Re: [ccp4bb] Ca or Zn
Dear John, Thanks for pointing to the long-wavelength MX beamline project here at Diamond. The core wavelength range will be from 3 to 8 keV (1.5 to 4.1 A) and mainly targeting anomalous phasing experiments from native proteins/DNA/RNA exploiting anomalous differences from S or P. But of course, the energy range will allow to unambiguously determine cations such as K+ or Ca++. We have just started X-ray commissioning of the beamline optics and will expect first users from autumn 2013. Best regards, Armin -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jrh Sent: 31 October 2012 08:20 To: ccp4bb Subject: Re: [ccp4bb] Ca or Zn Dear Ethan, Yes indeed. An exciting development underway at Diamond Light Source led by Armin Wagner will greatly improve the ease of measurement at eg the calcium edge but also extending that wavelength range. The furin paper I quoted did nevertheless successfully show structural detail from those measurements. The Ga and Zn edges wavelengths are an easier challenge to access, i agree of course, rather the interest I tried to stress was the getting of the sigmas on the occupancies as well as the occupancies themselves, and how we did that and checked them via more than one software is also hopefully of interest. Greetings from Taipei, John Prof John R Helliwell DSc On 31 Oct 2012, at 10:37, Ethan Merritt merr...@u.washington.edu wrote: On Tuesday, 30 October 2012, Jrh wrote: This paper describes use of data either side of the calcium edge:- http://dx.doi.org/10.1107/S0907444905002556 I think that counts as not amenable (which is not quite the same as impossible. From the Methods section of that paper: Measurements in the vicinity of the K absorption edge of calcium (3.07 Å) are close to or beyond the physical limit of most beamlines typically used for X-ray crystallography [...] It was not possible to observe interpretable diffraction patterns at λ = 3 Å with the weakly diffracting furin crystals using the MAR CCD detector and exposure times up to 20 min per degree of rotation. They did soldier on and managed to collect extremely weak data below the Ca edge and stronger but still very weak data above the edge where the Ca f term was appreciable. But this is far from a routine experiment. Another approach dating back to work in 1972 by Peter Coleman and Brian Matthews http://dx.doi.org/10.1016/0006-291X(72)90750-4 is to replace the Ca with a rare earth having similar chemistry (e.g. La, whose L-1 edge is at 1.98Å). This next paper describes a case of gallium and zinc mix at one site with occupancy AND sigmas estimated with different software. This example is however much better diffraction resolution than that you may have. But hopefully will still be of interest:- http://dx.doi.org/10.1107/S0108768110011237 Ga and Zn, sure. That's an easy one. The Ga edge is at 1.196Å and the Zn edge is at 1.284Å, both edges are nicely in range for data collection and they are close enough together that little or no beamline readjustment is needed when jumping from one to the other. Ethan Prof John R Helliwell DSc On 31 Oct 2012, at 04:53, Ethan Merritt merr...@u.washington.edu wrote: On Tuesday, October 30, 2012 01:44:43 pm Adrian Goldman wrote: The coordination is indicative but not conclusive but, as I responded to the original poster, I think the best approach is to use anomalous scattering. You can measure just below and above the Ca edge, Actually, you can't. The Ca K-edge is at 3.07Å, which is not a wavelength amenable to macromolecular data collection. cheers, Ethan and similarly with the Zn, and those maps will be _highly_ indicative of the relative amounts of metal ion present. In fact, you can deconvolute so that you know the occupancy of the metals at the various sites. Adrian On 30 Oct 2012, at 22:37, Chittaranjan Das wrote: Veerendra, You can rule out if zinc has replaced calcium ions (although I agree with Nat and others that looking at the coordination sphere should give a big clue) by taking a few crystals, washing them a couple of times and subjecting them to ICP-MS analysis, if you have access to this technique. You can learn how many zinc, if any, have bound per one protein molecule in the dissolved crystal. Best Chitta - Original Message - From: Veerendra Kumar veerendra.ku...@uconn.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, October 30, 2012 2:55:33 PM Subject: [ccp4bb] Ca or Zn Dear CCP4bb users, I am working on a Ca2+ binding protein. it has 4-5 ca2+ binding sites. I purified the protein in presence of Ca2+ and crystallized the Ca2+ bound protein. I got crystal and solved the structure by SAD phasing at 2.1A resolution. I can see the clear density in the difference map for metals
Re: [ccp4bb] Ca or Zn
