When the JCSG was in production, we would routinely collect X-ray emission
spectra from our samples to look for metals. We also saved all crystals
after data collection and if we saw extra anomalous peaks in the maps
during refinement, we would put the crystal up again and collect
quick low resolution data sets ~50 eV above and below the absorption
edges of the candidate elements. Then by comparing the resulting
anomalous maps, we could identify the metal by a large decrease
in anomalous peak height / integrated peak intensity between the above
and the below edge data sets. (one example is described in Acta Cryst.
(2010). F66, 1198-1204 https://doi.org/10.1107/S1744309109025196 )
We even identified a few cases where we had sites with mixed occupancy
of different metals which showed changes in the peak at a particular
site across more than one absorption edge.
There was a recent Acta D paper by Julia Griese and Martin Högbom
(Acta Cryst. (2019). D75, 764-771 https://doi.org/10.1107/S2059798319009926 )
where they undertake a more formal derivation of site specific
quantification based on anomalous maps taken just at the
50 eV above edge location.
While the above/below edge method takes twice as many data sets, I think
it simplifies the analysis. When comparing changes in anomalous
maps across 100 eV pairs of data sets, that small energy shift does not
change f" very much except for the metal whose edge you are crossing. Thus,
you can cancel out the f" contribution from other metals. As others already
mentioned, using S or Se atoms from the protein can still be a useful control.
Regards,
Mitch
Quoting Sarah Bowman <sbow...@hwi.buffalo.edu>:
This conversation brings up the question of whether it would be good
practice to always do a fluorescence scan of a crystal sample,
whether it is known to be a metalloprotein or not...
Energy dispersive X-ray fluorescence spectra (accessible at most
synchrotron sources at this point) can often tell you whether there
is metal in your sample and, because the transitions are element
specific, what the metal identity is (although it can't place the
metal - you need the anomalous difference for that).
Note also there is a recent paper out in JACS using PIXE to
investigate metalloproteins in the PDB which resolved a number of
misidentified or missing metal atoms in the structures:
High-Throughput PIXE as an Essential Quantitative Assay for Accurate
Metalloprotein Structural Analysis: Development and Application J.
Am. Chem. Soc. 2020, 142, 1, 185-197
https://doi.org/10.1021/jacs.9b09186
Metals are everywhere!!
Cheers,
Sarah
Sarah EJ Bowman, PhD
Associate Research Scientist, Hauptman-Woodward Medical Research Institute
Director, High-Throughput Crystallization Screening Center
Research Associate Professor, Department of Biochemistry, University
at Buffalo
Research Webpage<https://hwi.buffalo.edu/scientist-directory/sbowman/>
www.getacrystal.org<http://www.getacrystal.org>
sbow...@hwi.buffalo.edu<mailto:sbow...@hwi.buffalo.edu>
716-898-8623
From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of
Christian Roth <christianroth...@gmail.com>
Reply-To: Christian Roth <christianroth...@gmail.com>
Date: Wednesday, January 22, 2020 at 12:17 PM
To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK>
Subject: Re: Unusual monomer-monomer interface in crystal
Hi Chris,
it could be all Ni besides Zn. I've seen Ni carried over from the
initial metal affinity chromatography I presume.
Cheers Christian
Chris Fage <fage...@gmail.com<mailto:fage...@gmail.com>> schrieb am
Di., 21. Jan. 2020, 21:23:
Thanks to Guenter and Eleanor for their replies. I mentioned that
there is not adequate space for a metal ion at the described
interfaces. Nevertheless, placement of a metal ion, followed by
refinement in Phenix, repositions the side chains significantly so
as to make room for the ion without distorting geometry. There is
also a very strong difference signal centered between the four side
chains. This agrees with the MS data, which indicate a monomeric
state without a disulfide linkage. Now, I just need to identify the
metal. A Zn2+ seems to fit well based on coordination number and
interatomic distances, as Guenter exemplified.
Best wishes,
Chris
On Tue, Jan 21, 2020 at 6:33 PM Eleanor Dodson
<0000176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk<mailto:0000176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>>
wrote:
Easy to check whether it is a metal by looking at an anomalous
difference map..
But there are examples of di-sulphides formed between symmetry
related molecules..
Query the wwwpdb -
On Tue, 21 Jan 2020 at 18:06, Guenter Fritz
<guenter.fritz.phenix.c...@gmail.com<mailto:guenter.fritz.phenix.c...@gmail.com>>
wrote:
Dear Chris, are there any metal ions in your buffer or in your
protein. We had a similar looking case. A Zn2+ ion bridged two
monomers. Our protein is a Zn2+ binding protein. The Zn2+ originated
from some denatured protein in the drop. No extra Zn2+ was in the
crystallization buffer.
http://www.rcsb.org/structure/5CHT
https://www.nature.com/articles/nsmb.3371
HTH
Guenter
Dear CCP4BB Users,
I've recently solved the ~2.2 angstrom structure of a protein. In my
electron density there are unusual monomer-monomer interfaces
involving pairs of His and Cys residues (see
https://ibb.co/wdWBcdk). Note the positive Fo-Fc density between the
four side chains. As there is not adequate space for a water
molecule or metal ion, perhaps the Cys residues are partially tied
up disulfide bonds? However, the protein looks to be fully monomeric
based on LC-MS measurements. Has anyone else observed crystal-driven
formation of disulfide bridges?
Aside from this region, there is no extensive interface between
momoners, and PDBePISA suggests a monomeric state.
Thanks in advance for any advice!
Best wishes,
Chris
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