Hi @cbeck,
Thanks for your questions. Below are some of recommendations based on our experiences, so “your mileage may vary” as there are often many exceptions that will be target or dataset specific.
For your questions about when to do the above items:
- Local CTF Refinement ~4Å. For smaller particles, try limiting the defocus range to 500 to avoid deviating too much but at 3Å the default is usually good
- Global CTF Refinement (Tilt & Trefoil): ~4Å
- Global CTF Refinement (Spherical Aberration & TetraFoil) ~3-3.5Å
- Global CTF Refinement (Aniso mag): ~3-4Å
- RBMC: ~5-6Å
We would recommend a workflow like the following:
- NU-ref +/- PPS
- Take the best result and perform Local CTF Refinement and Global CTF refinement (all options enabled based on resolution of volumes) as separate, independent jobs (ie not within NU-ref).
- Examine results of the CTF Refinement jobs and if changes in defoci (local CTF ref) or plots from Global CTF look reasonable (strong red and blue features with fits that match the data and low residuals) then re-run Global CTF with just the parameters that adequately correct for aberrations.
- Run homogeneous reconstruct on all three particle sets (before, after local, after global) to check if things got better or worse.
- If any of the above are better, add them those CTF refinements to any further NU-ref or homogeneous refinement jobs.
- RBMC
- Once everything is corrected for (CTF refinements and RBMC), proceed with local refinements.
If resolution improves with RBMC, it is sometimes worth assessing if higher order CTF aberrations (e.g. tetrafoil) can now be fit with more confidence. So, it might be advantageous to run another Global CTF refinement with all options enabled to see if there is any improvement.
Best,
Kye