Re: [ccp4bb] distorted phosphate molecule geometry after refinement
The metal ion is looking reasonable, and it is also chemically sensible based on the crystallization conditions. Add another water around the remaining positive difference density and see if the resulting geometry looks remotely octahedral. From the one, view provided, it does appear to be an approximately octahedral coordination sphere. If so, this interpretation might be a winner. If this is a surface site, it is very possible that the nickel ion (and associated waters) are at less than 100% occupancy. Depending on the wavelength selected and the quality of data collected, you might have some anomalous scattering that would help confirm the presence of nickel. Cheers, ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 6/22/2015 7:27 PM, Keller, Jacob wrote: Definitely Ni, and maybe add another two waters to fill in the density. Regarding B-factors, it depends on those of the surrounding side chains, and should be a bit higher than theirs. Also, since it’s probably not a biologically-relevant Ni site, it would have low affinity and therefore you could plausibly lower the occupancy to make it work (b-factor would decrease.) JPK *From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *ansuman biswas *Sent:* Monday, June 22, 2015 7:04 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] distorted phosphate molecule geometry after refinement I tried refining with a phosphorylated His (NEP, attached figures 1 and 2 ). After refinement the geometry looks fine, there are no short contacts and the B-factors on the attached phosphate are ~30A2, except for one O-atom which is at 50 A2. However, some positive density is showing up in the Fo-Fc map. I also tried refining with unmodified His, but with a Ni-ion and 2 water molecules. Ni was present in the crystallization condition. After refinement (3rd Fig attached), there is hardly much positive density in Fo- Fc map. However, the B-factors of the added Ni and water molecules are ~50A2. The Ni coordination site can have both His (predominant) and Lys. The data was collected at wavelength 0.9A and has resolution 2.3A Please suggest. Regards, Ansuman On Tuesday, 23 June 2015 1:13 AM, Shane Caldwell mailto:shane.caldwel...@gmail.com>> wrote: It's probably much less likely than metal coordination and it's hard to judge from only one angle, but phospho-histidine might be something else to consider. http://www.jbc.org/content/276/5/3247.full Shane On Mon, Jun 22, 2015 at 2:15 PM, Roger Rowlett <mailto:rrowl...@colgate.edu>> wrote: I agree...one possibility is a M-His(2)-Lys-(OH2) site. Possible metal ions would include Zn(II), although Lys is a relatively rare ligand in zinc-metalloenzyme sites. Cheers, ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu <mailto:rrowl...@colgate.edu> On 6/22/2015 11:20 AM, Keller, Jacob wrote: Looks to me like a metal binding site with those histidines, perhaps--any chance of that? That might also explain the weird geometry issues. JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of Dale Tronrud Sent: Monday, June 22, 2015 11:17 AM To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> Subject: Re: [ccp4bb] distorted phosphate molecule geometry after refinement It is possible that your PO4 has its atoms labeled with the wrong chirality. Yes, I know that PO4 is not chiral when you ignore hydrogen atoms and single/double bonds but adding labels creates an unnatural chriality. Try your refinement again after switching the labels on two oxygen atoms. Dale Tronrud On 6/22/2015 7:48 AM, ansuman biswas wrote: Dear CCP4 users, I am working on a protein from a hyperthermophilic archaeon. I have collected mutliple X-Ray datasets, both from home source and synchrotron and always found a clear density for tetrahedral geometry, co-ordinated by two histidines and one lysine. I tried fitting phosphate there, but its geometry always gets distorted after each refinement cycle (Refmac 5.8.0073). Also I found some short contacts between the coordinated residues and phosphate which were very difficult to remove. I am attaching a figure with the density and phosphate. Kindly suggest - 1. if this may be a possible modification of any of the associated residues, and the code of the modified residue to be used. 2. If the ligand requires separate restraints during refinement, I am using the "restrained refin
Re: [ccp4bb] distorted phosphate molecule geometry after refinement
