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 <[log in to unmask]> 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 <[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
>>>>
>>>>
>>>
>>
>
> --
> Dr Tim Gruene
> Institut fuer anorganische Chemie
> Tammannstr. 4
> D-37077 Goettingen
>
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>
>