Overfitting in refinements

Hi all,
I am new to cryoEM and cryoSPARC. I am working with a small protein complex (100 kDa) in my master thesis and I am having some problems with the 3D reconstruction. The dataset was collected with volta phase plates.
I have only about 70-100k particles.
The ab initio models don’t look bad, but when I turn into the refinement, the “new” homogeneous refinement overrefines the model and “breaks it” completely. If I use the “legacy” algorithm or the non-uniform refinement, it also produces overfitting.
Example of ab initio model (71k particles):


“New” homogeneous refinement:


Non-uniform refinement “New”:

![P4_J1060_fsc_iteration_004_after_fsc_mask_auto_tightening|496x283]P4_J1060_viewing_direction_distribution_iteration_004 (upload://5imET93ikf1DFbR8KEVEcMR2Vp0.png)

“Legacy” homogeneous refinement:


If tried tweaking the parameters of the dynamic mask to make it softer, but it has not helped. I also tried limiting the alignment resolution, performing multiple ab initio classes and doing heterogeneous refinement. But I always get overfitting. Is it that this dataset is not good enough or I do not have enough particles?
I should also say that the 2D classes look “grainy” unless I turn Force max over shifts/poses off.
Thank you!

I don’t have a direct answer to your questions, but here are my observations:

  1. The elevation/azimuth plots of the ab initio refinement indicate you have a restricted orientation at (±pi,0), which may be problematic. You can sometimes overcome this if you collect enough data and then discard a large fraction of the particles in those views, so that the angular distribution is more uniform.
  2. Your FSC plots indicate you collected this data at (or down-sampled it to) 1.6 Å/pixel. I suspect this may prove too course for a small particle like this. You’d probably do better to take the micrographs at 2x the mag, which requires collecting 4x as many images. Yes, it’s a trade off.
  3. I’ve no experience with Volta plate data, but when I run into problems I always want to check whether the CTF correction is right, and whether the particle picking is decent (i.e. not a lot of background picks). I assume you did 2D classification to get rid of junk particle picks?


Thank you for your answer!
Yes, the data was collected at 0.82 A/pix, I down sampled to 1.6 A/pix. I removed junk particles with 2D classification, but still the classes look “grainy” (overfitted but not with streaks), unless I turn off Force max over poses/shifts.
This is one example of one of the last 2D classification:

And this is if I turn off the Force max over poses/shifts:

The final classes I retained were these:

I think the CTF estimation is not that bad, with an average fit of about 4-5A.
Thank you again for your answer!

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