GSFSC curve and map resolution


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!

Dear @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.

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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.

Thanks for the useful suggestion! I will try the CTF refinement and collect the dataset again.

Yes I would first try CTF refinement - if it is just an issue of large beam tilt you can often correct that pretty effectively computationally

Also double check that you entered the correct dose when importing the data