Re: [ccp4bb] How to identify unknow heavy atom??
Dear All, re identifying metals, It's possible to calculate crystallographic element-specific anomalous difference maps if you can collect data on either side of the absorption edge. I have used this successfully for Sr, Zn and Mn. http://www.ncbi.nlm.nih.gov/pubmed/15858259 I have a set of scripts to conveniently calculate such a map, which I can send off list. James -- Dr. James W. Murray David Phillips Research Fellow Division of Molecular Biosciences Imperial College, LONDON Tel: +44 (0)20 759 48895 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Frank Murphy [frankvmur...@gmail.com] Sent: Wednesday, July 25, 2012 3:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] How to identify unknow heavy atom?? Dear Haytham, It should be relatively simple (and quick) to determine the metal in your sample employing either crystallized sample or solution using X-ray fluorescence (http://en.wikipedia.org/wiki/X-ray_fluorescence) at any beamline with a silicon drift detector (http://www.amptek.com/drift.html) set up for energy dispersive spectrometry (EDS). We perform these analyses regularly at NE-CAT and I am sure there are a number of other beamlines set up for the experiment. Frank Murphy Beamline Scientist NE-CAT / Cornell University frankvmur...@gmail.com
Re: [ccp4bb] How to identify unknow heavy atom??
Shameless self plug: http://scripts.iucr.org/cgi-bin/paper?fw5057 That was done with a crappy crystal in a relatively weak beamline. With a good crystal one can get cleaner data. Cheers. Jose. Jose Antonio Cuesta-Seijo, PhD Carlsberg Laboratory Gamle Carlsberg Vej 10 DK-1799 Copenhagen V Denmark Tlf +45 3327 5332 Email josea.cuesta.se...@carlsberglab.dk From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Haytham Wahba Sent: Tuesday, July 24, 2012 7:14 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] How to identify unknow heavy atom?? 1- if i have anomalous peak of unknown heavy atom, How can i identify this heavy atom in general. (different methods) 2- in my case, i see anomalous peak in heavy atom binding site (without any soaking). preliminary i did mass spec. i got Zn++ and Cu, How can i know which one give the anomalous peak in my protein. 3- there is way to know if i have Cu+ or Cu++. Haytham UdeM Biochemistry
Re: [ccp4bb] How to identify unknow heavy atom??
On Tue, Jul 24, 2012 at 10:14 AM, Haytham Wahba haytham_wa...@yahoo.com wrote: 1- if i have anomalous peak of unknown heavy atom, How can i identify this heavy atom in general. (different methods) 2- in my case, i see anomalous peak in heavy atom binding site (without any soaking). preliminary i did mass spec. i got Zn++ and Cu, How can i know which one give the anomalous peak in my protein. 3- there is way to know if i have Cu+ or Cu++. You may be able to identify the element based on the coordination geometry - I'm assuming (perhaps incorrectly) that it is actually different for Cu and Zn. Marjorie Harding has written extensively on the geometry of ion binding: http://tanna.bch.ed.ac.uk/ The only way to be certain crystallographically, if you have easy access to a synchrotron, is to collect data above and below the K edge of any candidate element, and compare the difference maps. (For monovalent ions it is more complicated, since they don't have accessible K edges.) On a home source, Cu should have a larger anomalous map peak, but I'm not sure if this will be enough to identify it conclusively. -Nat
Re: [ccp4bb] How to identify unknow heavy atom??
