Kavya,
Presumably your enzyme is turning over the ATP? Driving it towards ADP?
In that case I suspect that you may have a mixture of ATP and ADP (and possibly contaminating AMP).
You could either model both ATP and ADP, and set relative occupancies of both (add up to 1).
Or, you could assign different occupancies to the phosphate groups alone.
I personally think the former is probably the correct method? Comments CCP4 community?
Many regards,
Antony.
On 25 May 2013, at 11:29, <[log in to unmask]>
wrote:
> Dear Sir,
>
> That is true the ligand is ATP, the occupancy problem
> comes only for the phosphates. This ATP tends to get
> cleaved to ADP and AMP in other complexes I got. So in
> this case do you suggest keeping different occupancies
> for nucleoside and phosphate groups? I am not aware of
> any publication with this scenario so I am not sure whether
> it is right.
>
>
> Thank you
> Regards
> Kavya
>
>> Dear Kavyashree,
>>
>> It is possible that your bound ligand (for which you have strong electron
>> density) is actually a break-down of the parent compound? We have seen
>> this a couple of times now.
>>
>> Also - are the poorly refining areas (those with negative density) part of
>> a pendant ring connected by a conformationally unrestricted bond? These
>> quite often have poor density.
>>
>> Hard to judge without seeing the actual density - but understand why!
>>
>> Regards,
>> Antony.
>>
>>
>> Sent from my iPhone
>>
>> On 25 May 2013, at 10:40, "Kavyashree Manjunath" <[log in to unmask]>
>> wrote:
>>
>>> Dear Sir,
>>>
>>> Thank you all for your kind advice and clarifications.
>>> I will keep the occupancy 1.0 for both the ligands and
>>> refine without considering the negative density in this
>>> case.
>>>
>>> Thanking you
>>> Regards
>>> Kavya
>>>
>>>
>>>
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>>>> Dear Kavya,
>>>>
>>>> I don't see much sense in having different occupancies within the same
>>>> molecule (unless one atoms sits on a special position, but then refmac
>>>> will take care of it).
>>>>
>>>> If positive density comes up it's a good sign the ligand really is
>>>> there. At 2.2A I would not be too surprised some atoms show less
>>>> density than others (but don't look too much at the map with a sigma
>>>> level < 1: you are going to see what you want to see).
>>>>
>>>> It's difficult to judge without sitting next to you, so my advice is
>>>> try to model it as good as your knowledge permits, and do take your
>>>> chemical knowledge into account when doing so!
>>>>
>>>> Best,
>>>> Tim
>>>>
>>>> On 05/25/2013 06:55 AM, Kavyashree Manjunath wrote:
>>>>> Dear users,
>>>>>
>>>>> I tried giving occupancy of 0.65 and 0.6 respectively for all atoms
>>>>> of the two ligands and refined. Now after refinement, the atoms of
>>>>> ligand does not have negative density but those which did not have
>>>>> negative density previously appear positive. So what do I need to
>>>>> do? under what circumstances does a ligand have different
>>>>> occupancies for different atoms or for a group of atoms. Any such
>>>>> references are very much welcome.
>>>>>
>>>>> Thank you Regards Kavya
>>>>>
>>>>>> Sir,
>>>>>>
>>>>>> Yes it is around ligand. The average B-factor of one of the
>>>>>> ligand is 36.78, of which one of the atom has occupancy B
>>>>>> factor 0.58 39.37 0.56 38.77 0.87
>>>>>> 37.00 Three atoms are of same type. The other ligand's overall
>>>>>> Bfactor is 17.64. occupancy B factor 1.00 19.29
>>>>>> 0.64 23.90 Two atoms are of same type.
>>>>>>
>>>>>> So in the present case should I put the occupancy of 0.56 for all
>>>>>> atoms of ligand-1 and 0.64 for all atoms of ligand-2 and refine?
>>>>>>
>>>>>> I mean will it be wrong to put different occupancies for
>>>>>> different atoms of same ligand?
>>>>>>
>>>>>> I could not see any alternate densities coming up for those
>>>>>> atoms which did not have densities. But 2FoFc map would appear
>>>>>> around these atoms at 0.7 sigma at the same position where the
>>>>>> atoms are present.
>>>>>>
>>>>>> Thank you Regards Kavya
>>>>>>
>>>>>>> Hi Kavya,
>>>>>>>
>>>>>>> If I understand you correctly (from title and text), you meant
>>>>>>> your negative FoFc was around your ligand, is that right? I
>>>>>>> wonder if this is a case in which the ligand has an occupancy
>>>>>>> below 1, but was modeled as 1, so the refinement program had to
>>>>>>> give it a high B factor to compensate that, which results in
>>>>>>> the electron density bleeding into nearby space where there
>>>>>>> should not be so much of it. If you want to test this
>>>>>>> hypothesis, one thing you can try is to set the occupancy to
>>>>>>> 0.25, 0.5,0.75 and so on, and refine a few cycles to see what
>>>>>>> happens to the maps. Also, what's the B factor of the ligand,
>>>>>>> and what's the B of the nearby protein atoms? The difference
>>>>>>> between them can also give you some hint for guessing the
>>>>>>> ligand's occupancy. Some refinement programs(phenix.refine and
>>>>>>> shelx) can also let you refine the ligand's occupancy.
>>>>>>>
>>>>>>> As to the "missing" atoms, that could be caused by alternative
>>>>>>> conformations of the ligand - assuming you have already done a
>>>>>>> thorough refinement.
>>>>>>>
>>>>>>> Zhijie
>>>>>>>
>>>>>>> -------------------------------------------------- From:
>>>>>>> "Kavyashree Manjunath" <[log in to unmask]> Sent: Friday,
>>>>>>> May 24, 2013 12:50 PM To: <[log in to unmask]> Subject:
>>>>>>> [ccp4bb] Negative FoFc around ligand
>>>>>>>
>>>>>>>> Dear users,
>>>>>>>>
>>>>>>>> I am using refmac 5.7.0029 for refining a structure
>>>>>>>> (resolution 2.2 Ang) bound to 2 ligands. After MR There is a
>>>>>>>> very clear density of ligands but after refinement, I get
>>>>>>>> negative fofc map near one of the ligand upto 5 sigma.
>>>>>>>> However its 2fofc map covers the whole ligand. Also for the
>>>>>>>> other ligand, I do not see any 2fofc density (at 3 sigma) for
>>>>>>>> 2 atoms, without these atoms the ligand is unrealistic. But
>>>>>>>> the density comes up around these at around 0.7 sigma.
>>>>>>>> Overall completeness is 99.9% Rmerge 7.5%
>>>>>>>>
>>>>>>>> What else I need to check in the data. Kindly provide some
>>>>>>>> suggestions.
>>>>>>>>
>>>>>>>> Thanking you Regards Kavya
>>>>>>>>
>>>>>>>>
>>>>>>>>
>>>>>>>>
>>>>>>>>
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>>>>>>>
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>>>>>>
>>>>>>
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>>>> - --
>>>> Dr Tim Gruene
>>>> Institut fuer anorganische Chemie
>>>> Tammannstr. 4
>>>> D-37077 Goettingen
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