Merge different Subtracted particles from the same initial set from te ,recenter and crop

Utilities
1

Dear community,

I am struggling to merge two sets of signal-subtracted particles. I found a previous post ( Combining two sets of signal subtracted particles for refinement ) where this topic is discussed to some extent, but I still have several doubts.

  • After particle subtraction, can I recenter the region of interest and re-extract the particles with a new box size?
  • If I re-extract the particles, does this generate a new particle stack with new particle IDs? In that case, could I re-extract different regions from the initial stack and end up with multiple (X-times) more particles?
  • Once I have the newly extracted dataset, can I process it as an entirely new dataset?

Thanks in advance

Best

Sergio

2

Hi @baratachencho,

Here are some answers to your inquiry, please let me know if you need any additional info!

After particle subtraction, can I recenter the region of interest and re-extract the particles with a new box size?

Yes this is possible via the following jobs in this order:

  1. Particle subtraction (at the largest realistic physical extent)

  2. Volume Alignment Tools (VAT) - recentering on the ROI, reassign UIDs ON

  3. Downsample Particles - new box size specified, re-centering enabled.

NOTE: Do not use the job “Extract from Micrographs” to re-extract the particles. Re-extracting particles from micrographs will “undo” the particle subtraction since the particle subtraction job operates on the particle images, not on the micrographs.

If I re-extract the particles, does this generate a new particle stack with new particle IDs? In that case, could I re-extract different regions from the initial stack and end up with multiple (X-times) more particles?

New particle UIDs are not generated during extraction, rather, they can be generated during volume alignment tools. Note step 2 above to enable “Reassign particle UIDs” in VAT.

Once I have the newly extracted dataset, can I process it as an entirely new dataset?

Yes, you can treat these new particle images as a “new” protein. You shouldn’t need to perform ab-initio reconstruction since you already have particle poses that get things close. Good steps to follow-up with would include homogeneous reconstruction followed by a local refinement.

Best,

Kye

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