Hi,
Consider a situation like this (denoised & not denoised):
The little black dots are (I think) small gold particles shed from the grid. Where they end up in an extracted box, these can perturb alignment, and lead to 2D classes like this:
Relion has an option to deal with this kind of situation, by which pixels above and below a user-configurable number of std. devs from the mean are replaced with the mean value (or noise distributed around the mean value - not sure).
I understand from previous posts on the forum that particle extraction in CS does something similar, but it is not user-configurable. Would it be possible to make it user adjustable, in order to allow rescuing more usable data in situations like this? Would also help with proteins bound to gold nanoparticles, metal clusters etc.
Cheers
Oli
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Hi Oli,
thanks for reporting, it’s interesting. It’s quite a lot of gold, do you see this often?
Does it impact 3D reconstruction if you go directly to ab-initio?
Thanks
Vincent
Not often but enough that I would like a better way to deal with such data! Yes, high contrast junk like this affects ab initio badly.
Hi @olibclarke thanks for the report, definitely the presence of strong contrast “dots” would affect abinitio and pose alignment generally.
In CryoSPARC, we do hot pixel rejection at the movie frame stage, but not within micrographs after motion correction. Since these dots are not detector defects, they probably wouldn’t get picked up by the frame-level hot pixel detection (because these pixels would be dark, i.e. lower electron counts, not hot).
We’ve recorded this topic internally for investigation.
Have you tried removing the dots in Relion using the stdev pixel replacement you mentioned? It would be good to know if you found that to be a workable solution.
Thanks!
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Hi Ali - I haven’t tried re-extracting in relion in this instance to compare, but it is a good point - I will give it a go and do a direct comparison by Class2D!
Cheers
Oli
