Hello @hai,
The micrograph above doesn’t look good to me. I cannot locate particles with ~150 Å diameter that look similar to the 2d class averages calculated from your template-picked “particles”.
Which brings up the elephant in the room: I’d be cautious of the template bias you could introduce with template picking. While there is nothing wrong with generating templates from published structures, an unbiased method, such as blob picking, should first be able to produce something that looks like your protein of interest in 2D classification.
If the majority of the micrographs looks like the one you posted above, I would go back to obtaining a better set of micrographs.
Can you manually pick some particles from your micrographs? Do manually picked particles yield realistic 2d class averages? You can also try different parameters for blob picker or blob picker tuner. I’d also try removing the noise particles as much as I can with Inspect Particle Picks.
Best,
Kookjoo