Dear Ethan, Yes indeed. An exciting development underway at Diamond Light Source led by Armin Wagner will greatly improve the ease of measurement at eg the calcium edge but also extending that wavelength range. The furin paper I quoted did nevertheless successfully show structural detail from those measurements. The Ga and Zn edges wavelengths are an easier challenge to access, i agree of course, rather the interest I tried to stress was the getting of the sigmas on the occupancies as well as the occupancies themselves, and how we did that and checked them via more than one software is also hopefully of interest. Greetings from Taipei, John Prof John R Helliwell DSc On 31 Oct 2012, at 10:37, Ethan Merritt merr...@u.washington.edu wrote: On Tuesday, 30 October 2012, Jrh wrote: This paper describes use of data either side of the calcium edge:- http://dx.doi.org/10.1107/S0907444905002556 I think that counts as not amenable (which is not quite the same as impossible. From the Methods section of that paper: Measurements in the vicinity of the K absorption edge of calcium (3.07 Å) are close to or beyond the physical limit of most beamlines typically used for X-ray crystallography [...] It was not possible to observe interpretable diffraction patterns at λ = 3 Å with the weakly diffracting furin crystals using the MAR CCD detector and exposure times up to 20 min per degree of rotation. They did soldier on and managed to collect extremely weak data below the Ca edge and stronger but still very weak data above the edge where the Ca f term was appreciable. But this is far from a routine experiment. Another approach dating back to work in 1972 by Peter Coleman and Brian Matthews http://dx.doi.org/10.1016/0006-291X(72)90750-4 is to replace the Ca with a rare earth having similar chemistry (e.g. La, whose L-1 edge is at 1.98Å). This next paper describes a case of gallium and zinc mix at one site with occupancy AND sigmas estimated with different software. This example is however much better diffraction resolution than that you may have. But hopefully will still be of interest:- http://dx.doi.org/10.1107/S0108768110011237 Ga and Zn, sure. That's an easy one. The Ga edge is at 1.196Å and the Zn edge is at 1.284Å, both edges are nicely in range for data collection and they are close enough together that little or no beamline readjustment is needed when jumping from one to the other. Ethan Prof John R Helliwell DSc On 31 Oct 2012, at 04:53, Ethan Merritt merr...@u.washington.edu wrote: On Tuesday, October 30, 2012 01:44:43 pm Adrian Goldman wrote: The coordination is indicative but not conclusive but, as I responded to the original poster, I think the best approach is to use anomalous scattering. You can measure just below and above the Ca edge, Actually, you can't. The Ca K-edge is at 3.07Å, which is not a wavelength amenable to macromolecular data collection. cheers, Ethan and similarly with the Zn, and those maps will be _highly_ indicative of the relative amounts of metal ion present. In fact, you can deconvolute so that you know the occupancy of the metals at the various sites. Adrian On 30 Oct 2012, at 22:37, Chittaranjan Das wrote: Veerendra, You can rule out if zinc has replaced calcium ions (although I agree with Nat and others that looking at the coordination sphere should give a big clue) by taking a few crystals, washing them a couple of times and subjecting them to ICP-MS analysis, if you have access to this technique. You can learn how many zinc, if any, have bound per one protein molecule in the dissolved crystal. Best Chitta - Original Message - From: Veerendra Kumar veerendra.ku...@uconn.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, October 30, 2012 2:55:33 PM Subject: [ccp4bb] Ca or Zn Dear CCP4bb users, I am working on a Ca2+ binding protein. it has 4-5 ca2+ binding sites. I purified the protein in presence of Ca2+ and crystallized the Ca2+ bound protein. I got crystal and solved the structure by SAD phasing at 2.1A resolution. I can see the clear density in the difference map for metals at the expected binding sites. However I had ZnCl2 in the crystallization conditions. Now i am not sure whether the observed density is for Ca or Zn or how many of them are ca or zn? Since Ca (20 elctron) and Zn (30 electron), is this value difference can be used to make a guess about different ions? is there any way we can find the electron density value at different peaks? Thank you Veerendra
Re: [ccp4bb] Ca or Zn
calculate an anomalous map, you should see the Zn signal even if you collected at the SeMet peak. Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu On Oct 30, 2012, at 2:55 PM, Kumar, Veerendra wrote: Dear CCP4bb users, I am working on a Ca2+ binding protein. it has 4-5 ca2+ binding sites. I purified the protein in presence of Ca2+ and crystallized the Ca2+ bound protein. I got crystal and solved the structure by SAD phasing at 2.1A resolution. I can see the clear density in the difference map for metals at the expected binding sites. However I had ZnCl2 in the crystallization conditions. Now i am not sure whether the observed density is for Ca or Zn or how many of them are ca or zn? Since Ca (20 elctron) and Zn (30 electron), is this value difference can be used to make a guess about different ions? is there any way we can find the electron density value at different peaks? Thank you Veerendra
