Hi,
Your FSC curve indeed looks like particle duplication.
When you remove duplicates in a Subset Selection job,
did you specify the right pixel size?
For refinement, did you apply sufficiently strong low pass filtering to
the initial model? Didn't you follow Class3D with alignment by
Refine3D local search?
(Class3D does not separate half sets, so angles from it can be over-fitted,
especially for low resolution, hard-to-align cases. Starting Refine3D with
global search is safer.)
Best regards,
Takanori Nakane
On 2020/12/21 9:21, Aghil Soman wrote:
> Dear EM experts,
>
> I am pretty new to Cryo-EM and would like to seek expert opinion on an issue I am facing.
>
> I was refining the structure my first refinement gave a resolution of 5.9A (65K pcls), FSC attached. As some views were missing I manually selected
> those classes and refined again (80K pcls) to a resolution of 4.4A. But the FSC of 4.4A does not drop to zero. The map indeed is better in the 4.4A
> with better resolution of helices.
>
> Is the FSC acceptable?
>
> What could be the issue? And how can I sort this?
>
> I read in forum archive that it could be duplicate particles, I tried reextracting-First round I detected around 5k duplicate particles but the FSC
> doesn’t change much and doesn’t drop to zero.
>
> Many thanks in advance.
>
> Regards,
>
> Aghil
>
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