Hi,
I am a new user of cryosparc and I have quite a lot of problems with my refinement. Currently I am working with a couple of GPCR complexes. I have one case like that: the map quality looks quite OK to build a model, but the resolution is around 4.2 A. I noticed that all the FSC curves overlap to each other, so is there something wrong when I do the refinement?
PS: I followed the workflow of the GPCR case in the cryosparc video on the Youtube and for the NU refinement I used the default parameters.
Thanks for help!
Geng
How large is the box size relative to the protein? The fact that the curves are all closely spaced indicates to me the box could be a bit too small: applying the mask eliminates a lot of noise from the reconstruction, so the masked FSC scores are significantly higher than unmasked. If the initial box size is less than around 2.5-3 times the max diameter of the protein, could you try re-doing everything post extraction with a larger box size?’
We have also seen similar results in the event of large higher order optical aberrations in the dataset - I would also try Global CTF refinement as a diagnostic step.
Thanks a lot for your kind reply! The box size is 400A and my protein complex diameter is 150A, so I think it might not be the box size’s problem. I will try to collect data again to see whether it could be solved or not.
