Recenter and rebox after subtraction

Local Refinement
1

I am wondering if it is possible to add the functions of “align 3D volume” and rebox into particle subtraction. For particle subtraction, we sometimes only want a small part of map and would like it to be in the center and with smaller box.
Thanks,
Lei

2

Hi @rainfieldcn,

Thanks for the query – the recommended way to accomplish this in CryoSPARC is by performing the steps separately. For example, one possible workflow looks like:

  1. Volume alignment tools (re-center):
    • Supply a volume, particles, and a mask covering the part of the map of interest; re-center to the center of this mask
  2. Extract from micrographs (re-extract)
    • Re-extract particles from Step 1 with “re-centering based on aligned shifts” enabled
  3. Create a mask around the area to subtract from the output volume from Step 1
  4. Particle subtraction (subtract)
    • Connect the inputs below and run the job:
      • mask from Step 3,
      • particles from Step 2, and
      • volume from Step 1
  5. Downsample particles (crop)
    • Set the real-space crop size to a reasonable number, based on the desired final refinement.

Best,
Michael

2 Likes
3

Hi Michael,
Thank you for reply!
Is Step #5 carried out in Step #2? Or is there another function for this cropping process?
Best,
Lei

4

Dear @rainfieldcn,

In step 2 you have the choice to choose a new box size. This box size must be big enough to cover both the centered region of interest, as well as the region that is being subtracted in step 4. In fact it should still be at least 2x the size of the full protein (as per our standard recommendation of the box size). This is because particle subtraction uses the CTF of the subtracted region, and if this region is near the edge of the box, signal can be cut off (see this reference for some discussion). It is only after particle subtraction occurs that we would recommend cropping to the final smallest box size, via downsample particles

Best,
Michael

5

Hi @mmclean,

Thank you for summarizing the necessary steps here, but I noticed a couple of potential issues of the workflow that i want to share here.

  1. The volume output of step1 is masked. So to get an unmasked, but translated volume for subtraction, we have to repeat step1, but this time without a mask, using the calculated number of mask center. Can cryosparc also output the unmasked volume here so we don’t have to repeat?
  2. If the particle box is not large enough, then the shifted volume from step 1 will be truncated. In this case, we have to do a reconstruction job using particles from step2. Therefore the box size of the particles in this step has to be big enough.

Best,
Hui