Dear Eleanor,
many thanks for all your help and suggestions. We finally have solved
the mutant structure. The trick was to use the pseudo translation (there
was not any 2 fold axis in the self rotation) with MOLREP which found
two molecules and then applied the pseudo translation to provide us with
the other two molecules yielding a tetramer. I think that initially the
programs and also ourselves missed the pseudo translation because it
is very close to the crystallographic one (0.49 0 0.48). Many thanks
to Peter Zwart who also advised us to follow the same procedure.
Demetres
Eleanor Dodson wrote:
> Oh dear - this is tricky!
> You could have a dimer in the asymmetric unit, or two monomers in the
> asymm unit which generate dimers using the crystallographic 2 folds.
>
> Things worth checking -
> What does the self rotation show? Is there a clear 2 fold axis which
> is different from the crystallographic one?
> If so you can use all of MOLREPs cleverness to use the self rotation
> definition and the pseudo translation.
> (Under search parameters you can give the self rotation list of
> solutions - just use self rotation with 1 or 2 solutions and edit out
> the top one which will be the crystallographic 2 fold)
>
> It will find the pseudo trans vector automatically.
>
> If the only 2 fold is the cryst one then you need to search for 2
> molecules with the pseudo translation.
>
> Eleanor
>
> What is the solvent content of the native - the mutant has a smaller
> volume so less solvent - is that feasible?
>
>
>
>
> I think I would generate P1 data Demetres D. Leonidas wrote:
>> Dear Eleanor,
>>
>> Yes the native is a dimer and we did the search using the dimer as a
>> model but we had similar results (i.e. all programs find one
>> molecule). The graphs from TRUNCATE show rather "normal" and I am
>> attaching a gif file with the plot for the cumulative intensity.
>>
>> As for pseudo-translation running the "Analyse Data for MR" option in
>> ccp4 in the patterson map we are getting a significant peak at
>> fractional coordinates 0.4897 0.0 0.4767. How this can help ? Do we
>> need to apply this pseudo translation to the solution we are getting
>> from molrep ?
>>
>> many thanks
>>
>> Demetres
>>
>> P.S. I will summarize for the members of the list all the suggestions
>> I will get at the end
>>
>>
>> Eleanor Dodson wrote:
>>> You dont say whether the molecules in the native cell form a dimer -
>>> if so I would search with that (you may need to turn off the packing
>>> search)
>>>
>>> Or whether there is a pseudo translation vector in the mutant form..
>>>
>>> Or what the data analysis graphs from TRUNCATE show - are they
>>> "normal"?
>>>
>>> Eleanor
>>>
>>> Demetres D. Leonidas wrote:
>>>> Dear all,
>>>>
>>>> we have encountered a problem in solving one mutant structure. The
>>>> mutant protein crystallizes in the same space group as the native
>>>> (C2) but the unit cell dimensions are different. These for the
>>>> native structure are 101.2 33.1 73.4 90 90.3 90 and for the mutant
>>>> 160.4 32.3 107.0 90 125.7 90. As a result the mutant structure has
>>>> four molecules in the asymmetric unit while the native had two.
>>>> When we run molecular replacement all programs (CNS, molrep, and
>>>> amore) find only two molecules. Phaser finds four but when we try
>>>> to refine the Rfree does not drop below 0.44 if we use four
>>>> molecules and 0.53 if we only use two no matter how well we built
>>>> the molecule and regardless of any addition of water molecules (the
>>>> resolution of the data is 2.1). The interesting thing is that in
>>>> the electron density map we can clearly see density for a substrate
>>>> analog that was included in the crystallization media. Do you
>>>> thing that we have a case of twinning here ? We have to mention
>>>> that Tod Yates served did not indicate any perfect merohedral
>>>> twinning (partial merohedral twinning for this space group is not
>>>> possible).
>>>>
>>>> We would appreciate any comments
>>>>
>>>> Many thanks
>>>>
>>>> Demetres
>>>>
>>>
>>>
>>
>>
>> ------------------------------------------------------------------------
>>
>
>
--
Demetres D. Leonidas, Ph.D.
Structural Biology & Chemistry Group
Institute of Organic and Pharmaceutical Chemistry
The National Hellenic Research Foundation
48, Vassileos Constantinou Avenue
Athens 116 35, Greece
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