Many thanks for all of the suggestions. Ni2+ or Zn2+ seem to be the most likely culprits, but I plan to carry out a fluorescence scan at my next beam session.
While it’s interesting that others have observed crystallization-driven formation of disulfides, the binding of a metal ion at the given position looks much more probable. Best wishes, Chris On Wed, Jan 22, 2020 at 17:56 Sarah Bowman <sbow...@hwi.buffalo.edu> wrote: > 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 > > > > 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> 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> 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> 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 > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
