Hi Tim, That's a good question. The negative density vanished and the model itself has not changed much. I still plan to set up soaking/cocrystallization experiments with the metal ion. I am hoping this will increase occupancy. That is the only solution that comes to mind.
Regards, Chris On 5/8/14, Tim Gruene <[email protected]> wrote: > Hi Chris, > > this certainly improved your model with respect to the data including > all their errors. How do you know this did not make your model worse > with respect to chemistry, respectively to what is inside your crystal? > > Best, > Tim > > On 05/08/2014 07:26 PM, Chris Fage wrote: >> 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 >>>> >>>> >>> >> > > -- > Dr Tim Gruene > Institut fuer anorganische Chemie > Tammannstr. 4 > D-37077 Goettingen > > GPG Key ID = A46BEE1A > >