The metal ion is looking reasonable, and it is also chemically sensible based on the crystallization conditions. Add another water around the remaining positive difference density and see if the resulting geometry looks remotely octahedral. From the one, view provided, it does appear to be an approximately octahedral coordination sphere. If so, this interpretation might be a winner. If this is a surface site, it is very possible that the nickel ion (and associated waters) are at less than 100% occupancy. Depending on the wavelength selected and the quality of data collected, you might have some anomalous scattering that would help confirm the presence of nickel. Cheers, ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 6/22/2015 7:27 PM, Keller, Jacob wrote: Definitely Ni, and maybe add another two waters to fill in the density. Regarding B-factors, it depends on those of the surrounding side chains, and should be a bit higher than theirs. Also, since it’s probably not a biologically-relevant Ni site, it would have low affinity and therefore you could plausibly lower the occupancy to make it work (b-factor would decrease.) JPK *From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *ansuman biswas *Sent:* Monday, June 22, 2015 7:04 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] distorted phosphate molecule geometry after refinement I tried refining with a phosphorylated His (NEP, attached figures 1 and 2 ). After refinement the geometry looks fine, there are no short contacts and the B-factors on the attached phosphate are ~30A2, except for one O-atom which is at 50 A2. However, some positive density is showing up in the Fo-Fc map. I also tried refining with unmodified His, but with a Ni-ion and 2 water molecules. Ni was present in the crystallization condition. After refinement (3rd Fig attached), there is hardly much positive density in Fo- Fc map. However, the B-factors of the added Ni and water molecules are ~50A2. The Ni coordination site can have both His (predominant) and Lys. The data was collected at wavelength 0.9A and has resolution 2.3A Please suggest. Regards, Ansuman On Tuesday, 23 June 2015 1:13 AM, Shane Caldwell mailto:shane.caldwel...@gmail.com>> wrote: It's probably much less likely than metal coordination and it's hard to judge from only one angle, but phospho-histidine might be something else to consider. http://www.jbc.org/content/276/5/3247.full Shane On Mon, Jun 22, 2015 at 2:15 PM, Roger Rowlett <mailto:rrowl...@colgate.edu>> wrote: I agree...one possibility is a M-His(2)-Lys-(OH2) site. Possible metal ions would include Zn(II), although Lys is a relatively rare ligand in zinc-metalloenzyme sites. Cheers, ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu <mailto:rrowl...@colgate.edu> On 6/22/2015 11:20 AM, Keller, Jacob wrote: Looks to me like a metal binding site with those histidines, perhaps--any chance of that? That might also explain the weird geometry issues. JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of Dale Tronrud Sent: Monday, June 22, 2015 11:17 AM To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> Subject: Re: [ccp4bb] distorted phosphate molecule geometry after refinement It is possible that your PO4 has its atoms labeled with the wrong chirality. Yes, I know that PO4 is not chiral when you ignore hydrogen atoms and single/double bonds but adding labels creates an unnatural chriality. Try your refinement again after switching the labels on two oxygen atoms. Dale Tronrud On 6/22/2015 7:48 AM, ansuman biswas wrote: Dear CCP4 users, I am working on a protein from a hyperthermophilic archaeon. I have collected mutliple X-Ray datasets, both from home source and synchrotron and always found a clear density for tetrahedral geometry, co-ordinated by two histidines and one lysine. I tried fitting phosphate there, but its geometry always gets distorted after each refinement cycle (Refmac 5.8.0073). Also I found some short contacts between the coordinated residues and phosphate which were very difficult to remove. I am attaching a figure with the density and phosphate. Kindly suggest - 1. if this may be a possible modification of any of the associated residues, and the code of the modified residue to be used. 2. If the ligand requires separate restraints during refinement, I am using the "restrained refin
Re: [ccp4bb] distorted phosphate molecule geometry after refinement
