Hi @ruiruigo, cryosparc is assuming you have cryo data and inverting the templates. Here’s a dark version of the template you can try (and then select “negative stain images” during particle extraction), however I found that cryosparc doesn’t work as well with the dark template and the options for data sign are confusing or buggy.
What I do is to simply invert my micrographs before I import, and treat them like cryo data throughout the cryosparc processing. Here’s my full negative stain pipeline, I do this mechanically for new datasets and it basically works well.
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for i in *.mrc; do e2proc2d.py --mult=-1 $i ${i%.mrc}_inv.mrc; doneORparallel -j 8 e2proc2d.py --mult=-1 {} {.}_inv.mrc ::: *.mrc - Import the _inv.mrc files to cryosparc.
- CTF estimation, set amplitude contrast 0.15 - 0.4.
- Template pick with the imported white Gaussian, set particle diameter as desired and angular sampling to 360 deg. (no reason to rotate isotropic template).
- Extract particles with desired box size and bin to ~6 A / px (e.g. 200 px -> 64 px for 2 - 3 A/px from T12 at 46kx mag), or use low magnification so pixel size is already ~6 A.
- Run 2D classification, set desired no. classes and resolution limit 12 - 20 A.
