Poor resolution of 2D classes

Dear all,
I am trying to solve the structure of a protein with molecular weight of approximately 300kDa, that is predicted to be flexible and probably contains disordered regions. The particle size should be around 13 nm. Our micrographs look great, the particles are nicely visible and can be picked easily, but they fail to form reasonable 2D classes. They are fuzzy and the resolution is poor.
Below, you can find an example of one of our best 2D classifications. This result was obtained already after two rounds of 2D classification, from 40000 best behaving particles.

We used following parameters:
Circular mask diameter (A): 200
Circular mask diameter outer (A): 240
Force max over poses/shifts: off
Other parameters on default

Previously, I also tried to increase the batch size to 400 and number of iterations to 40, but this only led to overfitting.

Do you have any tips what could help us to improve our results?
Thank you!

hi @HelleborusViridis , what about trying to update the binning factor?

Hi and thank you for your answer. What do you mean by “update the binning factor”? I don’t think we did any binning throughout the process…

Are ctf estimated to be high resolution? Also for flexible smallish protein this is somewhat expected… take all classes and move forward, hoping something “latches on” to high res rigid detail in 3D. Might have 50kDa domain similar in all particles and can assess extending variability later.