Hi Everyone, Thank you for the advice, especially Pavel's. My issue has been resolved. I lowered the occupancy and B-factors of the metal ions in the .pdb and ran phenix.refine for 10 cycles. This removed most of the negative Fo-Fc density. Anisotropic refinement of the metal ion B-factors was also effective, dropping Rwork and Rfree by ~1% each. To do this, I edited the "Individual B-factor refinement" values as follows. Isotropic atoms: not (element Zn) Anisotropic atoms: element Zn
Best, Chris On 5/6/14, Chris Fage <[email protected]> wrote: > I have used CNS before, but not for this sort of refinement. I see in > the bindividual.inp file that I can "select atoms to be included"--it > is defaulted at "known and not hydrogen". Do you know the proper > nomenclature for selecting a Zn ion in chains A and B? > > Thanks, > Chris > > On 5/6/14, Steven Herron <[email protected]> wrote: >> >> Refining the occupancy will help your R-factor and flatten your density, >> but you need to be careful to also refine the B-factor of the metal >> ion. Don't refine both the occupancy and the B-factor during the same >> run (the two are correlated at this resolution), refine the occupancy of >> just the metal ion and then refine the B-factor of just the metal ion >> (repeat as needed). I used X-plor/CNS to do my refinements, so it was >> easy to refine the occupancy (or B-factor) of just the metal ion. After >> a few rounds of refinement both parameters will stop changing and you >> will have your answer. The final B-factor of the metal ion should be >> similar to the amino acid residues that are coordinated to it. >> >> Soaking in several different ion concentrations and collecting >> additional datasets is also a good idea (if you have the time). I did >> this type of experiment once before (see: JBC 278(14):12271-7. [ Apr 4, >> 2003]) (or: http://www.ncbi.nlm.nih.gov/pubmed/12540845). I soaked in >> several different Ca2+ ion concentrations and was able to determine the >> binding affinity for that calcium ion using crystallography. >> >> To make sure I was not stuck in a local minima, I would modify either >> the occupancy of the B-factor of the metal while keeping the other fixed >> and do a refinement. I even tried both large and small changes (both >> increases and decreases in value). It always came back to the earlier >> answer. >> >> Different Ca2+ ion concentrations can give some additional insight into >> the metal binding site. Between the no-Ca2+ structure and the high-Ca2+ >> structure there was a conserved Asp-residue that changed conformation. >> So, I soaked in the appropriate amount of Ca2+ to see the residue in >> both positions. There was a high correlation between the asp residue >> orientation and the Ca2+ ion occupancy. >> >> Steven Herron >> [email protected] >> >> >> >> >> On 5/6/2014 11:02 AM, Chris Fage wrote: >>> Hi Everyone, >>> >>> In my 2.5-angstrom structure, there is negative Fo-Fc density >>> surrounding a metal ion after refining in Phenix. From anomalous >>> diffraction I am certain of the metal's identity and position in each >>> monomer. Also, the ion is appropriately coordinated by nearby side >>> chains. Should I be refining the occupancy of the ion in attempt to >>> "flatten" the negative density? I am considering soaking the metal ion >>> into crystals or cocrystallizing and collecting additional datasets. >>> >>> Thanks for your help! >>> >>> Regards, >>> Chris >> >> >
