I want to merge two datasets by CryoSparc. However, these datasets have been collected from two different centres with different magnifications and pixel size, number of frames in each movie and total dose. Is it possible to mix them? If yes at which step can I do that?
So far only of all image processing programs, only Relion directly supports using particles with mixed pixel sizes. Even then, whether this is completely safe or if you would see, e.g. 3D classification dominated by the pixel size difference is not well tested.
Therefore you have to match the pixel sizes somehow. It can be done at two steps - particle extraction or motion correction.
For particle extraction, you have to find particular values of raw and Fourier cropped box sizes that result in matched pixel and box sizes. For example, I had one dataset collected with a pixel size of 1.08, and another with a pixel size of 1.14. I was able to extract the first one at 456px, and the second at 432px, and by luck 432/456 = 0.9473684211 times 1.14 = 1.08 exactly. There are scripts floating around that can help you search for pairs of box size that satisfy this relationship within a given amount of error.
Really, though, the best way is to do match micrograph pixel sizes during motion correction. Because the micrographs are so large compared to particles, the spectral resolution of the Fourier transform is much higher. That means much finer increments in the final pixel size may be obtained, and also the interpolation errors are less severe. All you have to do is re-run motion correction, with a precise F-crop factor. For example, if you have movies with a pixel size of 0.84 Å and another set with size 0.66 Å, you could run Patch Motion on the 0.66 Å/px set with F-crop = 0.7857142857. After that the output micrographs will have the same 0.84 Å/px and you can forget about the difference. Just make sure they are in different exposure groups for any CTF refinement.
PS All the other parameters you mention are fine, differences are already allowed/accounted for.
The second method seems promising.
However, in cryoSPARC, only a set of F-crop can be selected. User can`t specify a certain value.
Yes, I would recommend Motioncor2 for that.
The second method is promising. By ‘F-crop’ you mean the ‘FtBin’ right?
BTW, 1, I processed my data using Patch motion correction and Patch CTF estimation in the CryoSPARC and extracted particles.
2, Now, after Motioncor2, Import Micrographs and Patch CTF estimation in the CryoSPARC, do you think the particle extraction will works or not when I apply particles from 1 to micrographs from 2? I am worrying how the CryoSPRAC know go to the right micrographs to extract particles (I thought the UID already changed).
Thanks a lot,
Right, in MotionCor3 (2 is sooooo 2023) the parameter is -FtBin and output pixel size is the argument times the input pixel size. In CS the parameter is F-crop and it is the reciprocal (also only certain rational numbers are allowed).