How to apply .mrc gain reference file for .eer images?


I’m using cryosparc live to import .eer files collected from falcon 4 camera and pick particles.
Although the images are in .eer format, the gain reference file is in .mrc format.
I’m using upsampling factor of 2 and 40 frames.

I have processed the same data set twice, once with the gain reference and once without. The reconstruction calculated from images without the gain reference applied achieved a higher resolution by 0.2 Å, indicating that the reference had been applied incorrectly. The gain reference file has already been accounted for flip or rotation, so I don’t think that would have been the problem.

What is the correct way to apply .mrc gain reference file for .eer file?
It seemed like other tools (ex. Relion) requires multiplying or dividing pixel values of the gain reference file depending on the upsampling factor. Does Crysparc also require something like that too?

Thanks in advance for your help!


Hi @Jecy,

When using upsampled EER data, CryoSPARC automatically scales the gain reference and divides by the squared upsampling factor, which should correct for the size change. When you say the gain reference file has already been accounted for flip or rotation, are you referring to using the “flip in x/y” toggles within CryoSPARC, or do you mean it was corrected externally? If the latter, I’d recommend having a careful look at the plots from your import job to make sure that the orientation is still correct and doesn’t need to be flipped or rotated after importing.