Dear Martin, For well-refined structures with low residuals, +/- 3 sigma levels (the standard coot contour levels) are very small on an absolute scale. How does your 2Fo-Fc map look like? Are there really big holes in the FAD for the missing atoms with almost zero 2Fo-Fc density?
As others have mentioned, the most likely explanation will be that your ringsystem lost some planarity, which may be a butterfly motion, but also a twisting. I would edit your FAD cif file and reduce the planarity restraints at least with a factor of 10 and better even more, do a real space refinement and see what happens. For testing purposes, you could also remove the planarity restraints completely. At 1.65 Å you may have to move the atoms manually into the positive difference density since they otherwise may remain stuck in the nearby false minimum. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Martin Malý Gesendet: Montag, 6. November 2017 12:02 An: CCP4BB@JISCMAIL.AC.UK Betreff: [EXTERNAL] [ccp4bb] Radiation damage to the FAD in enzyme structure Dear colleagues, I am investigating a structure of a FAD-dependent enzyme. The electron density map suggests radiation damage to the FAD. It apparently is different from simple change of the redox state and "butterfly"-like structure. We did not find in literature possible products of radiation damage, like a removal of several atoms of the FAD. Has anyone observed such effect? To describe it in more detail, I can observe negative difference map of C2, N3, C4, and O4 atoms of flavine. Moreover, there is positive difference map close to O2 and O4 atoms thus it looks as water molecules are bound there instead of the missing FAD atoms. I am attaching parameters of the experiment: performed at synchrotron, exposition time 210 s, high resolution diffraction limit 1.65 A (<I/sigma> = 2 at shell 1.75-1.65 A). We could see a decrease of diffraction data statistics during the experiment hence we think there is significant radiation damage to the crystal. Thank you very much for ideas. Regards, Martin Maly
