Re: [ccp4bb] Ambiguous metal ion at active site.
Dilip, Depending upon your data and the wavelength you collected it at - can you generate an anomalous difference map? First off - if there is a significant anomalous peak, this will exclude Magnesium at it's K-edge is at ~9.5Å You could also compare the peak heights of your unknown metal ions with those of identifiable anomalous scatterers, such as well defined CYS or MET Sulphur atoms (again, depending upon the wavelength you collected at), and obtain a rough* value for the f value of the anomalous scatterers responsible for the peaks. You can then use the data here http://skuld.bmsc.washington.edu/scatter/ to come up with a list of potential metal ions. Also, you might check distances from the centre of the peak to the co-ordinating atoms and compare with known co-ordinating distances for metal-ions (see http://mespeus.bch.ed.ac.uk/tanna/ for target distances). You could also look at the co-ordination geometry. Chimera has a fantastic tool for doing this (just ctrl-double click on the metal ion and select co-ordination geometry). HTH, Dave *rough, but better than nothing. [image: David Briggs on about.me] David Briggs about.me/david_briggs http://about.me/david_briggs On 9 July 2015 at 10:35, Dilip Kumar dku...@igib.in wrote: Dear All I have solved a structure of a metal-ion dependent exonuclease enzyme. In homologous structures, two or three Manganese ions are present at catalytic center. However, I have used 2 mM MgCl2 in protein purification buffer. I tried to fit both of these metal ions at catalytic center but in both cases it still shows green density (Sigma level ~ 7) in difference map and low b-factor (10) for these metal ions. For better understanding I have attached the screenshot of metal ions with difference map on. Please suggest me the possible reasons or methods to validate the presence of any other metal ions at catalytic center. Thanks in advance. Regards Dilip Kumar Research Associate Chemical and Systems Biology Unit CSIR-Institute of Genomics Integrative Biology Delhi-110025
Re: [ccp4bb] Ambiguous metal ion at active site.
Hi Dilip, There are a few things you might try. You have to make sure that all coordinating atoms (including waters) are in your model. If you refined with TLS for the protein, try resetting the B-factors for all atoms and refine without TLS. If this reduces the difference density, you could also try including your metal atoms in the TLS group of the protein. In some cases the metal ions have anisotropic density. You could try refining anisotropic B-factor for just the metal ions. Cheers, Robbie From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dilip Kumar Sent: Thursday, July 9, 2015 11:35 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Ambiguous metal ion at active site. Dear All I have solved a structure of a metal-ion dependent exonuclease enzyme. In homologous structures, two or three Manganese ions are present at catalytic center. However, I have used 2 mM MgCl2 in protein purification buffer. I tried to fit both of these metal ions at catalytic center but in both cases it still shows green density (Sigma level ~ 7) in difference map and low b-factor (10) for these metal ions. For better understanding I have attached the screenshot of metal ions with difference map on. Please suggest me the possible reasons or methods to validate the presence of any other metal ions at catalytic center. Thanks in advance. Regards Dilip Kumar Research Associate Chemical and Systems Biology Unit CSIR-Institute of Genomics Integrative Biology Delhi-110025
Re: [ccp4bb] Ambiguous metal ion at active site.
If the metal center is a stable complex then ICP-OES or XRF (e.g. TXRF) methods can easily identify and quantify metals present in a small sample of protein. Roger Rowlett Dear All I have solved a structure of a metal-ion dependent exonuclease enzyme. In homologous structures, two or three Manganese ions are present at catalytic center. However, I have used 2 mM MgCl2 in protein purification buffer. I tried to fit both of these metal ions at catalytic center but in both cases it still shows green density (Sigma level ~ 7) in difference map and low b-factor (10) for these metal ions. For better understanding I have attached the screenshot of metal ions with difference map on. Please suggest me the possible reasons or methods to validate the presence of any other metal ions at catalytic center. Thanks in advance. Regards Dilip Kumar Research Associate Chemical and Systems Biology Unit CSIR-Institute of Genomics Integrative Biology Delhi-110025
Re: [ccp4bb] Ambiguous metal ion at active site.
