Dear Martin, Now it makes more sense. Is there any evidence from biochemical studies with your flavoprotein that there is any partial FAD dissociation from the enzyme? Can you reconstitute the FAD to the apoenzyme in solution or in the crystal? For example, in figure 2 of this article: https://www.ncbi.nlm.nih.gov/pubmed/16511173
You may have a portion of the proteins in the crystalline lattice in the holoenzyme (with FAD bound) and a portion of apoenzyme with water molecules filling the space ‘normally’ occupied by the flavin. Prior to crystallisation, was there any FAD that came through in the filtrate during the final concentration step? This would be a good indication that you have some apoenzyme in the protein which was crystallised – it is possible to selectively crystallise only the holoenzyme, but likely not unprecedented to have a mixed population in the crystal, although I do not have a good reference for that off the top of my head. 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: Tuesday, 7November, 2017 at 05:16 To: "[email protected]" <[email protected]> Subject: Re: [ccp4bb] Radiation damage to the FAD in enzyme structure Dear Ilme, thank you for your answer. We are sure that we have full occupancy. However, we do not know the dose exactly. Nevertheless, after reading the other answers, we consider that the X-ray radiation is not the reason but the damage is related to the age of crystal. Best regards, Martin Maly ________________________________ Od: Ilme Schlichting <[email protected]> Odesláno: 6. listopadu 2017 12:38:17 Komu: Martin Malý Předmět: Re: [ccp4bb] Radiation damage to the FAD in enzyme structure Hello, Are you sure you had full occupancy to begin with? Can be checked very easily by absorption spectroscopy. It seems much more likely that you have partial occupancy only and waters filling the void. What is your dose? That is the critical parameter, not the exposure time. Regards, Ilme Schlichting On 11/6/2017 12:01 PM, Martin Malý wrote: > 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 > -- Ilme Schlichting Max-Planck Institute for Medical Research Department of Biomolecular Mechanisms Jahn-Str. 29, D-69120 Heidelberg, Germany fon : +49 6221 486 500; fax: +49 6221 486 585 e-mail direct: [email protected] e-mail secretary: [email protected] (please cc. communications concerning scheduling)