Re: [ccp4bb] Ca or Zn
How would you distinguish between a mixture of Ca and Zn in the same locations? From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Kumar, Veerendra [veerendra.ku...@uconn.edu] Sent: Tuesday, October 30, 2012 1:55 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Ca or Zn Dear CCP4bb users, I am working on a Ca2+ binding protein. it has 4-5 ca2+ binding sites. I purified the protein in presence of Ca2+ and crystallized the Ca2+ bound protein. I got crystal and solved the structure by SAD phasing at 2.1A resolution. I can see the clear density in the difference map for metals at the expected binding sites. However I had ZnCl2 in the crystallization conditions. Now i am not sure whether the observed density is for Ca or Zn or how many of them are ca or zn? Since Ca (20 elctron) and Zn (30 electron), is this value difference can be used to make a guess about different ions? is there any way we can find the electron density value at different peaks? Thank you Veerendra
Re: [ccp4bb] Ca or Zn
On Tue, Oct 30, 2012 at 12:12 PM, Jim Pflugrath jim.pflugr...@rigaku.com wrote: How would you distinguish between a mixture of Ca and Zn in the same locations? How often would they be likely to bind in the same place? Some of the other transition metals are difficult to tell apart, but Ca and Zn have very different coordination preferences. -Nat
Re: [ccp4bb] Ca or Zn
On 10/30/12 15:02, Bosch, Juergen wrote: calculate an anomalous map, you should see the Zn signal even if you collected at the SeMet peak. Jürgen .. Ca can have a noticeable anomalous signal of its own, if your data are good. If the possibility to collect new data exists, collecting above and below the Zn edge should be informative. (Zn K edge 1.28 A = 9.6586 keV) -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Ca or Zn
If you calculate an edgeplot via Ethan's server: http://skuld.bmsc.washington.edu/scatter/AS_form.html you'll see that Zn @1Å has about 3 anomalous electrons whereas Ca less than 1, so assuming occupancy of 1 the stronger anomalous signal should give you a hint, second looking at the refined B-values if you placed the wrong metal should also coincide with your correct choice of metal. The question with the mixture, well lets wave some hands and bring in biological relevance. If the protein is supposed to do something with Ca then I would say so, even if Zn is bound. If the anomalous signal is comparable to the SeMet sites then I would model Zn there but say it's likely not physiological as Ca is required by this enzyme. Jürgen On Oct 30, 2012, at 3:12 PM, Jim Pflugrath wrote: How would you distinguish between a mixture of Ca and Zn in the same locations? From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Kumar, Veerendra [veerendra.ku...@uconn.edu] Sent: Tuesday, October 30, 2012 1:55 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Ca or Zn Dear CCP4bb users, I am working on a Ca2+ binding protein. it has 4-5 ca2+ binding sites. I purified the protein in presence of Ca2+ and crystallized the Ca2+ bound protein. I got crystal and solved the structure by SAD phasing at 2.1A resolution. I can see the clear density in the difference map for metals at the expected binding sites. However I had ZnCl2 in the crystallization conditions. Now i am not sure whether the observed density is for Ca or Zn or how many of them are ca or zn? Since Ca (20 elctron) and Zn (30 electron), is this value difference can be used to make a guess about different ions? is there any way we can find the electron density value at different peaks? Thank you Veerendra .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] Ca or Zn
Do occupancy refinement. Especially if you use the new anomalous refinement option available in refmac, phenix.refine and other packages as well, you should be able to get a pretty reliable number for how many real electrons as well as anomalous electrons are at each site. At almost any reasonable B-factor a Ca+2 looks a LOT like a Zn+2 at 64% occupancy, and the f will also be very different (depends on your wavelength). If the results are not abundantly clear, then you can get a kinda-sorta error bar by starting refinement with different occupancies, and perhaps kicking the starting model coordinates around (to remove bias). If the occupancy keeps refining back to the same value, then you can be reasonably confident that it is well-determined by your data. If the occupancy doesn't appear to stably return to the same value, or at least values different enough to say it is Zn vs Ca, then the identity of your metal sites is NOT well-determined by your data. -James Holton MAD Scientist On 10/30/2012 7:55 PM, Kumar, Veerendra wrote: Dear CCP4bb users, I am working on a Ca2+ binding protein. it has 4-5 ca2+ binding sites. I purified the protein in presence of Ca2+ and crystallized the Ca2+ bound protein. I got crystal and solved the structure by SAD phasing at 2.1A resolution. I can see the clear density in the difference map for metals at the expected binding sites. However I had ZnCl2 in the crystallization conditions. Now i am not sure whether the observed density is for Ca or Zn or how many of them are ca or zn? Since Ca (20 elctron) and Zn (30 electron), is this value difference can be used to make a guess about different ions? is there any way we can find the electron density value at different peaks? Thank you Veerendra
