Hello @yurotakagi,
When doing a local refinement, the mask supplied to the local refinement should cover only the region of the volume that you are interested in refining.
The reason that the tutorial mentions you may also want to create a mask that covers the rest of the volume (i.e. a mask covering the entire protein, except for your selected region of interest), is because local refinement often has improved results when the input particle stack has undergone partial signal subtraction. This is basically an attempt to remove all the signal coming from the undesired portion of the volume, such that the signal remaining in the particle stack corresponds only to the region of interest. In cryoSPARC, the “Particle Subtraction (BETA)” job implements this, and it takes in this “inverse mask”, as well as the consensus refinement, and the input particles. A typical workflow would be:
- Use homogeneous or Non-Uniform refinement to obtain a consensus refinement, and a particle stack with near-correct alignments
- Feed the consensus refinement volume and particles into the “Particle Subtraction (BETA)” job, along with the “inverse mask”, to create a stack of particles – these particles will ideally only contain signal from the region of interest
- Use the “Local Refinement (BETA)” job with the other mask, covering your region of interest, and the signal subtracted particles
Let me know if you have any additional questions!
Best,
Michael