Ab initio 3D reconstruction worked beautifully in my case, however, there’s a small issue associated: the final map has opposite chirality as it’s supposed to be, I flipped the map in Chimera using vop zflip command, I guess for people who have little knowledge about the chirality, it could easily guide towards a wrong map or a map that’s stuck at low resolution? I don’t know if this issue can be automatically fixed in 3D refinement as it brings in more higher resolution data.
This is not a cryosparc issue - it’s a fundamental issue with reconstruction in cryoEM in general.
Your particle images are projections of the molecule of interest, and hence have no inherent chirality - both the correct and the inverse hand will give equally good reconstructions, there will be no effect on resolution.
It is something one needs to be aware of, and if you are at too low resolution to determine handedness based on the chirality of alpha helices, then determination of handedness via some other means, e.g. tilt pair analysis, is required.
Nothing to add.
If you have a completely new structure without reference you need high resolution to basically “see” the chirality.
If the data is good enough, you will obtain this regardless of the handedness.
Otherwise a new data collection is indispensable.
Hi Tarek & Oli,
Can I just forget about the chirality and only to fix the chirality before the final step (building atomic model)? Or should I fix it as soon as I find out? Will these make any difference?
Yes, you can ignore until the end.