Re: [ccp4bb] Ca or Zn
Veerendra, You can rule out if zinc has replaced calcium ions (although I agree with Nat and others that looking at the coordination sphere should give a big clue) by taking a few crystals, washing them a couple of times and subjecting them to ICP-MS analysis, if you have access to this technique. You can learn how many zinc, if any, have bound per one protein molecule in the dissolved crystal. Best Chitta - Original Message - From: Veerendra Kumar veerendra.ku...@uconn.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, October 30, 2012 2:55:33 PM Subject: [ccp4bb] Ca or Zn Dear CCP4bb users, I am working on a Ca2+ binding protein. it has 4-5 ca2+ binding sites. I purified the protein in presence of Ca2+ and crystallized the Ca2+ bound protein. I got crystal and solved the structure by SAD phasing at 2.1A resolution. I can see the clear density in the difference map for metals at the expected binding sites. However I had ZnCl2 in the crystallization conditions. Now i am not sure whether the observed density is for Ca or Zn or how many of them are ca or zn? Since Ca (20 elctron) and Zn (30 electron), is this value difference can be used to make a guess about different ions? is there any way we can find the electron density value at different peaks? Thank you Veerendra
Re: [ccp4bb] Ca or Zn
This doesn't really give a useful answer. It tells you the overall composition, but there is no means of knowing whether the metal ions are equally present at all sites: some sites can favour Zn over Ca and vice versa. Flame spectroscopy also works but has the same issue - and both have the problem that you don't really know whether you have got rid of all metal ions bound weakly to the surface of the protein/crystal and most crystals, being small, have a humongous surface-to-volume ratio. The coordination is indicative but not conclusive but, as I responded to the original poster, I think the best approach is to use anomalous scattering. You can measure just below and above the Ca edge, and similarly with the Zn, and those maps will be _highly_ indicative of the relative amounts of metal ion present. In fact, you can deconvolute so that you know the occupancy of the metals at the various sites. Adrian On 30 Oct 2012, at 22:37, Chittaranjan Das wrote: Veerendra, You can rule out if zinc has replaced calcium ions (although I agree with Nat and others that looking at the coordination sphere should give a big clue) by taking a few crystals, washing them a couple of times and subjecting them to ICP-MS analysis, if you have access to this technique. You can learn how many zinc, if any, have bound per one protein molecule in the dissolved crystal. Best Chitta - Original Message - From: Veerendra Kumar veerendra.ku...@uconn.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, October 30, 2012 2:55:33 PM Subject: [ccp4bb] Ca or Zn Dear CCP4bb users, I am working on a Ca2+ binding protein. it has 4-5 ca2+ binding sites. I purified the protein in presence of Ca2+ and crystallized the Ca2+ bound protein. I got crystal and solved the structure by SAD phasing at 2.1A resolution. I can see the clear density in the difference map for metals at the expected binding sites. However I had ZnCl2 in the crystallization conditions. Now i am not sure whether the observed density is for Ca or Zn or how many of them are ca or zn? Since Ca (20 elctron) and Zn (30 electron), is this value difference can be used to make a guess about different ions? is there any way we can find the electron density value at different peaks? Thank you Veerendra
Re: [ccp4bb] Ca or Zn
On Tuesday, October 30, 2012 01:44:43 pm Adrian Goldman wrote: The coordination is indicative but not conclusive but, as I responded to the original poster, I think the best approach is to use anomalous scattering. You can measure just below and above the Ca edge, Actually, you can't. The Ca K-edge is at 3.07Å, which is not a wavelength amenable to macromolecular data collection. cheers, Ethan and similarly with the Zn, and those maps will be _highly_ indicative of the relative amounts of metal ion present. In fact, you can deconvolute so that you know the occupancy of the metals at the various sites. Adrian On 30 Oct 2012, at 22:37, Chittaranjan Das wrote: Veerendra, You can rule out if zinc has replaced calcium ions (although I agree with Nat and others that looking at the coordination sphere should give a big clue) by taking a few crystals, washing them a couple of times and subjecting them to ICP-MS analysis, if you have access to this technique. You can learn how many zinc, if any, have bound per one protein molecule in the dissolved crystal. Best Chitta - Original Message - From: Veerendra Kumar veerendra.ku...