[ccp4bb] AW: [ccp4bb] help identifying ligand
Dear Ed, What is the pH of your crystallization buffer? If it is acidic, either the azide or the carboxylate may be protonated. Also the local environment of the carboxylate can make a hugh difference in PKa. You could also use some Bayesian logic: given the elongated linear density, what else of the available components of your crystallization drop would fit? Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Edward A. Berry Gesendet: Sonntag, 7. Juli 2013 22:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] help identifying ligand In a structure I'm refining, there are a couple of oblong blobs associated with carboxylates. (screenshots at http://sb20.lbl.gov/berry/ccp4/azide/) If I modeled with two waters, they refine too close together for normal H-bond, 2.3 to 2.5 A; and their density is connected. I considered one water with alternate locations, but the distal position wouldn't make much sense if the proximal water wasn't there. The density is the right size for azide, which was present in the medium, but I expect a chemist would find it unreasonable to have anionic azide (pKa of hydrazoic acid ~4.6) associating with a carboxylate. Would that make sense? or does anyone have other suggestions? (resolution is 2.2A, contour 0.25 e/A^3 or about 1.3 sigma) Thanks, Ed
Re: [ccp4bb] AW: [ccp4bb] help identifying ligand
Dear CCP4BB, The most likely components are those at the highest concentration in the crystallization or cryosolution. And a few wild ideas to continue the discussion that is very important as the ligands are always very difficult to identify. Example: If you have 1.5 M ammonium sulfate you should consider hydrated ammonium ions H3O+ + NH3 in equilib. H2O + NH4+ The pH will determine the equilibrium point and NH4+ would be a good ligand for a carboxylate. Assuming 200mM Li2SO4: A lithium ion (H20-Li-H20 with a Li-O distance of 2.14 Ang) Li+ is often associated with more that two H2O molecules with an angle of 105° not 180° but cannot be excluded in proximity of a carboxylate where the environ ment could be distorted (not very believable). (H2O, Na+ and Mg++ 10 electrons) water is always the most probable. 2 H2O in equilib. OH- + H3O+ Carboxylates are often destroyed by radiation damage. The most probable ligand will be at high concentration in the mother liquor the moment the crystal was flashcooled. This is rarely the case for typically 0.02% azide (I would made an exception in proximity to Cu++, Fe++ or Zn++ ions). Azide -N=N+=N- is also suspitious as a negative ion is a bad counterion for a carboxylate. Enrico. On Mon, 08 Jul 2013 11:19:46 +0200, herman.schreu...@sanofi.com wrote: Dear Ed, What is the pH of your crystallization buffer? If it is acidic, either the azide or the carboxylate may be protonated. Also the local environment of the carboxylate can make a hugh difference in PKa. You could also use some Bayesian logic: given the elongated linear density, what else of the available components of your crystallization drop would fit? Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Edward A. Berry Gesendet: Sonntag, 7. Juli 2013 22:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] help identifying ligand In a structure I'm refining, there are a couple of oblong blobs associated with carboxylates. (screenshots at http://sb20.lbl.gov/berry/ccp4/azide/) If I modeled with two waters, they refine too close together for normal H-bond, 2.3 to 2.5 A; and their density is connected. I considered one water with alternate locations, but the distal position wouldn't make much sense if the proximal water wasn't there. The density is the right size for azide, which was present in the medium, but I expect a chemist would find it unreasonable to have anionic azide (pKa of hydrazoic acid ~4.6) associating with a carboxylate. Would that make sense? or does anyone have other suggestions? (resolution is 2.2A, contour 0.25 e/A^3 or about 1.3 sigma) Thanks, Ed -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] AW: [ccp4bb] help identifying ligand
Thanks all, the pH is 6.7, azide is 3 mM, and there is no added ammonium. I could get away with modeling as two waters since the separation is well above the 2.2A that gets flagged as a clash in the PDB, still it's close enough to suggest that two waters is not really what's there. Enrico Stura wrote: Dear CCP4BB, The most likely components are those at the highest concentration in the crystallization or cryosolution. And a few wild ideas to continue the discussion that is very important as the ligands are always very difficult to identify. Example: If you have 1.5 M ammonium sulfate you should consider hydrated ammonium ions H3O+ + NH3 in equilib. H2O + NH4+ The pH will determine the equilibrium point and NH4+ would be a good ligand for a carboxylate. Assuming 200mM Li2SO4: A lithium ion (H20-Li-H20 with a Li-O distance of 2.14 Ang) Li+ is often associated with more that two H2O molecules with an angle of 105° not 180° but cannot be excluded in proximity of a carboxylate where the environ ment could be distorted (not very believable). (H2O, Na+ and Mg++ 10 electrons) water is always the most probable. 