[3dem] 2D classification resolution problem

[Dario]

Thanks a lot. I'll have a look at the 0 and 30degree data separately and
see if I get a difference on resolution.

Takanori Nakane <tnakane.protein at osaka-u.ac.jp> schrieb am Do., 7. Sept.
2023, 11:57:

> Hi,
>
> Tilted data collection means that defoci vary a lot within a micrograph.
> RELION uses CTFFIND, which finds only one defocus value per micrograph.
> If the dataset is good enough, you can repeat CtfRefine and Refine3D
> to bootstrap (see Fig 3 of
> https://urldefense.com/v3/__https://elifesciences.org/articles/42166__;!!Mih3wA!AnimFSo7qGvWrVPMUf3pJDzBeoDKRfRjvihyb6Diktr0NQutZuEqaIb6gXZVx8uSCtu5m9NBsxsf1BiZCFr5NAC1H3AmRizEXqA$
> ).
> Otherwise, you might need local CTF estimation by other programs such
> as Warp.
>
> Even if you manage to get local CTF, a tilted grid means a longer
> path electrons have to pass through; so this has the same effect
> as thicker ice.
>
> In my opinion, tilted data collection is the last resort against
> preferred orientation problems. If you can resolve the issue by
> other means (e.g. optimization of blotting parameters, additives,
> slightly different construct), that would be much better.
>
> Best regards,
>
> Takanori Nakane
>
> On 9/7/23 18:48, Dario Saczko-Brack wrote:
> > Dear all,
> >
> > I have acquired a small cryo EM data set of a protein (ca 120kDa) and
> > processed it with Relion and Cryosparc. Since the 2D classes looked
> > promising, but the orientation bias was very strong and the number of
> > usable holes was very small, I made new grids and acquired another, much
> > larger data set of the same protein at 0° and 30°.
> >
> > For some reason, I dont get to a similar resolution as the first data
> > set. Any idea why? Would you say that is rather due to not optimal
> > processing parameters or is it due to bad ice quality? Meaning, is it
> > worth to invest more time into processing or into making new grids?
> >
> > Best
> >
> >
> > Dario
> >
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