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CCP4BB  March 2009

CCP4BB March 2009

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Subject:

Re: Could I improve an inconsecutive experimental map?

From:

Eleanor Dodson <[log in to unmask]>

Reply-To:

[log in to unmask]

Date:

Thu, 12 Mar 2009 09:28:33 +0000

Content-Type:

text/plain

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I presume you have done density modification after calculating the exptl
phases?
If you use REFMAC to refine your partial model with the exptl/density
modified phase restraints your FWT PHWT fourier coefficients already use
the combined phase.

I am not sure how to choose a damping factor. If you think the exptl/dm
phases are good and have realistic weights  I guess there shouldnt be one..
SHARP claims to get the weights more or less right; parrot is meant to
do a better job of weighting than DM.
Eleanor





 Fengyun Ni wrote:
> Hi,
>
> I have tried SHARP, but no positive results could be obtained.
>
> Do you have some articles on how to combine the model phase and experimental
> phases?
> I could only seen the consecutive density in mir 8 to 6 A resolution map, so
> I could only locate roughly the position of helix.
> After that I use this helix to calculate the structure factor in SFALL.
> In SIGMAA, what's the DAMP coefficient should I use?
> After combination, do I need to do density modification?
> Hope for your help!
>
> Regards,
> Alphar
>
>
> 2009/2/21 Charlie Bond <[log in to unmask]>
>
>
>> Hi Alphar,
>> A while ago I had success with a poor MIR map by building in a few
>> fragments I could identify and then combining phases from this model with
>> the MIR phases using SIGMAA (CCP4 program) and then calculating new maps. A
>> couple of cycles of this resulted in phases in which I had confidence and I
>> was able to build the complete structure. (There's probably a more modern
>> way to do this now ...)
>> Cheers,
>> Charlie
>>
>>
>> Quoting Fengyun Ni <[log in to unmask]>:
>>
>> Thanks, Ezra!
>>
>>> I have a native dataset.
>>>
>>> I'll try what you said, Hope it works in my case.
>>>
>>> Thanks!
>>>
>>> Alphar
>>>
>>> 2009/2/21 Ezra Peisach <[log in to unmask]>
>>>
>>> Personally, I would try to build into what you see and then do
>>>
>>> phase
>>>
>>> recombination.  I had a SeMet crystal in which I could only see
>>>
>>> part of a
>>>
>>> sheet and helix after DM... Things were not connected - until I
>>>
>>> improved
>>>
>>>   the phases with my model.   Also - do you have a native dataset?
>>>
>>> You do not
>>>
>>> mention it.
>>>
>>>> Ezra
>>>>
>>>>
>>>>
>>>> Fengyun Ni wrote:
>>>>
>>>> Hi everyone!
>>>>
>>>>> I have a question on my poor MIR map.  Four datasets (two AU and
>>>>>
>>>>>
>>>> two HG)
>>>>
>>>  were used for phasing upto 3 A resolution in my case. I could  locate
>>>
>>>>> several
>>>>> heavy atom sites for each dataset with occupancy finally refined
>>>>>
>>>>>
>>>> to about
>>>>
>>>  0.3 to 0.4, and with B value refined to about 50 to 80. I did not
>>>
>>>> include
>>>>
>>>  the anomalous data because once I refine against anomalous data,
>>>
>>>> the
>>>>
>>>  anomalous occupancy was only about 0.02, and even smaller as
>>>
>>>> 0.004 for some
>>>>
>>>  sites. I guess the anomalous data are not good enough, so I did  not use
>>>
>>>>> them
>>>>> right now (Could I do this? Or must I include them somehow?).
>>>>>   The FOM I got is about 0.6, the Cullis-R factors were about 0.6
>>>>>
>>>>>
>>>> for all
>>>>
>>>  four data sets, and the phasing power was about 2.5 in each  dataset.
>>>
>>>>> After I
>>>>> did the density modification, the FOM could increas to about 0.8.
>>>>>   My problem is that the protein should form a long helix
>>>>>
>>>>>
>>>> structure as
>>>>
>>>  indicated by other homology protein, but in the map after density
>>>
>>>>> modification, the densities are not consecutive though the
>>>>>
>>>>>
>>>> overall shape
>>>>
>>>  seemed to be a long helix. The good thing is that some short
>>>
>>>> helix-like
>>>>
>>>  densities could be observed in the current map, but they are not
>>>
>>>> connected
>>>>
>>>  to each other.
>>>
>>>>>   Right now, I am totally lost whether I should believe in the
>>>>>
>>>>>
>>>> mir-map I
>>>>
>>>  have. Could I improve this map with some other method? Or should
>>>
>>>> I
>>>>
>>>  re-do the
>>>
>>>>> phasing?
>>>>> BTW, the space group for my crystal is R3 or R32 with a=50, b=50,
>>>>>
>>>>>
>>>> c=380,
>>>>
>>>  alpha=90, beta=90 and gamma=120. The c-axis is much longer than
>>>
>>>> a- and
>>>>
>>>  b-axis, so the reflection data suffered from the anisotroy
>>>
>>>> effect.
>>>>
>>>    Any suggestion on my phasing problem is welcome.
>>>
>>>>> --
>>>>> Alphar Ni
>>>>> Call me alphar~
>>>>> :)
>>>>>
>>>>>
>>>>>
>>>>
>>> --
>>> Fengyun Ni
>>> Call me alphar~
>>> :)
>>>
>>>
>>>
>> --
>> Charlie Bond
>> Professorial Fellow
>> University of Western Australia
>> School of Biomedical, Biomolecular and Chemical Sciences
>> M310,  35 Stirling Highway
>> Crawley WA 6009, Australia
>>
>>
>>
>>
>
>
>

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