[ccp4bb] Metal ion differentiation - reg
Dear all, I have solved a structure (using molecular replacement) of metallo-enzyme which may have Zn or Ni at its active site. I collected data at in-house CuKa radiation. Now, I am able to locate the active site metal ion preciously but I am not able to differentiate whether it is Zn or Ni. I computed anomalous difference map and I got good anomalous map also. But still there is ambiguity, since Zn and Ni have closest F values at CuKa radiation ( For Zn = 0.68 and For Ni = 0.51). But both, Zn and Ni have different F' values at CuKa radiation ( For Zn = -1.61 and For Ni = -3.07). My question is that, *Can we used F' value information to differentiate metal ions?.Is it possible to find whether I have Zn or Ni at active site of my enzyme using crystallographic technique?. * Thanks in advance -- *Dhanasekaran Varudharasu* Post-Doctoral Fellow Department of Oral Biology Rutgers school of Dental Medicine Rutgers Biomedical and Health Sciences Newark, NJ 07103 USA
Re: [ccp4bb] Metal ion differentiation - reg
It is difficult! Ni Zn are rather interchangable.. You dont say what resolution you have: There will be a small difference in the number of electrons you expect to see at the metal site, depending on whether it is Zn Zn2+ etc etc, and you can correct that to take account of the f' as well. So you would expect a slightly smaller peak for Ni than Zn but it will be hard to give a definitive answer unless you have high resolution data. First Q - was there Zn or Ni or both around in the crystallisation mix? Second Q: consider the coordination: This article discusses examples. Journal of Inorganic Biochemistry http://www.sciencedirect.com/science/journal/01620134 Volume 71, Issues 3–4 http://www.sciencedirect.com/science/journal/01620134/71/3, September 1998, Pages 115–127 Eleanor On 1 July 2014 16:10, Dhanasekaran Varudharasu dhana...@gmail.com wrote: Dear all, I have solved a structure (using molecular replacement) of metallo-enzyme which may have Zn or Ni at its active site. I collected data at in-house CuKa radiation. Now, I am able to locate the active site metal ion preciously but I am not able to differentiate whether it is Zn or Ni. I computed anomalous difference map and I got good anomalous map also. But still there is ambiguity, since Zn and Ni have closest F values at CuKa radiation ( For Zn = 0.68 and For Ni = 0.51). But both, Zn and Ni have different F' values at CuKa radiation ( For Zn = -1.61 and For Ni = -3.07). My question is that, *Can we used F' value information to differentiate metal ions?.Is it possible to find whether I have Zn or Ni at active site of my enzyme using crystallographic technique?. * Thanks in advance -- *Dhanasekaran Varudharasu* Post-Doctoral Fellow Department of Oral Biology Rutgers school of Dental Medicine Rutgers Biomedical and Health Sciences Newark, NJ 07103 USA
Re: [ccp4bb] Metal ion differentiation - reg
If you have a access to a synchrotron you can try double difference anomalous DDANO maps see Than et al. Acta Cryst. (2005). D61, 505–512 for an example Best Roberto On 1 Jul 2014, at 16:10, Dhanasekaran Varudharasu dhana...@gmail.commailto:dhana...@gmail.com wrote: Dear all, I have solved a structure (using molecular replacement) of metallo-enzyme which may have Zn or Ni at its active site. I collected data at in-house CuKa radiation. Now, I am able to locate the active site metal ion preciously but I am not able to differentiate whether it is Zn or Ni. I computed anomalous difference map and I got good anomalous map also. But still there is ambiguity, since Zn and Ni have closest F values at CuKa radiation ( For Zn = 0.68 and For Ni = 0.51). But both, Zn and Ni have different F' values at CuKa radiation ( For Zn = -1.61 and For Ni = -3.07). My question is that, Can we used F' value information to differentiate metal ions?. Is it possible to find whether I have Zn or Ni at active site of my enzyme using crystallographic technique?. Thanks in advance -- Dhanasekaran Varudharasu Post-Doctoral Fellow Department of Oral Biology Rutgers school of Dental Medicine Rutgers Biomedical and Health Sciences Newark, NJ 07103 USA Roberto A. Steiner Group Leader Randall Division of Cell and Molecular Biophysics King's College London roberto.stei...@kcl.ac.ukmailto:roberto.stei...@kcl.ac.uk Room 3.10A New Hunt's House Guy's Campus SE1 1UL London Phone 0044 20 78488216 Fax0044 20 78486435
Re: [ccp4bb] Metal ion differentiation - reg