On Tuesday, July 24, 2012 10:22:18 am Nat Echols wrote: On Tue, Jul 24, 2012 at 10:14 AM, Haytham Wahba haytham_wa...@yahoo.com wrote: 1- if i have anomalous peak of unknown heavy atom, How can i identify this heavy atom in general. (different methods) 2- in my case, i see anomalous peak in heavy atom binding site (without any soaking). preliminary i did mass spec. i got Zn++ and Cu, How can i know which one give the anomalous peak in my protein. 3- there is way to know if i have Cu+ or Cu++. You may be able to identify the element based on the coordination geometry - I'm assuming (perhaps incorrectly) that it is actually different for Cu and Zn. Marjorie Harding has written extensively on the geometry of ion binding: http://tanna.bch.ed.ac.uk/ The only way to be certain crystallographically, if you have easy access to a synchrotron, is to collect data above and below the K edge of any candidate element, and compare the difference maps. (For monovalent ions it is more complicated, since they don't have accessible K edges.) On a home source, Cu should have a larger anomalous map peak, but I'm not sure if this will be enough to identify it conclusively. As to the SR experiment - yes. As to the home source - no. Neither Cu nor Zn has appreciable anomalous signal when excited with a Cu K-alpha home source. http://www.bmsc.washington.edu/scatter An element's emission edge (Cu K-alpha in this case) is about 1 keV below the corresponding absorption edge. This makes sense, because after absorbing a photon it can only emit at an equal or lower energy, not a higher energy. So you can't reach the Cu absorption edge, where the anomalous signal is, by exciting with Cu K-alpha. Ethan -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] How to identify unknow heavy atom??
On Tue, Jul 24, 2012 at 10:33 AM, Ethan Merritt merr...@u.washington.edu wrote: As to the home source - no. Neither Cu nor Zn has appreciable anomalous signal when excited with a Cu K-alpha home source. http://www.bmsc.washington.edu/scatter An element's emission edge (Cu K-alpha in this case) is about 1 keV below the corresponding absorption edge. This makes sense, because after absorbing a photon it can only emit at an equal or lower energy, not a higher energy. So you can't reach the Cu absorption edge, where the anomalous signal is, by exciting with Cu K-alpha. Oops, sorry, I was of course comparing the wrong numbers. -Nat
Re: [ccp4bb] How to identify unknow heavy atom??
Hello Actually, if the home source uses a copper tube, neither copper nor zinc have much of an anomalous signal at that wavelength (the energy is below the absorption edge for both). The best way is to check the location of the absorption edge at the synchrotron. Cu+ and Cu++ can be distinguished this way, but make sure the absorption scan is done before you collect data since copper(II) can be photoreduced to copper(I) in the synchrotron x-ray beam. Whether or not you can get a clue from geometry depends upon the resolution of the structure. Sue On Jul 24, 2012, at 10:22 AM, Nat Echols wrote: On Tue, Jul 24, 2012 at 10:14 AM, Haytham Wahba haytham_wa...@yahoo.com wrote: 1- if i have anomalous peak of unknown heavy atom, How can i identify this heavy atom in general. (different methods) 2- in my case, i see anomalous peak in heavy atom binding site (without any soaking). preliminary i did mass spec. i got Zn++ and Cu, How can i know which one give the anomalous peak in my protein. 3- there is way to know if i have Cu+ or Cu++. You may be able to identify the element based on the coordination geometry - I'm assuming (perhaps incorrectly) that it is actually different for Cu and Zn. Marjorie Harding has written extensively on the geometry of ion binding: http://tanna.bch.ed.ac.uk/ The only way to be certain crystallographically, if you have easy access to a synchrotron, is to collect data above and below the K edge of any candidate element, and compare the difference maps. (For monovalent ions it is more complicated, since they don't have accessible K edges.) On a home source, Cu should have a larger anomalous map peak, but I'm not sure if this will be enough to identify it conclusively. -Nat Dr. Sue A. Roberts Dept. of Chemistry and Biochemistry University of Arizona 1041 E. Lowell St., Tucson, AZ 85721 Phone: 520 621 8171 or 520 621 4168 s...@email.arizona.edu http://www.cbc.arizona.edu/xray or http://www.cbc.arizona.edu/facilities/x-ray_diffraction
Re: [ccp4bb] How to identify unknow heavy atom??
