This doesn't really give a useful answer. It tells you the overall composition, but there is no means of knowing whether the metal ions are equally present at all sites: some sites can favour Zn over Ca and vice versa. Flame spectroscopy also works but has the same issue - and both have the problem that you don't really know whether you have got rid of all metal ions bound weakly to the surface of the protein/crystal and most crystals, being small, have a humongous surface-to-volume ratio.
The coordination is indicative but not conclusive but, as I responded to the original poster, I think the best approach is to use anomalous scattering. You can measure just below and above the Ca edge, and similarly with the Zn, and those maps will be _highly_ indicative of the relative amounts of metal ion present. In fact, you can deconvolute so that you know the occupancy of the metals at the various sites. Adrian On 30 Oct 2012, at 22:37, Chittaranjan Das wrote: > Veerendra, > > You can rule out if zinc has replaced calcium ions (although I agree with Nat > and others that looking at the coordination sphere should give a big clue) by > taking a few crystals, washing them a couple of times and subjecting them to > ICP-MS analysis, if you have access to this technique. You can learn how many > zinc, if any, have bound per one protein molecule in the dissolved crystal. > > Best > Chitta > > > > ----- Original Message ----- > From: "Veerendra Kumar" <[email protected]> > To: [email protected] > Sent: Tuesday, October 30, 2012 2:55:33 PM > Subject: [ccp4bb] Ca or Zn > > Dear CCP4bb users, > > I am working on a Ca2+ binding protein. it has 4-5 ca2+ binding sites. I > purified the protein in presence of Ca2+ and crystallized the Ca2+ bound > protein. I got crystal and solved the structure by SAD phasing at 2.1A > resolution. I can see the clear density in the difference map for metals at > the expected binding sites. However I had ZnCl2 in the crystallization > conditions. Now i am not sure whether the observed density is for Ca or Zn or > how many of them are ca or zn? Since Ca (20 elctron) and Zn (30 electron), > is this value difference can be used to make a guess about different ions? > is there any way we can find the electron density value at different peaks? > > Thank you > > Veerendra
