Regarding the box size and cropping

Particle Picking
1

Hi there, I have a question regarding box cropping after particle subtraction in CryoSPARC. I performed particle subtraction using a large box size (560 px), followed by a homogeneous refinement as a sanity check. It now seems necessary to crop the particles and volume down to 360 px to achieve better resolution and for further downstream processing.

I’m unsure how to do this properly. I came across a thread suggesting that the Volume Alignment Tools job could help, but I’m not confident about the required parameters for re-centering, such as the 3D coordinates and rotation settings.

If I go this route, would I also need to re-extract the particles with the new desired box size (360 px)? And if so, would this re-extraction apply only to the subtracted particles or to the original particles as a whole?

Any advice or clarification would be appreciated—thank you!

2

Hi @rimonparves!

Thanks for your question, we think that a workflow that might work for you is:

  1. Create a focus mask for your volume region of interest
  2. Use Volume Alignment Tools with your subtracted particles and focus mask, enabling Re-center to mask center of mass

This will ensure that your region of interest is in the centre of the box, so that when you crop it should not get cut off.

  1. Use Downsample Particles to crop the box as desired

Then you should be able to continue with your processing without the need to re-extract (re-extracting will undo the subtraction).

Should you wish to re-extract first (this can sometimes be helpful to capture delocalised signal if your region of interest is near the edge of the box), you can instead run steps 1 and 2 followed by particle extraction with your desired box size, and then particle subtraction.