Definitely Ni, and maybe add another two waters to fill in the density. Regarding B-factors, it depends on those of the surrounding side chains, and should be a bit higher than theirs. Also, since it’s probably not a biologically-relevant Ni site, it would have low affinity and therefore you could plausibly lower the occupancy to make it work (b-factor would decrease.) JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ansuman biswas Sent: Monday, June 22, 2015 7:04 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] distorted phosphate molecule geometry after refinement I tried refining with a phosphorylated His (NEP, attached figures 1 and 2 ). After refinement the geometry looks fine, there are no short contacts and the B-factors on the attached phosphate are ~30A2, except for one O-atom which is at 50 A2. However, some positive density is showing up in the Fo-Fc map. I also tried refining with unmodified His, but with a Ni-ion and 2 water molecules. Ni was present in the crystallization condition. After refinement (3rd Fig attached), there is hardly much positive density in Fo- Fc map. However, the B-factors of the added Ni and water molecules are ~50A2. The Ni coordination site can have both His (predominant) and Lys. The data was collected at wavelength 0.9A and has resolution 2.3A Please suggest. Regards, Ansuman On Tuesday, 23 June 2015 1:13 AM, Shane Caldwell mailto:shane.caldwel...@gmail.com>> wrote: It's probably much less likely than metal coordination and it's hard to judge from only one angle, but phospho-histidine might be something else to consider. http://www.jbc.org/content/276/5/3247.full Shane On Mon, Jun 22, 2015 at 2:15 PM, Roger Rowlett mailto:rrowl...@colgate.edu>> wrote: I agree...one possibility is a M-His(2)-Lys-(OH2) site. Possible metal ions would include Zn(II), although Lys is a relatively rare ligand in zinc-metalloenzyme sites. Cheers, ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu<mailto:rrowl...@colgate.edu> On 6/22/2015 11:20 AM, Keller, Jacob wrote: Looks to me like a metal binding site with those histidines, perhaps--any chance of that? That might also explain the weird geometry issues. JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of Dale Tronrud Sent: Monday, June 22, 2015 11:17 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: Re: [ccp4bb] distorted phosphate molecule geometry after refinement It is possible that your PO4 has its atoms labeled with the wrong chirality. Yes, I know that PO4 is not chiral when you ignore hydrogen atoms and single/double bonds but adding labels creates an unnatural chriality. Try your refinement again after switching the labels on two oxygen atoms. Dale Tronrud On 6/22/2015 7:48 AM, ansuman biswas wrote: Dear CCP4 users, I am working on a protein from a hyperthermophilic archaeon. I have collected mutliple X-Ray datasets, both from home source and synchrotron and always found a clear density for tetrahedral geometry, co-ordinated by two histidines and one lysine. I tried fitting phosphate there, but its geometry always gets distorted after each refinement cycle (Refmac 5.8.0073). Also I found some short contacts between the coordinated residues and phosphate which were very difficult to remove. I am attaching a figure with the density and phosphate. Kindly suggest - 1. if this may be a possible modification of any of the associated residues, and the code of the modified residue to be used. 2. If the ligand requires separate restraints during refinement, I am using the "restrained refinement" option available at the top of the GUI for refmac. Thanking you, yours sincerely, Ansuman Biswas, PhD student, Dept. of Physics, IISc
Re: [ccp4bb] distorted phosphate molecule geometry after refinement
It's probably much less likely than metal coordination and it's hard to judge from only one angle, but phospho-histidine might be something else to consider. http://www.jbc.org/content/276/5/3247.full Shane On Mon, Jun 22, 2015 at 2:15 PM, Roger Rowlett wrote: > I agree...one possibility is a M-His(2)-Lys-(OH2) site. Possible metal > ions would include Zn(II), although Lys is a relatively rare ligand in > zinc-metalloenzyme sites. > > Cheers, > > ___ > Roger S. Rowlett > Gordon & Dorothy Kline Professor > Department of Chemistry > Colgate University > 13 Oak Drive > Hamilton, NY 13346 > > tel: (315)-228-7245 > ofc: (315)-228-7395 > fax: (315)-228-7935 > email: rrowl...