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

[Ashley Pike]

Hi all,
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. 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

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) - oddly it is always the same solvent molecules that hit 3.00. 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). 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). Hints for what to look for to diagnose the problem?

Suggestions welcome,
Many thanks,
Ashley
--
Dr. Ashley Pike

Membrane Protein Crystallography | Structural Genomics Consortium | University of Oxford
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