Dear Sjors and all the others,
thank you very much for the very helpful input!
We now expanded the star file and made a masked classification which
resulted in one class nicely showing the expected conformational
change. However, refinement of this class has led to a bad result.
We will now do the particle substraction as you suggested and try again.
For my understanding, could you clarify the need of many classes and
the higher-than usual T-factor?
Best,
Julian
Zitat von Sjors Scheres <[log in to unmask]>:
> Dear Julian,
> It is as Bayly says:
>
> relion_particle_symmetry_expand --i refine3D/jobXXX/run_data.star
> --sym C3 --o expanded_particles.star
>
> Then there are multiple options. I would suggest making a mask
> around 1 monomer and another mask around the other 2 monomers. Then
> subtract 2 monomers from all expanded particles, and feed the
> resulting images into a 3D classification (without re-alignment)
> that uses the single-monomer mask. Use many classes (10?) and a
> higher-than usual T-factor, say 10-20.
>
> The original reference is :
> S.H.W. Scheres (2016) "Processing of Structurally Heterogeneous
> Cryo-EM Data in RELION" Meth. Enzym. 579, 125-157 (section 4.7)
>
> HTH,
> Sjors
>
>
> Charles Bayly-Jones wrote:
>> Yes, the relion_particle_symmetry_expand function will allow you to
>> accomplish this. Then perform masked 3D classification as you
>> described. It is very useful indeed :)
>>
>> You could also use the localised reconstruction method from Ilca et
>> al. (2015) doi:10.1038/ncomms9843
>> <http://www.nature.com/ncomms/2015/151104/ncomms9843/full/ncomms9843.html>
>> Best of luck,
>> Charlie
>> _________________*
>> *
>>
>> *Charles Bayly-Jones*
>> BSc(ScSchProg)(Hons)
>>
>> *PhD Candidate*
>> *Teaching Associate*
>> *
>> *
>> *Monash Biomedicine Discovery Institute*
>> *Department of Biochemistry and Molecular Biology*
>> Level 2, Building 77
>> 23 Innovation Walk
>> Clayton VIC 3800
>> Australia
>>
>>
>> On Tue, 5 Mar 2019 at 21:01, Julian Reitz <[log in to unmask]
>> <mailto:[log in to unmask]>> wrote:
>>
>> Dear all,
>>
>> we are working on a dataset from a homo-trimeric protein. Until
>> now we have very successfully refined the structure to good
>> resolution applying C3 symmetry in RELION.
>>
>> However, we also have a sample from this protein that contains a
>> low affinity binding substrate and we would like to do a
>> classification to see if there are some subunits showing the
>> expected conformational change due to substrate binding. The point
>> is, that we do not expect the ligand to bind to all three monomers
>> of one particle but that the conformational change might only
>> happen in one of the monomers of a particle. Therefore, normal
>> 3D-classification seems unrewarding and a masked classification
>> would suffer from the aspect, that we always only look on one of
>> the monomers ignoring two-thirds of the data.
>>
>> We were thinking that it might be helpful if we somehow triplicate
>> the particles, each rotated by 120° to get all of the three
>> monomers from one particle inside the mask. Is there any clever
>> way to do this in RELION or does anybody else has a good idea how
>> we could proceed?
>>
>> Best,
>> Julian
>>
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>
> --
> 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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--
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
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