Hi Julian,
If you extract 1 new AU per box upon extraction of helical segments in
RELION, then you can do this fully inside RELION already. In that case
you wouldn't need the expansion of helical symmetry: just take the
central AU. Particle subtraction and masked classification can then all
be done from the GUI. In our experience, this remains difficult, but
I'll be happy to hear yours.
:-)
Sjors
On 12/07/2016 03:46 PM, Julian Reitz wrote:
> Thanks to everybody who contributed to the discussion. There are a lot
> of good ideas and I will have to give them a try to see what I can
> achieve.
>
> Dear Juha, I have read your paper and your method sounds indeed very
> interesting. I would absolutely like to try this with my data. What do
> you think how long it will take until you implemented it so that we
> can at least try it?
>
> Thank you very much,
> Julian
>
>
> Zitat von Juha Huiskonen <[log in to unmask]>:
>
>> Dear Julian,
>>
>> Your problem could be addressed with our localized reconstruction method
>> (Ilca et al. 2015 Nat Commun) which allows dealing with all the
>> sub-structures at the same time. The method works nicely with
>> icosahedral
>> and lower point group symmetries, allowing 3D classification of
>> different
>> states. We don't support helical symmetry yet but are working on
>> adding it,
>> please get in touch if this is something you'd like to try.
>>
>> Best wishes,
>> Juha
>>
>> On Wed, Dec 7, 2016 at 11:35 AM, Julian Reitz <[log in to unmask]>
>> wrote:
>>
>>> Dear Sjors,
>>>
>>> thank you for your answer. Indeed the chapter in Meth Enzym was from
>>> great
>>> help.
>>>
>>> But I figured out a problem for our case that for me seems to be more
>>> severe.
>>>
>>> We are working on a helical reconstruction.
>>> Considering the relevant parameters we have approx. 22 subunits in our
>>> particle boxes.
>>> If I now mask out a single region in the reference map I only use the
>>> information of one of these 22 subunits per particle. And if I mask
>>> out the
>>> region in all the subunits I might have the problem that they are
>>> not all
>>> in the same state and that the structural heterogeneity is not
>>> consistent
>>> for all subunits in one particle.
>>>
>>> What would be your advise to deal with this?
>>>
>>> Thanks,
>>> Julian
>>>
>>>
>>> Zitat von Sjors Scheres <[log in to unmask]>:
>>>
>>>
>>> Dear Julian,
>>>> Hopefully our most recent chapter in Meth Enzym will help.
>>>> http://dx.doi.org/10.1016/bs.mie.2016.04.012
>>>> Indeed, it is the 'Reference mask' option that is needed, possibly
>>>> together with the subtraction jobtype.
>>>> For making masks, you can use relion_mask_create, but we also often
>>>> use
>>>> other software (e.g. volume eraser in Chimera, a fitted PDB-file, etc)
>>>> HTH,
>>>> Sjors
>>>>
>>>>
>>>> On 12/05/2016 01:10 PM, Julian Reitz wrote:
>>>>
>>>>> Hi all,
>>>>>
>>>>> we would like to do some 3D-classifications of our data.
>>>>> However we have reasons to believe, that there are positions in the
>>>>> structure that show independent heterogeneity. Meaning, that it is
>>>>> difficult to cover this variety with a normal classification.
>>>>>
>>>>> We would like to do something as follows:
>>>>> We would like to apply a mask that masks out a single of this regions
>>>>> and that the classification (and the following refinement) puts
>>>>> the focus
>>>>> on this region.
>>>>>
>>>>> As far as there is no misunderstanding on my site this is not what
>>>>> the
>>>>> "Reference mask" option for 3D-Classification is made for, since
>>>>> it should
>>>>> be used to mask out protein against the solvent.
>>>>>
>>>>> Any suggestions about how to do this?
>>>>>
>>>>> Best,
>>>>> Julian
>>>>>
>>>>
>>>> --
>>>> Sjors Scheres
>>>> MRC Laboratory of Molecular Biology
>>>> Francis Crick Avenue, Cambridge Biomedical Campus
>>>> Cambridge CB2 0QH, U.K.
>>>> tel: +44 (0)1223 267061
>>>> http://www2.mrc-lmb.cam.ac.uk/groups/scheres
>>>>
>>>
>>>
>>> --
>>> Julian Reitz, Dipl.-Biochem.
>>> J.W. Goethe Universität Frankfurt am Main
>>> BMLS - Buchmann Institut for Molecular Life Sciences
>>> Frangakis Group
>>> Max-von-Laue-Str. 15 Raum 1.658, D-60438
>>> Tel: +49 (0)69 798-46429
>>> e-mail: [log in to unmask]
>>> http://fcem.uni-frankfurt.de/em
>>>
>
>
--
Sjors Scheres
MRC Laboratory of Molecular Biology
Francis Crick Avenue, Cambridge Biomedical Campus
Cambridge CB2 0QH, U.K.
tel: +44 (0)1223 267061
http://www2.mrc-lmb.cam.ac.uk/groups/scheres
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