[ccp4bb] ligand density

[Karolina Michalska]

Hi all,

I have a ligand-corresponding density which I cannot identify:

http://s1194.photobucket.com/albums/aa376/dziuba22/?action=view&current=coot1.png

Here is the story. I collected a couple of datasets, three of them belong
to methylated protein and one to non-methylated. The protein is putative
chitinase.
Methylation reaction was carried out in a buffer composed of HEPES, NaCl,
BME, imidazole, glycerol. For the reaction, formaldehyde and
dimethylamine borane complex were added and reaction was quenched with
glycine. After that, buffer was exchanged to HEPES, NaCl and DTT. The
non-methylated protein was stored in the same buffer. Both proteins are
His-tagged.
The density that I'm showing here is present only in the crystals of
methylated protein, both were grown form PEG3350 and either NaI or NaSCN.
I have been trying to model something and histidine nicely fits on the
left side, but right fragment seems to be more complicated. In terms of
geometry, D-Thr-L-His would fit:

http://s1194.photobucket.com/albums/aa376/dziuba22/?action=view&current=coot4.png

But the problem is that instead of CB from Thr I need a proton donor to
interact with a neighboring Asp (unless it is C-H...O interaction).
Moreover, if such a compound were grabbed from bacterial cells I would
expect it to be present also in non-methylated
crystals, which is not the case. This made me think of some sort of
intermediate of methylation reaction as dimethylamine borane complex
would fit in Thr position with nitrogen atom replacing CB, but with
borane I could not find an explanation for an equivalent of Thr amino
group. Moreover, I do not know what was the source of free histidine in
the methylated protein prep.

A separated big peak on right corresponds to an anion and it is also
conserved in methylated crystals. In case of crystals obtained from NaI
it is an iodide anion.

I will appreciate any suggestions,

Karolina