Hi Martin, I am not sure which class of flavoprotein you are working with, but in many cases, one can alter the redox state of the FAD (or FMN) in the crystalline lattice and this sometimes changes the conformation of the isoalloxazine ring of the flavin and deviates from a simple planar isoalloxazine. Is the data set that you measured with a straightforward oxidized FAD enzyme form, no funky-stuff kind of experiment? Or is this a more complicated experiment where you have attempted to bind a ligand or alter the redox state of the enzyme?
We did this with a few different flavoproteins and infrequently saw changes in the electron density for the FAD, but more often conformational changes in active-site amino acid side chains. A couple of examples here from NADH Oxidase: https://www.ncbi.nlm.nih.gov/pubmed/26506002 and Coenzyme A Disulfide Reductase: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2819376/ Item 2: Have you collected a full data set using your crystals and a home lab Cu X-ray source? X-ray induced reduction and radiation damage can be probed by comparing electron density maps from your beamline data and that from a home laboratory. This was productive to probe X-ray induced reduction of an active-site disulfide in the enzyme CoADR-RHD: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2758330/ This was all inspired by the excellent work that Carrie Wilmot and Janos Hajdu had done with a copper amine oxidase published in ’99: https://www.ncbi.nlm.nih.gov/pubmed/10576737 Good luck. -Conn — Conn Mallett, PhD SCX SBU Sales Rigaku Oxford Diffraction Carlsbad, CA 92008 Cell: +1 713 614 6891 From: CCP4 bulletin board <[email protected]> on behalf of Martin Malý <[email protected]> Reply-To: Martin Malý <[email protected]> Date: Monday, 6November, 2017 at 03:01 To: "[email protected]" <[email protected]> Subject: [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
