Hi everyone,
I’m processing a K3 dataset collected at 130 kx nominal magnification with super-resolution mode enabled (factor 2).
The EPU metadata gives a physical pixel size of 0.6719 Å (6.7187e-11 m). The raw movie files are 11520×8184 pixels.
I’m confused about which value to use in the CryoSPARC “Import Movies” job:
The physical pixel size 0.67 Å
The super-resolution pixel size 0.67 Å / 2 ≈ 0.335 Å (or 0.336 Å)
Any experience would be much appreciated. Thanks in advance!
Since your movies are superresolution, cryosparc wants the pixel size to be 1/2 of the physical pixel size, so use 0.336 here. I would typically apply an F-crop of 0.5 during motion correction so that the output micrographs are at 0.67 Å, but that depends on your application.
When in doubt, if your pixel size is off by a factor of 2, you will see weird results in the CTF fitting, like very high or very low resolution.
Thanks for your generous answer.
I have another question, my gain file is in dm4 format, when converting it to mrc format, should the pixel value in the converted gain file be 0.67 or 0.336?
Thanks again!
Hopefully your gain file is super resolution, and you should keep it that way. Gain subtraction happens before F cropping.
So the pixel value in the gain file should be 0.336?
Thanks again for your answer!
Right! At my facility the superres gain file name includes “m1”, but I’m not sure if that’s universal
With K3 data, I usually prefer to generate a gain reference from the movie frames. It’s always been a lot more robust doing that.
Can I ask how you go about generating gain files like this?
relion_estimate_gain from RELION or sum_all_tif_files from cisTEM. The RELION program is more flexible, but the cisTEM one requires less setup.
Thanks for your generous help！