Perhaps also exafs should be mentioned--I believe the various ion species, redox states, and even binding geometry can be determined. JPK On Tue, Jul 24, 2012 at 12:37 PM, Roberts, Sue A - (suer) s...@email.arizona.edu wrote: Hello Actually, if the home source uses a copper tube, neither copper nor zinc have much of an anomalous signal at that wavelength (the energy is below the absorption edge for both). The best way is to check the location of the absorption edge at the synchrotron. Cu+ and Cu++ can be distinguished this way, but make sure the absorption scan is done before you collect data since copper(II) can be photoreduced to copper(I) in the synchrotron x-ray beam. Whether or not you can get a clue from geometry depends upon the resolution of the structure. Sue On Jul 24, 2012, at 10:22 AM, Nat Echols wrote: On Tue, Jul 24, 2012 at 10:14 AM, Haytham Wahba haytham_wa...@yahoo.com wrote: 1- if i have anomalous peak of unknown heavy atom, How can i identify this heavy atom in general. (different methods) 2- in my case, i see anomalous peak in heavy atom binding site (without any soaking). preliminary i did mass spec. i got Zn++ and Cu, How can i know which one give the anomalous peak in my protein. 3- there is way to know if i have Cu+ or Cu++. You may be able to identify the element based on the coordination geometry - I'm assuming (perhaps incorrectly) that it is actually different for Cu and Zn. Marjorie Harding has written extensively on the geometry of ion binding: http://tanna.bch.ed.ac.uk/ The only way to be certain crystallographically, if you have easy access to a synchrotron, is to collect data above and below the K edge of any candidate element, and compare the difference maps. (For monovalent ions it is more complicated, since they don't have accessible K edges.) On a home source, Cu should have a larger anomalous map peak, but I'm not sure if this will be enough to identify it conclusively. -Nat Dr. Sue A. Roberts Dept. of Chemistry and Biochemistry University of Arizona 1041 E. Lowell St., Tucson, AZ 85721 Phone: 520 621 8171 or 520 621 4168 s...@email.arizona.edu http://www.cbc.arizona.edu/xray or http://www.cbc.arizona.edu/facilities/x-ray_diffraction -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] How to identify unknow heavy atom??
Sent using BlackBerry® from Orange -Original Message- From: Jacob Keller j-kell...@fsm.northwestern.edu Sender: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK Date: Tue, 24 Jul 2012 12:55:31 To: CCP4BB@JISCMAIL.AC.UK Reply-To: Jacob Keller j-kell...@fsm.northwestern.edu Subject: Re: [ccp4bb] How to identify unknow heavy atom?? Perhaps also exafs should be mentioned--I believe the various ion species, redox states, and even binding geometry can be determined. JPK On Tue, Jul 24, 2012 at 12:37 PM, Roberts, Sue A - (suer) s...@email.arizona.edu wrote: Hello Actually, if the home source uses a copper tube, neither copper nor zinc have much of an anomalous signal at that wavelength (the energy is below the absorption edge for both). The best way is to check the location of the absorption edge at the synchrotron. Cu+ and Cu++ can be distinguished this way, but make sure the absorption scan is done before you collect data since copper(II) can be photoreduced to copper(I) in the synchrotron x-ray beam. Whether or not you can get a clue from geometry depends upon the resolution of the structure. Sue On Jul 24, 2012, at 10:22 AM, Nat Echols wrote: On Tue, Jul 24, 2012 at 10:14 AM, Haytham Wahba haytham_wa...@yahoo.com wrote: 1- if i have anomalous peak of unknown heavy atom, How can i identify this heavy atom in general. (different methods) 2- in my case, i see anomalous peak in heavy atom binding site (without any soaking). preliminary i did mass spec. i got Zn++ and Cu, How can i know which one give the anomalous peak in my protein. 3- there is way to know if i have Cu+ or Cu++. You may be able to identify the element based on the coordination geometry - I'm assuming (perhaps incorrectly) that it is actually different for Cu and Zn. Marjorie Harding has written extensively on the geometry of ion binding: http://tanna.bch.ed.ac.uk/ The only way to be certain crystallographically, if you have easy access to a synchrotron, is to collect data above and below the K edge of any candidate element, and compare the difference maps. (For monovalent ions it is more complicated, since they don't have accessible K edges.) On a home source, Cu should have a larger anomalous map peak, but I'm not sure if this will be enough to identify it conclusively. -Nat Dr. Sue A. Roberts Dept. of Chemistry and Biochemistry University of Arizona 1041 E. Lowell St., Tucson, AZ 85721 Phone: 520 621 8171 or 520 621 4168 s...@email.arizona.edu http://www.cbc.arizona.edu/xray or http://www.cbc.arizona.edu/facilities/x-ray_diffraction -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] How to identify unknow heavy atom??
