of a PDB file and bulk-solvent in form of a
mask) - one of which is a low-resolution object (solvent mask) and the
other a high-resolution object (atoms). They need to be combined
through some clever scaling: if there are issues with the
low-resolution reflections this scaling can go wrong - sometimes
really badly.
Hope that helps a bit.
Cheers
Clemens
>
>
> ho
> UC Berkeley
>
> > Date: Fri, 13 Feb 2009 17:14:38 +0000
> > From: Clemens Vonrhein <[email protected]>
> > Subject: Re: unstable refinement
>
> > * resolution limits: are you suddenly including all those poorly
> > measured or non existent reflections at the low resolution end (10A
> > and lower) that are only present because the beamstop masking wasn't
> > don properly during data processing/integration?
> >
> > These bogus reflections can mess up your bulk-solvent correction and
> > scaling with weird effects. Better check the scale factors you're
> > getting during refinement and if they make sense.
> >
> >
> > Cheers
> >
> > Clemens
> >
> > ***************************************************************
> > * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
> > *
> > * Global Phasing Ltd.
> > * Sheraton House, Castle Park
> > * Cambridge CB3 0AX, UK
> > *--------------------------------------------------------------
> > * BUSTER Development Group (http://www.globalphasing.com)
--
***************************************************************
* Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
*
* Global Phasing Ltd.
* Sheraton House, Castle Park
* Cambridge CB3 0AX, UK
*--------------------------------------------------------------
* BUSTER Development Group (http://www.globalphasing.com)
***************************************************************
