Hello, I’ve been using cryoSparc since last week.
And weirdly my membrane protein is mirror image of previously determined structures.
This protein is homotetramer and expected to have ‘L’ shape for each protomer, however i am having half of the particle reconstituting into ‘_l’ shape. This shape is further refined into the same shape.
Has anyone been through this kind of weird result?
Should I just ignore mirror image particles for further refining.
This is the handedness issue of cryo-EM - you can’t determine the handedness (chirality) of a 3D object from its 2D projection. Think about the shadow of a hand, can you tell whether it is a left or right hand from the shadow?
This will have no impact on processing, and you can “flip” the map at the end in the Volume Tools job (and others like “Homogeneous reconstruction only”).
Welcome to the forum. Have fun with cryo-EM.
Hello, and Thanks!
As you suggested volume tools and flipping the density results in much more reasonable structure.
I’ll just comment for future reference; due to the methods used to avoid overfitting noisy data, refinements start with low resolution models. CryoSPARC by default uses 30 Å, RELION by default uses 60 Å. As these low resolution models lose almost all features, you can sometimes (normally with higher symmetry objects) find that you will have one handedness to start, but end up with the other. I’ve seen it with icosahedral viruses quite a bit, occasionally with octahedral things like apoferritin and it can be a bit confusing. It’s nothing to worry about, but had me really confused the first time I saw it happen.