Dear Dilip, it's difficult to exactly analyse the coordination of your metal in 2D. But, my suggestion is : Try another metal like Zn, which maybe is a contamination (from the plastic of eppendorf for example). Zn has more electron than Mg maybe that's the reason why it's still green. I suggest that you calculate anomalous difference map, maybe it could help, depending of the wavelenght used for data-collection. And you can also try this phenix.refine which has an algorithm to build solvent. Hope to help. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Dilip Kumar [dku...@igib.in] Envoyé : jeudi 9 juillet 2015 11:35 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Ambiguous metal ion at active site. Dear All I have solved a structure of a metal-ion dependent exonuclease enzyme. In homologous structures, two or three Manganese ions are present at catalytic center. However, I have used 2 mM MgCl2 in protein purification buffer. I tried to fit both of these metal ions at catalytic center but in both cases it still shows green density (Sigma level ~ 7) in difference map and low b-factor (10) for these metal ions. For better understanding I have attached the screenshot of metal ions with difference map on. Please suggest me the possible reasons or methods to validate the presence of any other metal ions at catalytic center. Thanks in advance. Regards Dilip Kumar Research Associate Chemical and Systems Biology Unit CSIR-Institute of Genomics Integrative Biology Delhi-110025
Re: [ccp4bb] Ambiguous metal ion at active site.
Dear Dilip, I was wondering if the problem was more basic, just about setting the occupancy in your input pdb file. Try setting the occupancy of Mg to 1 in your input coordinate file (pdb file) and refine it. If its Mg, it should turn blue. If its not Mg, the density should turn red instead of staying green, or may be partially green. Next, try Mn with occupancy 1 in the input file. Did you have Calcium chloride in your crystallization condition? You could try Ca as well. -Gyan On Thu, Jul 9, 2015 at 4:35 AM, Dilip Kumar dku...@igib.in wrote: Dear All I have solved a structure of a metal-ion dependent exonuclease enzyme. In homologous structures, two or three Manganese ions are present at catalytic center. However, I have used 2 mM MgCl2 in protein purification buffer. I tried to fit both of these metal ions at catalytic center but in both cases it still shows green density (Sigma level ~ 7) in difference map and low b-factor (10) for these metal ions. For better understanding I have attached the screenshot of metal ions with difference map on. Please suggest me the possible reasons or methods to validate the presence of any other metal ions at catalytic center. Thanks in advance. Regards Dilip Kumar Research Associate Chemical and Systems Biology Unit CSIR-Institute of Genomics Integrative Biology Delhi-110025 -- Gyanendra Kumar, PhD St. Jude Children's Research Hospital, Department of Structural Biology, 262, Danny Thomas Place, MS-311 Memphis, TN 38105 Phone: 901-595-3839 Cell: 631-875-9189 ---
Re: [ccp4bb] Ambiguous metal ion at active site.
Hi Kumar, I suggest to try Fe in the active site since it binds with Asp and His which is typical binding motif of Fe. Best, Ping At 2015-07-09 17:35:10, Dilip Kumar dku...@igib.in wrote: Dear All I have solved a structure of a metal-ion dependent exonuclease enzyme. In homologous structures, two or three Manganese ions are present at catalytic center. However, I have used 2 mM MgCl2 in protein purification buffer. I tried to fit both of these metal ions at catalytic center but in both cases it still shows green density (Sigma level ~ 7) in difference map and low b-factor (10) for these metal ions. For better understanding I have attached the screenshot of metal ions with difference map on. Please suggest me the possible reasons or methods to validate the presence of any other metal ions at catalytic center. Thanks in advance. Regards Dilip Kumar Research Associate Chemical and Systems Biology Unit CSIR-Institute of Genomics Integrative Biology Delhi-110025
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Re: [ccp4bb] Ambiguous metal ion at active site.
Hi Dilip, More information is needed regarding the crystallization condition(s). Anyway, Mg2+ ions are always (most of the time) are surrounded by water molecules in their primary hydration sphere...(typical distance should be 2.2 A, if I remember correctly)...and the typical geometry would be octahedral (could be distorted sometime)...or very rarely tetrahedral (we found once..)..Try fitting Mg2+ along with waters...keeping the proper metal water distances and the coordination geometry...and then refine..Good luck...!!! HTH Sudipta. On Thu, Jul 9, 2015 at 9:41 AM, Gyanendra Kumar gyanendr...@gmail.com wrote: Dear Dilip, I was wondering if the problem was more basic, just about setting the occupancy in your input pdb file. Try setting the occupancy of Mg to 1 in your input coordinate file (pdb file) and refine it. If its Mg, it should turn blue. If its not Mg, the density should turn red instead of staying green, or may be partially green. Next, try Mn with occupancy 1 in the input file. Did you have Calcium chloride in your crystallization condition? You could try Ca as well. -Gyan On Thu, Jul 9, 2015 at 4:35 AM, Dilip Kumar dku...@igib.in wrote: Dear All I have solved a structure of a metal-ion dependent exonuclease enzyme. In homologous structures, two or three Manganese ions are present at catalytic center. However, I have used 2 mM MgCl2 in protein purification buffer. I tried to fit both of these metal ions at catalytic center but in both cases it still shows green density (Sigma level ~ 7) in difference map and low b-factor (10) for these metal ions. For better understanding I have attached the screenshot of metal ions with difference map on. Please suggest me the possible reasons or methods to validate the presence of any other metal ions at catalytic center. Thanks in advance. Regards Dilip Kumar Research Associate Chemical and Systems Biology Unit CSIR-Institute of Genomics Integrative Biology Delhi-110025 -- Gyanendra Kumar, PhD St. Jude Children's Research Hospital, Department of Structural Biology, 262, Danny Thomas Place, MS-311 Memphis, TN 38105 Phone: 901-595-3839 Cell: 631-875-9189 ---
Re: [ccp4bb] Ambiguous metal ion at active site.
