Local refinement artifacts, spikes, possibly over refinement

open

#1

Hello,

The results of my local refinements look very strange. Although some real features of the molecule emerge (i.e. I can see the backbone better in certain locations), the map is really distorted and shows a lot of “spikes” and disconnected parts. Can anyone please explain the origin of this problem; I’m somewhat new to cryo-EM and cryoSparc. I have seen similarly strange results in the past, when running global homogenous refinements or NU-refinements. The GSFSC resolution gets much better, but the map looks like it “splashed”. Any help is greatly appreciated. The molecule that I am working with is relatively small < 200 kDa.

Original mask:

After refinement:
image


#2

Hi @bonils,

Thanks for reporting.
Over-refinement definitely is a phenomenon that can occur, especially in local refinements and with smaller particles/thicker ice (i.e. lower SNR). It is also most heavily affected by the presence of junk particles.

Some questions:
In 2D classification, do you also see streaks and signs of erroneous signal?
For local refinement, how soft is the mask that you are creating? it should ideally be quite soft especially when the particle is small.