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 <[log in to unmask]> 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 <[log in to unmask]> 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
>> [log in to unmask]
>>
>>
>>
>>
>> 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
>>
>>
>
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