Hi,
This is difficult to investigate without access to the files.
Did you check CTF parameters and optics group table remain intact?
Best regards,
Takanori Nakane
On 2021/05/04 7:20, Piotr Dr ączkowski wrote:
> Dear Takanori and cryoEM community,
>
> I run into a problem when I tried to re-extract/un-bin a set of particles and I can't solve it by myself.
> Whenever I re-extract (with a full box size) a set of binned particles that were previously signal-subtracted (with automatic re-centering on the centre of the subtraction mask) and then reconstruct them using relion_reconstruct I am getting a completely messed up map as a result. The map roughly resembles the shape of my complex but is all fragmented and I can't see even a trace of secondary structure elements. Refining it sometimes can re-align the particles back but often I get a worse resolution than the one with binned particles. I tried to perform re-extraction using reverted signal-subtracted particles, with and without re-centring on refined coordinates, none of those helped.
> Earlier, with earlier Relion versions, I used to center the particles by myself at specific coordinates and then do the subtraction. I this case, when I later re-extract/un-bin and reconstruct the particles I was getting a map that looked more or less the same as the one with binned particles (of course plus the extra part that was earlier subtracted).
> I am puzzled why this is not the case when I re-extract/un-bin particles after signal subtraction done by Relion 3.1? Did anybody experienced the same problem or is it intrinsic to my pipeline? Could it be due to the automatic centering on the centre of the subtraction mask? If so, should I manually edit the off-sets/coordinates?
>
> Best regards,
> Piotr
>
> ______________________
> Piotr Drączkowski, PhD
> Academia Sinica
> Institute of Biological Chemistry
> 128, Sec. 2, Academia Road, Nankang, Taipei 115
> TAIWAN
>
> Faculty of Pharmacy with Division of Medical Analytics
> Medical University of Lublin
> ul. W. Chodzki 4a, 20-093 Lublin
> POLAND
>
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