Dear QL,
You could try to 'ignore CTFs until first peak' to get more classes in 2D
classification. Also, you could extract segments in larger boxes to
separate different types of filaments. Often though, we pick our filament
types separately by hand, which seems to work better than classification.
Unfortunately, amyloid reconstruction isn't yet as streamlined as
single-particle analysis. One problem is that the 3D
refinements/classifications have many local minima: a problem that is well
known in helical reconstruction in general.
HTH,
Sjors




> Hi all,
> We are an amyloid lab and just started working on cryoEM. Our own dataset
> was giving me a really hard time so I'm trying to go through the whole
> process by processing data deposited on EMPIAR. Particularly, #10230
> (Falcon B et al., Acta Neuropathol. 136 699-708 (2018)).
> I learned mostly from Relion tutorial, helical processing tutorial, Sjors'
> lecture online as well as some colleagues who do single particle. It seems
> to be not easy to repeat the literature results. For example, in 2D
> classification, I ended up with only one class (with clear beta-sheets and
> very blur edges) with T=2~8. How should I setup the parameters in this
> step? Or anything in earlier steps should be adjusted?
> Also just in general, would anyone be kind enough to share a general
> tutorial or tips for amyloid fibrils helical reconstruction? Or anywhere
> else I can find such specific helps? It would help the newcomers a lot!
> Thanks!
>
> QL
>
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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