I have a data set collected at 25° tilt in superresolution mode. I am processing it with cryoSPARC 4.2.1. When the micrographs were binned twofold in “Patch Motion Correction,” the following “Patch CTF Estimation” gave an average tilt angle of 23.7°, which is reasonable. However, when the micrographs were not binned in Patch Motion Correction, the Patch CTF gave an average tilt angle of 12.3°, which is close to half of the angle of the stage. Comparing the results of these two approaches, all statistics are comparable except the average intensity, which is ~4 times higher in the binned data and makes total sense.
What caused the tilt angle to be halved? Any suggestions? Thanks!
Maybe a small number of (likely bad) micrographs with very different outlier values?
GPU computation is non-deterministic, there are going to be some differences like this just by rerunning the same job with e.g. a slightly different resolution limit.
Thanks for the input. I am sorry I didn’t state that this is not a small data set (~3500 movies). There are some outliers, but I don’t think those made much of a difference.
Here is the tilt angle histogram from unbinned data.
I tested on a different 40° tilt data set and got the same result. Unbinned data gave an average tilt angle of half of the stage tilt, while the twofold binned data gave an average tilt angle close to the stage tilt. I am using cryoSPARC 4.2.1 as well.
BTW this calculated tilt isn’t going to be used for anything elsewhere in processing - it’s only relevant if you wanted to threshold micrographs by the estimated tilt (which I would recommend against doing anyway - it should still be calcuated correctly though!).
Yes, I agree with you. I don’t think the tilt angle is used anywhere else. However, I was rather surprised when I first saw it in cryoSPARC Live, and it made me double-check the stage angle in data collection. If it is a bug, it should be fixed. Otherwise, people could be thrown off by that.
Just to add @hsnyder - we see the same thing in v4.4. We are collecting a dataset with 40 degree sample tilt right now, and the value reported after Patch CTF (excluding outliers) is almost exactly 20degrees. This is for data processed in CS Live, v4.4. Was there any further info on this issue? Is the underlying defocus estimation wrong, or is it purely an incorrect calculation of the tilt?