Looks like Ni (II) in the 4-coordination tetrahedral config. Could it be loose Ni ions from the IMAC column if you’d used one? Ni and Mg have very close atomic radii and Ni (II) can form the 6-coordinate complex as well. On Jul 9, 2015, at 4:35 AM, Dilip Kumar dku...@igib.in wrote: Dear All I have solved a structure of a metal-ion dependent exonuclease enzyme. In homologous structures, two or three Manganese ions are present at catalytic center. However, I have used 2 mM MgCl2 in protein purification buffer. I tried to fit both of these metal ions at catalytic center but in both cases it still shows green density (Sigma level ~ 7) in difference map and low b-factor (10) for these metal ions. For better understanding I have attached the screenshot of metal ions with difference map on. Please suggest me the possible reasons or methods to validate the presence of any other metal ions at catalytic center. Thanks in advance. Regards Dilip Kumar Research Associate Chemical and Systems Biology Unit CSIR-Institute of Genomics Integrative Biology Delhi-110025 mn_ion.pngmg_ion
Re: [ccp4bb] Ambiguous metal ion at active site.
Calcium with a His bound, I do not think so. SVL From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Gyanendra Kumar [gyanendr...@gmail.com] Sent: Thursday, July 09, 2015 10:41 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Ambiguous metal ion at active site. Dear Dilip, I was wondering if the problem was more basic, just about setting the occupancy in your input pdb file. Try setting the occupancy of Mg to 1 in your input coordinate file (pdb file) and refine it. If its Mg, it should turn blue. If its not Mg, the density should turn red instead of staying green, or may be partially green. Next, try Mn with occupancy 1 in the input file. Did you have Calcium chloride in your crystallization condition? You could try Ca as well. -Gyan On Thu, Jul 9, 2015 at 4:35 AM, Dilip Kumar dku...@igib.inmailto:dku...@igib.in wrote: Dear All I have solved a structure of a metal-ion dependent exonuclease enzyme. In homologous structures, two or three Manganese ions are present at catalytic center. However, I have used 2 mM MgCl2 in protein purification buffer. I tried to fit both of these metal ions at catalytic center but in both cases it still shows green density (Sigma level ~ 7) in difference map and low b-factor (10) for these metal ions. For better understanding I have attached the screenshot of metal ions with difference map on. Please suggest me the possible reasons or methods to validate the presence of any other metal ions at catalytic center. Thanks in advance. Regards Dilip Kumar Research Associate Chemical and Systems Biology Unit CSIR-Institute of Genomics Integrative Biology Delhi-110025 -- Gyanendra Kumar, PhD St. Jude Children's Research Hospital, Department of Structural Biology, 262, Danny Thomas Place, MS-311 Memphis, TN 38105 Phone: 901-595-3839 Cell: 631-875-9189 ---
Re: [ccp4bb] Ambiguous metal ion at active site.
Hi, If you have a crystal you could do a x-ray fluorescence scan to see what might be in your crystal and collect anomalous data at higher wavelength close to the manganese edge to see if it is manganese. These enzymes can also deal with Zinc which is a bit heavier and might be still bound from the expression system during protein synthesis. You could also do a ICP-AAS analysis with your protein and see what you find if you don't have a crystal anymore. There is also PIXIE for such an analysis on some synchrotrons available. There are probably many more options to identify the metal ion. Christian Am 09.07.2015 um 10:35 schrieb Dilip Kumar: Dear All I have solved a structure of a metal-ion dependent exonuclease enzyme. In homologous structures, two or three Manganese ions are present at catalytic center. However, I have used 2 mM MgCl2 in protein purification buffer. I tried to fit both of these metal ions at catalytic center but in both cases it still shows green density (Sigma level ~ 7) in difference map and low b-factor (10) for these metal ions. For better understanding I have attached the screenshot of metal ions with difference map on. Please suggest me the possible reasons or methods to validate the presence of any other metal ions at catalytic center. Thanks in advance. Regards Dilip Kumar Research Associate Chemical and Systems Biology Unit CSIR-Institute of Genomics Integrative Biology Delhi-110025
Re: [ccp4bb] Industrial Positions of Associate Scientist Protein Biochemistry/crystallography at Amgen
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