[buster-discuss] Temp factors for solvent refining to very low values?
Clemens Vonrhein
vonrhein at globalphasing.com
Fri Apr 21 11:57:30 CEST 2017
Dear Ashley,
(sorry for the late reply).
On Wed, Apr 05, 2017 at 08:13:34PM +0000, Ashley Pike wrote:
> Have run into an issue with a structure I am refining (3.2A). The
> temperature factors for the refined solvent molecules show a very
> weird distribution with half having B's below 10 and the remainder
> around 30-50 (I have modelled around 30 solvent in total) - see
> excerpt below.
How many waters are we talking about: hundreds or a few dozen?
What is the source of that water model: are these waters from a
high-resolution native/apo structure or does it come fro automatic or
manual water update starting with an empty model?
> Average B for protein chains is around 45 and other "solvent"
> heterogroups (lipid) have B's around 30-70. The solvent is not part
> of a TLS group (single TLS group per protein chain). Refining with
> autoncs / TLSalternate
Sounds all sensible, yes.
> HETATM10043 O HOH C 1 37.384 85.915 48.227 1.00 3.00 O
> HETATM10044 O HOH C 2 41.165 85.554 55.195 1.00 33.13 O
> HETATM10045 O HOH C 3 38.207 79.024 102.082 1.00 40.10 O
> HETATM10046 O HOH C 4 31.928 83.353 106.446 1.00 3.00 O
> HETATM10047 O HOH C 5 59.895 83.126 112.069 1.00 53.13 O
> HETATM10048 O HOH C 6 71.980 110.118 85.303 1.00 41.10 O
> HETATM10049 O HOH C 7 35.067 77.191 94.667 1.00 23.21 O
> HETATM10050 O HOH C 8 58.834 101.563 100.861 1.00 7.26 O
> HETATM10051 O HOH C 9 62.444 104.317 96.908 1.00 3.00 O
> HETATM10052 O HOH C 10 31.634 95.839 49.139 1.00 6.41 O
> .......
>
> Solvent peaks return to +fofc maps if I leave them out. If I reset
> B's to 30.0 and re-refine, they converge to similar values. I am
> assuming 3.00 is a minimum Bvalue allowed (similar to Bmax of 300)
Correct.
> oddly it is always the same solvent molecules that hit 3.00.
If it were different waters hitting that, it would be even weirder I
think: then it would show some instability in parameter
refinement. Whereas the fact that it is always the same waters seem to
suggest there is something "special" about those.
> I do not think there are likely to be many additional ions about - I
> do have anomalous peaks for calcium sites (whose B's are in the
> range 25-40).
What other ions (apart from Ca) have you already fitted? Could it be
Mg or Na - which wouldn't show up in anomalous maps. These abnormal
B-factors could also arise because peaks corresponding to something
like disordered phosphate groups on the surface of the lipid membrane
have been assigned to waters, that then need a very low B-factor in
order to pretend to be a disordered phosphate.
Anything interesting or special about the environment of those waters
(H-bonding partners, geometry)?
> I am at the end of rebuilding with Rwork/Rfree of 25.0/27.0,
> fom 0.8.
>
> Any reason for this behaviour (maybe I am trying to push my luck
> refining B's but BUSTER has produced/refined sensible solvent
> molecules in the past with other datasets at similar
> resolutions).
3.2A doesn't seem to be too unusual when refining B's in
BUSTER. Unless of course that 3.2A label hides other data issues like
anisotropy, incompleteness, ice-rings etc.
> Hints for what to look for to diagnose the problem?
Cheers
Clemens & Gerard (for buster-develop)
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