I would like to start a discussion about common strategies for high resolution refinements within cryosparc. If anyone has an example where it was beneficial to change software packages feel free to chip in.
If not stated otherwise default parameters were used.
My basic workflow starts with MC2 aligned movies, Patch CTF estimation, iterative 2D/3D classification and finally NU refinement (per-particle scaling). Usually, switching from MC2 aligned mics to cryosparc patch correction improves the resolution by 0.3-0.5A (0.832 A/pix). That way I achieve for a 120 kDa protein a resolution of 2.4 A (=0.7 Nq).
The data was acquired with AFIS, which seems to be calibrated quite well.
Using Global CTF correction (Tilt/trefoil) only marginally improves resolution by 0.1 A. Refinement of Cs consistently results in ~2.8 instead of 2.7 mm with no meaningful improvement of the refinement. Tetrafoil refinement usually worsens the results therefore I refrain from using that.
Per-particle CTF estimation may improve by 0.1 A.
Local motion correction improved by additional 0.1 A.
Finally I’m stuck at 2.2 A and piqued to hit 1.X A.
The map indeed looks like 2.2 A, masks generated by cryosparc (refinement and FSC) are in this particular case quite generous.
I’m curious about your strategies and hope for active discussion.