Sorry for the late reply.
I previously used these commands in Relion:
relion_reconstruct --angpix 1.06 --i from_csparc_P7J258_AC00.star --ctf --o from_csparc_P7J258_reconstruct.mrc
relion_project --i from_csparc_P7J258_reconstruct.mrc --mask cryosparc_P7_J456_Cdommask.mrc --subtract_exp --angpix 1.06 --ctf --ang from_csparc_P7J258_AC00.star --o Ndom_tightmask_AC00
When you mentioned lowpass filter, I realized that the mask used for subtraction was created by taking the localfilter map, erasing the unwanted domain, lowpass filtering to 10A and binarizing in cryosparc.
I now used the volume from NU-refine with Segger to delete the unwanted domain and created a mask without lowpass filtering. This seemed to improve signal subtraction in cryosparc and I have done local refinement using those particles.
Could I please check the following procedure for local refine with you? I am working remotely from home and am unsure if my method, maps and FSC curves are correct.
I have a consensus map from NU refine at 4.4A res
After signal subtraction, I performed local refinement in CS which improved both domains (5A initial lowpass of first domain, 7A initial lowpass of second domain (otherwise alignment plots were empty), dynamic masking with 6-18 width, 10deg 10A search domain 1, 25deg 10A search domain 2, gaussian prior enabled):
I then ran those particles and volumes through Relion 3D classification w/o align (T=20, K=4) with single-domain masks and separated out a few remaining junk particles and lower res classes. These were again imported to CS and local resolution performed (force-redo split enabled, ini lowpass 5A for both domains, 5deg and 2A search with gaussian prior, dynamic masking 0.2 threshold 6-18 width)
Does anything look wrong in the method I followed? At 0.2 threshold the glycans are not included in the dynamic mask and using a lower threshold leads to poorer resolution, as expected. How can you tell if overfitting to noise is present?
This is domain 1 with volume_map and volume_mask_refine from local refine which shows the glycans being cut by the refinement mask. Is that alright to do? It does have a soft edge which covers them at the lowest threshold.
When I look at volume_map (left) vs volume_map_sharp (right) from local refine, the helices at the top in this image look stretched after sharpening. How can I improve this? Does it mean that they are of lower resolution than the rest and now over-sharpened?