-----BEGIN PGP SIGNED MESSAGE----- Hash: SHA1 Dear Gregg,
your post raised my curiosity and I look at the map after a round of refinement with shelxl. I thought you might be interested in two observations: 1) shelxl refines the occupancy of the 1Q1 ligand to 60% rather than 78% (I don't think shelxl fills in missing reflections) 2) the second observation is the reason why I am posting this publicly, because it illustrates the danger of automatic water placement to hover out important information: there is strong difference density for another ligand as you can see in the attached screenshot (the Met on the left is residue A2 in the deposited PDB file. The water network that was build instead has too high B-values compared to the surround protein structures. I assume you used an automated procedure to place these waters - the map from the EDS sever looks very different on does not reveal an extra ligand. Once you model this extra ligand you should further decrease the noise level and might get an even better indication of the two overlapping ligands. Did I mention I like shelxl ;-) (NB: these are 1.55A data!) Best regards, Tim On 11/28/2014 06:22 AM, Gregg Crichlow wrote: > My fault for not changing the thread subject! I'm sorry! Greetings > to ccp4bb readers! This is a long-delayed reply to requests that I > received after I posted an observation on the ccp4bb almost three > years ago. (I think it was early 2012). The paper had not yet been > published (pending finalization of experiments unrelated to > structural biology) and so I was not free to distribute any of the > structure factors to those who were interested. The paper now has > been published, and the coordinates and structure factors have been > released. The following is the peculiarity that made this of > interest. > > I refined a structure with 1.55 A data of a homotrimeric protein > bound to an inhibitor. Although the inhibitor added to the enzyme > contained a five-membered ring, the inhibitor we observed in two of > the three active sites had the ring opened. We therefore discovered > that the ring-opened form is also an inhibitor. However, in the > remaining active site, we discovered *both* the ring-opened and > ring-closed forms of the inhibitor. (The active sites are > identical, but crystal contacts create asymmetry among the three). > The two forms of the inhibitor were positioned slightly differently > in this active site, and so the electron density at this resolution > clearly identified the presence of both forms, even though there > was much overlap in their positions. I used the CNS occupancy > refinement script, and then normalized the results so that the two > occupancies added up to 1, and so I came up with occupancies of > 0.88 (ring-opened) and 0.12 (ring-closed). Then having all > inhibitor molecules in the model, I calculated maps with another > program, which substituted Fcalc for missing reflections, and could > no longer see the lower occupancy form in the electron density. I > re-calculated maps in CNS, and again saw both forms in the electron > density. So I calculated new maps in CNS the same way as before, > but turned on the option to substitute missing reflections with > Fcalc: and the lower occupancy form was missing from the density in > the resulting maps. So, this was the difference - when I used zero > for missing reflections, I was able to see both forms; but when I > substituted Fcalc for missing reflections in maps, I could only see > the predominant form. The data set was 99.6% complete overall, > 99.2% in the highest resolution shell; with a multiplicity of 5.8 > (5.3 in the highest shell). So there didn't seem to be many missing > reflections to be substituted. The test set contained roughly 2% of > the total number of observed unique reflections. > > Some had replied to me, saying that they were interested in using > the coordinates & structure factors for running tests; however, as > I said, I was not free to distribute them at the time. Now they > are publicly available: PDB ID 4K9G. I hope this helps all who are > interested. Thank you. > > Gregg > > *************************** >> >> *Gregg Crichlow *Visiting Research Scientist Department of >> Pharmacology Yale University School of Medicine >> > - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -----BEGIN PGP SIGNATURE----- Version: GnuPG v1.4.12 (GNU/Linux) iD8DBQFUeEE9UxlJ7aRr7hoRAnGEAKC6+kb6ZvfvD9EjecRsd8gPNUa28QCg6JPH XN7Zdg+Of2BEjhUtGPx8vHY= =4ZPS -----END PGP SIGNATURE-----
