Dear All,
Thank you for a solution. Probably this is the Best
solution for this problem.
Thank you
Regards
Kavya
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> Dear Antony,
>
> I agree, modelling entirely separate entities with one occupancy for
> all atoms inside make chemically much more sense and it has the
> advantage that the refinement program can move e.g. ADP to a slightly
> different position than ATP - it is unlikely that the common atoms
> between the two molecules are exactly in the same position.
>
> Best,
> Tim
>
> On 05/25/2013 12:34 PM, Antony Oliver wrote:
>> 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
>>>>>
>>>>>
>>>>>
>> 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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>
> - --
> Dr Tim Gruene
> Institut fuer anorganische Chemie
> Tammannstr. 4
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