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”:
“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.