2 H2O in equilib. OH- + H3O+ Carboxylates are often destroyed by radiation damage. The most probable ligand will be at high concentration in the mother liquor the moment the crystal was flashcooled. This is rarely the case for typically 0.02% azide (I would made an exception in proximity to Cu++, Fe++ or Zn++ ions). Azide -N=N+=N- is also suspitious as a negative ion is a bad counterion for a carboxylate. Enrico. On Mon, 08 Jul 2013 11:19:46 +0200, herman.schreu...@sanofi.com wrote: Dear Ed, What is the pH of your crystallization buffer? If it is acidic, either the azide or the carboxylate may be protonated. Also the local environment of the carboxylate can make a hugh difference in PKa. You could also use some Bayesian logic: given the elongated linear density, what else of the available components of your crystallization drop would fit? Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Edward A. Berry Gesendet: Sonntag, 7. Juli 2013 22:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] help identifying ligand In a structure I'm refining, there are a couple of oblong blobs associated with carboxylates. (screenshots at http://sb20.lbl.gov/berry/ccp4/azide/) If I modeled with two waters, they refine too close together for normal H-bond, 2.3 to 2.5 A; and their density is connected. I considered one water with alternate locations, but the distal position wouldn't make much sense if the proximal water wasn't there. The density is the right size for azide, which was present in the medium, but I expect a chemist would find it unreasonable to have anionic azide (pKa of hydrazoic acid ~4.6) associating with a carboxylate. Would that make sense? or does anyone have other suggestions? (resolution is 2.2A, contour 0.25 e/A^3 or about 1.3 sigma) Thanks, Ed
[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] help identifying ligand
Dear Ed, For me, 3 mM is a significant concentration. If you have another crystal left, you could transfer it to a storage buffer without azide and collect a data set and see if the density disappears. A very small molecule, non-covalently bound on the outside of the protein should disappear in minutes if the compound is not present in solution. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Edward A. Berry Gesendet: Montag, 8. Juli 2013 15:31 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] AW: [ccp4bb] help identifying ligand Thanks all, the pH is 6.7, azide is 3 mM, and there is no added ammonium. I could get away with modeling as two waters since the separation is well above the 2.2A that gets flagged as a clash in the PDB, still it's close enough to suggest that two waters is not really what's there. Enrico Stura wrote: Dear CCP4BB, The most likely components are those at the highest concentration in the crystallization or cryosolution. And a few wild ideas to continue the discussion that is very important as the ligands are always very difficult to identify. Example: If you have 1.5 M ammonium sulfate you should consider hydrated ammonium ions H3O+ + NH3 in equilib. H2O + NH4+ The pH will determine the equilibrium point and NH4+ would be a good ligand for a carboxylate. Assuming 200mM Li2SO4: A lithium ion (H20-Li-H20 with a Li-O distance of 2.14 Ang) Li+ is often associated with more that two H2O molecules with an angle of 105° not 180° but cannot be excluded in proximity of a carboxylate where the environ ment could be distorted (not very believable). (H2O, Na+ and Mg++ 10 electrons) water is always the most probable. 2 H2O in equilib. OH- + H3O+ Carboxylates are often destroyed by radiation damage. The most probable ligand will be at high concentration in the mother liquor the moment the crystal was flashcooled. This is rarely the case for typically 0.02% azide (I would made an exception in proximity to Cu++, Fe++ or Zn++ ions). Azide -N=N+=N- is also suspitious as a negative ion is a bad counterion for a carboxylate. Enrico. On Mon, 08 Jul 2013 11:19:46 +0200, herman.schreu...@sanofi.com wrote: Dear Ed, What is the pH of your crystallization buffer? If it is acidic, either the azide or the carboxylate may be protonated. Also the local environment of the carboxylate can make a hugh difference in PKa. You could also use some Bayesian logic: given the elongated linear density, what else of the available components of your crystallization drop would fit? Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Edward A. Berry Gesendet: Sonntag, 7. Juli 2013 22:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] help identifying ligand In a structure I'm refining, there are a couple of oblong blobs associated with carboxylates. (screenshots at http://sb20.lbl.gov/berry/ccp4/azide/) If I modeled with two waters, they refine too close together for normal H-bond, 2.3 to 2.5 A; and their density is connected. I considered one water with alternate locations, but the distal position wouldn't make much sense if the proximal water wasn't there. The density is the right size for azide, which was present in the medium, but I expect a chemist would find it unreasonable to have anionic azide (pKa of hydrazoic acid ~4.6) associating with a carboxylate. Would that make sense? or does anyone have other suggestions? (resolution is 2.2A, contour 0.25 e/A^3 or about 1.3 sigma) Thanks, Ed
