Dear James.
What small molecule programs report often looks like:
R1 = 0.1550 for 17413 Fo > 4sig(Fo) and 0.2058 for all 23715 data
R1(Free) = 0.2208 for 1938 Fo > 4sig(Fo) and 0.2766 for all 2635 data
from a well-known small molecule program being (mis)used to refine a
protein. This clearly shows the influence of the weak outer reflections,
however if properly weighted they still make a useful contribution to
the refinement. Although for historical reasons this quotes I>4sigma(F),
it corresponds to I>2sigma(I) and the structure was refined against
intensities, not Fs. In the above case, the large gap betwen R1 and
R1(free) also suggests that the structure has been a over-refined by
using anisotropic temperature factors with too lax restraints. R1 is
unweighted and based on Fs, not intensities, but in practice it seems to
be a more useful criterion than a weighted or unweighted R2 (i.e. based
on intensities).
Best wishes, George
On 10/16/2017 05:02 PM, James Holton wrote:
If you suspect that weak data (such as all the spot-free hkls beyond
your anisotropic resoluiton limits) are driving up your Rwork/Rfree,
then a good sanity check is to compute "R1". Most macromolecular
crystallographers don't know what "R1" is, but it is not only
commonplace but required in small-molecule crystallography. All you
do is throw out all the weak data, say everything with I/sigma < 2 or
3, and then re-compute your R factors. That is, use something like
"sftools" to select only clearly "observed" reflections, and feed that
data file back into your refinement program. In fact, refining only
against data with I/sigma>3 is the way macromolecular refinement was
done up until about 1990. These days, for clarity, you may want to
call the resulting Rwork/Rfree as R1work and R1free.
If you do this, and your R1work/R1free are still just as bad as
Rwork/Rfree, then weak data are not your problem. You'd be surprised
how often this is the case. Next on the list are things like wrong
symmetry choice, such as twinning masquerading as a symmetry operator,
or disorder, as in large regions of the molecule that are too fluttery
to peak above 1 sigma. The list goes on, but doing the weak-data
rejection test really helps narrow it down.
-James Holton
MAD Scientist
On 10/16/2017 3:55 AM, herman.schreu...@sanofi.com wrote:
Dear Michael,
Did you ask Phaser to check for all possible space groups? There are
still I422 and I4 you did not mention. If the space group that came
out of Phaser is different from the space group used for processing,
subsequent refinement programs may use the wrong space group from the
processing. This should be easy to check.
The other suggestion I have is to try a different processing program.
Although XDS is excellent, I find that sometimes it has difficulties
with ice rings, which reveal themselves not in the processing, but in
the subsequent refinement. You may want to try Mosflm or some other
processing program.
Best,
Herman
*Von:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag
von *Michael Jarva
*Gesendet:* Sonntag, 15. Oktober 2017 03:09
*An:* CCP4BB@JISCMAIL.AC.UK
*Betreff:* [EXTERNAL] [ccp4bb] Another troublesome dataset (High
Rfree after MR)
To add to the current anisotropic discussion I recently got a dataset
I’m unable to refine and I’m hoping I could get some help on figuring
out if there’s anything I can do.
I get a clear cut solution with Phaser using the same protein as
search model and got a TFZ of >16, LLG >200, and a packing that makes
sense, so I don’t doubt the solution. However, the maps look
terrible, more like something I would expect from a 3.65Å dataset
rather than the 2.65Å it supposedly is.
The dataset merges well in I4122 to 2.65Å with an overall Rmerge of
5% and a CC1/2 of >0.5 in the outer shell (see the bottom for full
summary). There is some minor radiation damage but I could cut out
most of it due to the high symmetry.
Xtriage reports no indication of twinning, but does say that the data
is moderately anisotropic, so I ran the unmerged data through the
StarAniso server, which reported the ellipsoidal resolution limits to
be 2.304, 2.893, and 3.039. Refining with the anisotropically
truncated data improves the maps somewhat, but I am still unable to
get the Rfree below 38%. I tried using both phenix.refine and buster
with similar results.
I’ve considered the choice of space group and tried I41, F222,
I212121 , and C2, but with the same results, and Zanuda tells me the
same thing.
Lastly, there is some minor ice rings, so my last try was to exclud
the ice ring resolutions, but this made little to no difference.
Normally I would just write this off as the data being bad but this
time all the statistics tell me this should be doable so I’m curious
what has gone wrong.
Cheers
Michael Jarva
Summary data for Project: XDSproject Crystal: XDScrystal Dataset:
XDSdataset
Overall InnerShell OuterShell
Low resolution limit 34.87 34.87 2.78
High resolution limit 2.65 8.79 2.65
Rmerge (within I+/I-) 0.052 0.026 1.595
Rmerge (all I+ and I-) 0.057 0.030 1.805
Rmeas (within I+/I-) 0.062 0.031 1.924
Rmeas (all I+& I-) 0.063 0.033 1.993
Rpim (within I+/I-) 0.032 0.017 1.042
Rpim (all I+& I-) 0.025 0.014 0.817
Rmerge in top intensity bin 0.030 - -
Total number of observations 19931 566 2681
Total number unique 3597 114 471
Mean((I)/sd(I)) 11.3 42.3 0.8
Mn(I) half-set correlation CC(1/2) 0.999 0.999 0.575
Completeness 97.9 93.1 99.6
Multiplicity 5.5 5.0 5.7
Anomalous completeness 92.4 92.1 96.8
Anomalous multiplicity 3.0 3.0 3.0
DelAnom correlation between half-sets 0.176 0.258 0.051
Mid-Slope of Anom Normal Probability 1.078 - -
Average unit cell: 82.39 82.39 69.73 90.00 90.00 90.00
Space group: I 41 2 2
Average mosaicity: 0.10
--
Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-33021 or +49-5594-227312