@colgate.edu > > > On 6/22/2015 11:20 AM, Keller, Jacob wrote: > >> Looks to me like a metal binding site with those histidines, perhaps--any >> chance of that? That might also explain the weird geometry issues. >> >> JPK >> >> -Original Message- >> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of >> Dale Tronrud >> Sent: Monday, June 22, 2015 11:17 AM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: Re: [ccp4bb] distorted phosphate molecule geometry after >> refinement >> >> It is possible that your PO4 has its atoms labeled with the wrong >> chirality. Yes, I know that PO4 is not chiral when you ignore hydrogen >> atoms and single/double bonds but adding labels creates an unnatural >> chriality. Try your refinement again after switching the labels on two >> oxygen atoms. >> >> Dale Tronrud >> >> On 6/22/2015 7:48 AM, ansuman biswas wrote: >> >>> Dear CCP4 users, >>> >>> I am working on a protein from a hyperthermophilic archaeon. >>> >>> I have collected mutliple X-Ray datasets, both from home source and >>> synchrotron and always found a clear density for tetrahedral geometry, >>> co-ordinated by two histidines and one lysine. >>> >>> I tried fitting phosphate there, but its geometry always gets >>> distorted after each refinement cycle (Refmac 5.8.0073). Also I found >>> some short contacts between the coordinated residues and phosphate >>> which were very difficult to remove. >>> >>> I am attaching a figure with the density and phosphate. >>> >>> Kindly suggest - >>> 1. if this may be a possible modification of any of the associated >>> residues, and the code of the modified residue to be used. >>> >>> 2. If the ligand requires separate restraints during refinement, I am >>> using the "restrained refinement" option available at the top of the >>> GUI for refmac. >>> >>> Thanking you, >>> yours sincerely, >>> Ansuman Biswas, >>> PhD student, >>> Dept. of Physics, >>> IISc >>> >>>
Re: [ccp4bb] distorted phosphate molecule geometry after refinement
I often say this, but check the anomalous difference Fourier. You don't say what resolution your data reaches to, or what wavelength the data is collected at, but if the model is well refined you often can see anomalous peaks for Ss, and they give you a relative scale for any anomalous feature which corresponds to that site. Eleanor Dodson On 22 June 2015 at 19:15, Roger Rowlett wrote: > I agree...one possibility is a M-His(2)-Lys-(OH2) site. Possible metal > ions would include Zn(II), although Lys is a relatively rare ligand in > zinc-metalloenzyme sites. > > Cheers, > > ___ > Roger S. Rowlett > Gordon & Dorothy Kline Professor > Department of Chemistry > Colgate University > 13 Oak Drive > Hamilton, NY 13346 > > tel: (315)-228-7245 > ofc: (315)-228-7395 > fax: (315)-228-7935 > email: rrowl...@colgate.edu > > > On 6/22/2015 11:20 AM, Keller, Jacob wrote: > >> Looks to me like a metal binding site with those histidines, perhaps--any >> chance of that? That might also explain the weird geometry issues. >> >> JPK >> >> -Original Message- >> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of >> Dale Tronrud >> Sent: Monday, June 22, 2015 11:17 AM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: Re: [ccp4bb] distorted phosphate molecule geometry after >> refinement >> >> It is possible that your PO4 has its atoms labeled with the wrong >> chirality. Yes, I know that PO4 is not chiral when you ignore hydrogen >> atoms and single/double bonds but adding labels creates an unnatural >> chriality. Try your refinement again after switching the labels on two >> oxygen atoms. >> >> Dale Tronrud >> >> On 6/22/2015 7:48 AM, ansuman biswas wrote: >> >>> Dear CCP4 users, >>> >>> I am working on a protein from a hyperthermophilic archaeon. >>> >>> I have collected mutliple X-Ray datasets, both from home source and >>> synchrotron and always found a clear density for tetrahedral geometry, >>> co-ordinated by two histidines and one lysine. >>> >>> I tried fitting phosphate there, but its geometry always gets >>> distorted after each refinement cycle (Refmac 5.8.0073). Also I found >>> some short contacts between the coordinated residues and phosphate >>> which were very difficult to remove. >>> >>> I am attaching a figure with the density and phosphate. >>> >>> Kindly suggest - >>> 1. if this may be a possible modification of any of the associated >>> residues, and the code of the modified residue to be used. >>> >>> 2. If the ligand requires separate restraints during refinement, I am >>> using the "restrained refinement" option available at the top of the >>> GUI for refmac. >>> >>> Thanking you, >>> yours sincerely, >>> Ansuman Biswas, >>> PhD student, >>> Dept. of Physics, >>> IISc >>> >>>
Re: [ccp4bb] distorted phosphate molecule geometry after refinement
