[buster-discuss] Temp factors for solvent refining to very low values?

[Clemens Vonrhein]

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)