On Jun 1, 2014, at 10:08 PM, Eze Chivi <[log in to unmask]> wrote:
> Hello, when I refine my structure, I see negative density around the disulfide bond. I have 7 copies per ASU, and I can see this density in many of them. In some cases, I see positive density also (negative in the center of the straight line linking S atoms, and positive in both sides). What can I try to solve it? Is it due to radiation damage? Alternative conformation (partial oxidation)? Incorrect disulfide geometry parameters? My resolution is 2.1 A, R/Rfree are around 0.220/0.243, and similar results with refmac5, phenix and PDBREDO. Please find two example pictures in attachment.
> Thanks for your help!
>
>
> Ezequiel
> <disulfide.jpg><disulfide2.jpg>
Dear Ezequiel:
I apologize if I missed some crucial fact, but the simplest naive explanation would be the disulfide bonds became reduced somewhere along the line, since that would push the non-bonded sulfur atoms apart, relative to where you have them. You should be able to tell with a denaturing gel (run in the absence of DTT or equivalent). I’ve found a bit of Cu(II)-1,10-phenanthroline can help keep the disulfides oxidized. Try re-refining without the disulfide linkages, and see if the difference densities go away.
Bill
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