I think it's done on a crystal itself, but others who know better than I can comment. JPK On Tue, Jul 24, 2012 at 1:02 PM, Theresa Hsu theresah...@live.com wrote: Does EXAFS requires same amount of samples as ICP-MS/ICP-AES? Theresa On Tue, 24 Jul 2012 12:55:31 -0500, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Perhaps also exafs should be mentioned--I believe the various ion species, redox states, and even binding geometry can be determined. JPK -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] How to identify unknow heavy atom??
Dear Haytham, may I address your points (although some nice hints have already come from previous replies) 1- if i have anomalous peak of unknown heavy atom, How can i identify this heavy atom in general. (different methods) To see anomalous peak I guess you have done the experiment at a synchrotron (Cu and Zn do not show much anomalous if measured at home sources). It is always good practice to run some fluorescence scan on a new sample (say, a novel protein). This can be done at all tunable beamlines (you might be able to have a broad scan of the entire range of wavelength, such as an MCA scan first. Once you have found a region of intrerest you can do a proper energy scan, exactly as if you were looking for peak remote and inflection wavelengths for a MAD experiment). XANES (the proper absorption peak scan) and EXAFS are complementary techniques. They can be performed in solution at dedicated beamlines and they can provide precious information on the first and second coordination spheres of the metal, but for the elemental recognition it might be sufficient what you measure from your crystal. Additional information can come from the coordination geometry of your ion, as mentioned above. 2- in my case, i see anomalous peak in heavy atom binding site (without any soaking). preliminary i did mass spec. i got Zn++ and Cu, How can i know which one give the anomalous peak in my protein. They might both give you anomalous signal... if you are working around 12.5 keV (near the Se edge), both metals have anomalous signal of 2-3 electrons... if you have only 1 heavy atom binding site you might consider collecting anomalous at both the peak of Zn and Cu (9.7 and 9.0 keV approximately)... however this experiment would not be necessary if you first find from an energy scan that only one is the metal binding your protein. If you have a mixture of the two, your final structure should take this into account (i.e. the appropriate relative occupancies for the two ions) 3- there is way to know if i have Cu+ or Cu++. Apart from the coordination geometry for the two oxidation states of Copper, I know of people doing UV-Vis spectroscopy on the crystals alongside their data collection. Micro-spectrophotometers are available on demand at most (if not all) of the synchrotron sources. If your species is radiation sensitive, a dose-dependent oxidation could be monitored by collecting multiple spectra. HTH Best regards, Marco
Re: [ccp4bb] How to identify unknow heavy atom??
Dear Haytham, It should be relatively simple (and quick) to determine the metal in your sample employing either crystallized sample or solution using X-ray fluorescence (http://en.wikipedia.org/wiki/X-ray_fluorescence) at any beamline with a silicon drift detector (http://www.amptek.com/drift.html) set up for energy dispersive spectrometry (EDS). We perform these analyses regularly at NE-CAT and I am sure there are a number of other beamlines set up for the experiment. Frank Murphy Beamline Scientist NE-CAT / Cornell University frankvmur...@gmail.com