I agree...one possibility is a M-His(2)-Lys-(OH2) site. Possible metal ions would include Zn(II), although Lys is a relatively rare ligand in zinc-metalloenzyme sites. Cheers, ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 6/22/2015 11:20 AM, Keller, Jacob wrote: Looks to me like a metal binding site with those histidines, perhaps--any chance of that? That might also explain the weird geometry issues. JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dale Tronrud Sent: Monday, June 22, 2015 11:17 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] distorted phosphate molecule geometry after refinement It is possible that your PO4 has its atoms labeled with the wrong chirality. Yes, I know that PO4 is not chiral when you ignore hydrogen atoms and single/double bonds but adding labels creates an unnatural chriality. Try your refinement again after switching the labels on two oxygen atoms. Dale Tronrud On 6/22/2015 7:48 AM, ansuman biswas wrote: Dear CCP4 users, I am working on a protein from a hyperthermophilic archaeon. I have collected mutliple X-Ray datasets, both from home source and synchrotron and always found a clear density for tetrahedral geometry, co-ordinated by two histidines and one lysine. I tried fitting phosphate there, but its geometry always gets distorted after each refinement cycle (Refmac 5.8.0073). Also I found some short contacts between the coordinated residues and phosphate which were very difficult to remove. I am attaching a figure with the density and phosphate. Kindly suggest - 1. if this may be a possible modification of any of the associated residues, and the code of the modified residue to be used. 2. If the ligand requires separate restraints during refinement, I am using the "restrained refinement" option available at the top of the GUI for refmac. Thanking you, yours sincerely, Ansuman Biswas, PhD student, Dept. of Physics, IISc
Re: [ccp4bb] distorted phosphate molecule geometry after refinement
Looks to me like a metal binding site with those histidines, perhaps--any chance of that? That might also explain the weird geometry issues. JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dale Tronrud Sent: Monday, June 22, 2015 11:17 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] distorted phosphate molecule geometry after refinement It is possible that your PO4 has its atoms labeled with the wrong chirality. Yes, I know that PO4 is not chiral when you ignore hydrogen atoms and single/double bonds but adding labels creates an unnatural chriality. Try your refinement again after switching the labels on two oxygen atoms. Dale Tronrud On 6/22/2015 7:48 AM, ansuman biswas wrote: > Dear CCP4 users, > > I am working on a protein from a hyperthermophilic archaeon. > > I have collected mutliple X-Ray datasets, both from home source and > synchrotron and always found a clear density for tetrahedral geometry, > co-ordinated by two histidines and one lysine. > > I tried fitting phosphate there, but its geometry always gets > distorted after each refinement cycle (Refmac 5.8.0073). Also I found > some short contacts between the coordinated residues and phosphate > which were very difficult to remove. > > I am attaching a figure with the density and phosphate. > > Kindly suggest - > 1. if this may be a possible modification of any of the associated > residues, and the code of the modified residue to be used. > > 2. If the ligand requires separate restraints during refinement, I am > using the "restrained refinement" option available at the top of the > GUI for refmac. > > Thanking you, > yours sincerely, > Ansuman Biswas, > PhD student, > Dept. of Physics, > IISc >
Re: [ccp4bb] distorted phosphate molecule geometry after refinement
It is possible that your PO4 has its atoms labeled with the wrong chirality. Yes, I know that PO4 is not chiral when you ignore hydrogen atoms and single/double bonds but adding labels creates an unnatural chriality. Try your refinement again after switching the labels on two oxygen atoms. Dale Tronrud On 6/22/2015 7:48 AM, ansuman biswas wrote: > Dear CCP4 users, > > I am working on a protein from a hyperthermophilic archaeon. > > I have collected mutliple X-Ray datasets, both from home source and > synchrotron and always found a clear density for tetrahedral geometry, > co-ordinated by two histidines and one lysine. > > I tried fitting phosphate there, but its geometry always gets distorted > after each refinement cycle (Refmac 5.8.0073). Also I found some short > contacts between the coordinated residues and phosphate which were very > difficult to remove. > > I am attaching a figure with the density and phosphate. > > Kindly suggest - > 1. if this may be a possible modification of any of the associated > residues, and the code of the modified residue to be used. > > 2. If the ligand requires separate restraints during refinement, I am > using the "restrained refinement" option available at the top of the GUI > for refmac. > > Thanking you, > yours sincerely, > Ansuman Biswas, > PhD student, > Dept. of Physics, > IISc >