@uconn.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, October 30, 2012 2:55:33 PM Subject: [ccp4bb] Ca or Zn Dear CCP4bb users, I am working on a Ca2+ binding protein. it has 4-5 ca2+ binding sites. I purified the protein in presence of Ca2+ and crystallized the Ca2+ bound protein. I got crystal and solved the structure by SAD phasing at 2.1A resolution. I can see the clear density in the difference map for metals at the expected binding sites. However I had ZnCl2 in the crystallization conditions. Now i am not sure whether the observed density is for Ca or Zn or how many of them are ca or zn? Since Ca (20 elctron) and Zn (30 electron), is this value difference can be used to make a guess about different ions? is there any way we can find the electron density value at different peaks? Thank you Veerendra -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] Ca or Zn
On Tuesday, 30 October 2012, Jrh wrote: This paper describes use of data either side of the calcium edge:- http://dx.doi.org/10.1107/S0907444905002556 I think that counts as not amenable (which is not quite the same as impossible. From the Methods section of that paper: Measurements in the vicinity of the K absorption edge of calcium (3.07 Å) are close to or beyond the physical limit of most beamlines typically used for X-ray crystallography [...] It was not possible to observe interpretable diffraction patterns at λ = 3 Å with the weakly diffracting furin crystals using the MAR CCD detector and exposure times up to 20 min per degree of rotation. They did soldier on and managed to collect extremely weak data below the Ca edge and stronger but still very weak data above the edge where the Ca f term was appreciable. But this is far from a routine experiment. Another approach dating back to work in 1972 by Peter Coleman and Brian Matthews http://dx.doi.org/10.1016/0006-291X(72)90750-4 is to replace the Ca with a rare earth having similar chemistry (e.g. La, whose L-1 edge is at 1.98Å). This next paper describes a case of gallium and zinc mix at one site with occupancy AND sigmas estimated with different software. This example is however much better diffraction resolution than that you may have. But hopefully will still be of interest:- http://dx.doi.org/10.1107/S0108768110011237 Ga and Zn, sure. That's an easy one. The Ga edge is at 1.196Å and the Zn edge is at 1.284Å, both edges are nicely in range for data collection and they are close enough together that little or no beamline readjustment is needed when jumping from one to the other. Ethan Prof John R Helliwell DSc On 31 Oct 2012, at 04:53, Ethan Merritt merr...@u.washington.edu wrote: On Tuesday, October 30, 2012 01:44:43 pm Adrian Goldman wrote: The coordination is indicative but not conclusive but, as I responded to the original poster, I think the best approach is to use anomalous scattering. You can measure just below and above the Ca edge, Actually, you can't. The Ca K-edge is at 3.07Å, which is not a wavelength amenable to macromolecular data collection. cheers, Ethan and similarly with the Zn, and those maps will be _highly_ indicative of the relative amounts of metal ion present. In fact, you can deconvolute so that you know the occupancy of the metals at the various sites. Adrian On 30 Oct 2012, at 22:37, Chittaranjan Das wrote: Veerendra, You can rule out if zinc has replaced calcium ions (although I agree with Nat and others that looking at the coordination sphere should give a big clue) by taking a few crystals, washing them a couple of times and subjecting them to ICP-MS analysis, if you have access to this technique. You can learn how many zinc, if any, have bound per one protein molecule in the dissolved crystal. Best Chitta - Original Message - From: Veerendra Kumar veerendra.ku...@uconn.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, October 30, 2012 2:55:33 PM Subject: [ccp4bb] Ca or Zn Dear CCP4bb users, I am working on a Ca2+ binding protein. it has 4-5 ca2+ binding sites. I purified the protein in presence of Ca2+ and crystallized the Ca2+ bound protein. I got crystal and solved the structure by SAD phasing at 2.1A resolution. I can see the clear density in the difference map for metals at the expected binding sites. However I had ZnCl2 in the crystallization conditions. Now i am not sure whether the observed density is for Ca or Zn or how many of them are ca or zn? Since Ca (20 elctron) and Zn (30 electron), is this value difference can be used to make a guess about different ions? is there any way we can find the electron density value at different peaks? Thank you Veerendra
