 |
 |
The geometry is difficult to see from a static image, but it looks like you possibly have a His(2)Glu(4) coordination environment around what looks like one likely metal ion, based on the difference density. Zn(II) is typcially (but not always) tetrahedral with Zn-O/N bond distances of approximately 2.0 A. You should look carefully at the coordination environment of the putative metal ion to determine if it is tetrahedral, pentacoordinate (like a trigonal bipyramid), or octahedral. Zn(II) can adopt all of those coordination environments, but octahedral environment could be a lot of different metals, e.g., Fe(II), etc. Octahedral bond lengths for metal-bound ligands will be different from tetrahedral or pentacoordinate bond lengths. BTW, both Zn and Fe are very common adventitious metal ions in protein preps, and are very difficult to exclude. You have options for directly identifying the metal in the home lab: If the metal is bound tight enough (and it may be stoichiometric) then (1) ICP-OES or (2) ICP-MS or (3) TXRF of a protein sample should be able to detect its presence. TXRF is really nice in that there is no prep and it will work on a few uL of sample, but the others are also quite sensitive, and will only require 10-50 uL of sample depending on your protein concentration. When you are a building a metalloenzyme model you should really have some solid evidence that a metal ion is present by (1) inclusion in the crystallization medium, (2) direct determination by an analytical technique, (3) UV-visible spectroscopy (when appropriate--obviously Zn(II) is d10 and silent in the visible d-d transition wavelength range) and/or (4) appropriate coordination geometry and bond lengths. Cheers, _______________________________________ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [log in to unmask] On 1/16/2013 2:15 PM, ruisher hu wrote: > Hi, Dear All, > > I recently got a dataset about 2.3 A resolution, however, I got some > trouble assigning the metal sites. It suppose to have multiple binding > site(possibly four) around those four glu residues in the center (see > the attached figure), however, it shows up a huge single positive > density ,clustered in the binding center. The signal is pretty strong > and I think zn is definitely there. When I tried to put four zns > around, the geometry doesn't look very good and there is still some > positive density in the center (although get weaker) and the bfactor > of metals are high like 100. Does anyone know what's going on?Does it > mean only one single site in the middle?Or maybe just metals are too > mobile? What's the best way to tell how many metal sites are actually > there?Which experiment can I use to test? Thanks very much. > > Best, > > R >