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 > >