Dear Dhanasekaran, I am not sure from your email whether is important for you to resolve this ambiguity with crystallographic techniques. If not, an easy and simple way would be to shoot your crystals at energies above the Zn absorption edge (which is at highest energy of the two metals) and then collect a fluorescence spectrum. This can be done at most synchrotrons with tuneable beamlines and fluorescent detector. In fact even a fixed beam with standard energy at around 12 KeV will do for Zn and Ni. Another possibility that springs to mind is inductively coupled plasma - atomic emission spectroscopy (or ICP-AES), usually available in chemistry departments. For this you don’t even need crystals, just a purified protein solution sample. HTH D From: Dhanasekaran Varudharasu [mailto:dhana...@gmail.com] Sent: 01 July 2014 16:11 To: ccp4bb Subject: [ccp4bb] Metal ion differentiation - reg Dear all, I have solved a structure (using molecular replacement) of metallo-enzyme which may have Zn or Ni at its active site. I collected data at in-house CuKa radiation. Now, I am able to locate the active site metal ion preciously but I am not able to differentiate whether it is Zn or Ni. I computed anomalous difference map and I got good anomalous map also. But still there is ambiguity, since Zn and Ni have closest F values at CuKa radiation ( For Zn = 0.68 and For Ni = 0.51). But both, Zn and Ni have different F' values at CuKa radiation ( For Zn = -1.61 and For Ni = -3.07). My question is that, Can we used F' value information to differentiate metal ions?. Is it possible to find whether I have Zn or Ni at active site of my enzyme using crystallographic technique?. Thanks in advance -- Dhanasekaran Varudharasu Post-Doctoral Fellow Department of Oral Biology Rutgers school of Dental Medicine Rutgers Biomedical and Health Sciences Newark, NJ 07103 USA
Re: [ccp4bb] Metal ion differentiation - reg
Dear Dhanasekaran Varudharasu, you won't be able to distinguish between these two metals with CuKa radiation. You should get access to a synchrotron and collect data 1.4A and 1.25A in addition to the CuKa set you already have (see http://skuld.bmsc.washington.edu/scatter/AS_form.html). The CuKa data set with no (significant) anomalous peak is your control. If there is an anomalous peak at the 1.25A data set but not at 1.4, then it should be Ni, if both data sets contain peaks, it should be Cu - provided of course you can definitely rule out any other metal. If you are sure about this, you might as well to a fluorescence scan for which of course you don't need a crystal. Best, Tim On Tue, Jul 01, 2014 at 11:10:52AM -0400, Dhanasekaran Varudharasu wrote: Dear all, I have solved a structure (using molecular replacement) of metallo-enzyme which may have Zn or Ni at its active site. I collected data at in-house CuKa radiation. Now, I am able to locate the active site metal ion preciously but I am not able to differentiate whether it is Zn or Ni. I computed anomalous difference map and I got good anomalous map also. But still there is ambiguity, since Zn and Ni have closest F values at CuKa radiation ( For Zn = 0.68 and For Ni = 0.51). But both, Zn and Ni have different F' values at CuKa radiation ( For Zn = -1.61 and For Ni = -3.07). My question is that, *Can we used F' value information to differentiate metal ions?.Is it possible to find whether I have Zn or Ni at active site of my enzyme using crystallographic technique?. * Thanks in advance -- *Dhanasekaran Varudharasu* Post-Doctoral Fellow Department of Oral Biology Rutgers school of Dental Medicine Rutgers Biomedical and Health Sciences Newark, NJ 07103 USA -- -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] Metal ion differentiation - reg
You should try X-ray fluorescence measurements on your crystals You'll see directly the presence of Zn or Ni You can also perform a data collection at energies at both sides of max f for Zn and Ni. - Christine Cavazza iRTSV/Laboratoire de Chimie et Biologie des Métaux CEA Grenoble 17 rue des Martyrs 38054 Grenoble cedex 09 Tel. : (33) 4 38 78 91 21 Fax : (33) 4 38 78 91 24 Email: christine.cava...@cea.fr De : Dhanasekaran Varudharasu dhana...@gmail.commailto:dhana...@gmail.com Répondre à : Dhanasekaran Varudharasu dhana...@gmail.commailto:dhana...@gmail.com Date : mardi 1 juillet 2014 17:10 À : CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Metal ion differentiation - reg Dear all, I have solved a structure (using molecular replacement) of metallo-enzyme which may have Zn or Ni at its active site. I collected data at in-house CuKa radiation. Now, I am able to locate the active site metal ion preciously but I am not able to differentiate whether it is Zn or Ni. I computed anomalous difference map and I got good anomalous map also. But still there is ambiguity, since Zn and Ni have closest F values at CuKa radiation ( For Zn = 0.68 and For Ni = 0.51). But both, Zn and Ni have different F' values at CuKa radiation ( For Zn = -1.61 and For Ni = -3.07). My question is that, Can we used F' value information to differentiate metal ions?. Is it possible to find whether I have Zn or Ni at active site of my enzyme using crystallographic technique?. Thanks in advance -- Dhanasekaran Varudharasu Post-Doctoral Fellow Department of Oral Biology Rutgers school of Dental Medicine Rutgers Biomedical and Health Sciences Newark, NJ 07103 USA
