How to make the 2D result from crySPARC a template for autopicking in Relion?

Particle Picking
#1

Hi all,

I have some trouble in analyzing negative stain micrographs.

  1. I manually picked ~1000 particles for 2D classification. But when I tried to use the selected 2D classes for template picking, it seems like Cryosparc picked the dark background instead of the white particles. I know this is how it works for autopicking particles from Cryo-images in which the particle is black. But is there any solution for the negative stain?

  2. When I tried to use the generated .mrc file to serve as a template for autopicking in relion, it seems like I need to convert the .mrc to .mrcs. How could I achieve that?

  3. Where can I find the information about the pixel size of the generated .mrc file by cryoSPARC?

Thanks,

#2

  1. Not sure about this one, but when you import your micrograph to cryoSPARC, you should tell what type of images you are importing (negative staining, black-on-white).

  2. Just rename it to .mrcs

  3. You can use “header” command from IMOD to display the pixel space information of the mrc file.

1 Like
#3

Hi Bing,

Thanks for the answer! But could you show me how to tell cryoSPARC that the image is negative staining?

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#4

Probably you should tell cryoSPARC when you extract your particles. see the last option:

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#5

I opt to simply invert my negative stain micrographs using e2proc2d.py (EMAN2):

e2proc2d.py *.DM4 @.mrc --mult=-1

Disclaimer: this is for quickly checking if the protein is good or not, not for publication.

1 Like
#6

Hi @ruiruigo, @bing, @ZhijieLi, @DanielAsarnow,

For now, we recommend the solution posted above and here: Easy template picking

We’re aware of the inconsistency with how negative stain data is handled and will work to fix it in an upcoming release.

Thanks,
Suhail

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