Re: [ccp4bb]: Identity of unknown metal in protein crystal

[Sarah Hymowitz]

Dear Wendy, I had a similar situation (Hymowitz et al, Biochemistry 2000).  We used ICP-AES (inductively coupled plasma atomic emission spectrometry) to identify the bound metal as zinc.   Sarah On May 9, 2006, at 4:30 PM, Wendy Gordon wrote: ***  For details on how to be removed from this list visit the  *** ***          CCP4 home page http://www.ccp4.ac.uk         *** Hello- Thanks for entertaining my non-ccp4 question... I am refining a crystal structure at ~2 Angstroms resolution in which I find a large, unexpected electron density that is potentially a Zinc ion because it is coordinated by 2 Histidines, 1 Glutamic acid, and possibly a water molecule. This site appears as a strong peak in anomalous difference Patterson maps (the data was collected at SelMet wavelength 0.979 Angstroms and I believe that Zn's absorption edge occurs around 1.2 Angstroms).  The problem is, I didn't add any zinc in any purification or crystallization conditions.  I DID affinity purify the protein with nickel beads, so potentially it could be a Nickel ion. I should also say that during refinement in refmac where my Zn occupancy is held at 1, that I obtain a negative peak in the 2Fo-Fc in this position, but if I leave the site unoccupied- I get a huge positive peak- so I either have the wrong species defined or my occupancy is not 100%- right? Is there any way short of biochemical means (ITC, mutation, etc.) to figure out what species is occupying this electron density?  I have thought of atomic absorption- has anyone tried it to determine the metal species in a protein?  Does it seem possible that I could have a Zn ion in my protein crystal where the Zn could only come from our standard DI water supply? Thanks so much! Wendy Ryan Gordon Sarah G. Hymowitz Department of Protein Engineering Genentech, Inc. 1 DNA Way, South San Francisco, CA 94080 E-mail:  [EMAIL PROTECTED] Tel:    (650) 225 7819 FAX:  (650) 225 3734