3D ab initio generate model with different hands

Cryo-EM Data Processing
1

Dear cryoSPARC users,

I am wondering is there anyway (or need) in flipping the hand of particles?
I have a flexible particle that tends to be reconstructed in different hand from time to time.
Recently, one of the Ab initio 3D reconstruction with four models yield two classes (#0 and #2 with 183K and 186K particles, respectively) that looked similar but are flipped image of each other (which is confirmed in Chimera). Homo- and NU-refinement of volume_0 using particles from both classes gave me a resolution of 3.6Å which is only slightly better than similar refinements of volume and particles from each individual class (3.75Å and 3.85Å for class_0 and class_2, respectively). Since there is a big difference in the number of particles (360K vs 180K) and only a slight improvement in the resolution, I am wondering whether we have to flip the hand of the two class of particles or CS would automatically take care of the difference.

Thank you so much for enlightenments.

2

You can flip the hand of volumes (not particles) using Volume Tools - flip hand in cryosparc.

3

@adrian, my problem is that half of the particles are in the right hand and half in the flipped hand. I would like to use both classes of particles for the refinement of one volume in either hand. I can alway flip the hand of the volume after refinement. I read in an earlier thread that poem could flip the hand of particles. @DanielAsarnow, could you please comment on this.
Thanks.

4

I do not think is It possible for a particle to have the wrong hand.
Having right or wrong hand after ab initio reconstruction is just random.

5

@Ricky Just refine all the particles together with one reference.

6

@DanielAsarnow @Adrian. Thank you both for the comments. That is exactly I did. But then I also refine each reference with the respective class of particle. My concern was that doubling the number of particles from ~185k to 390K only improved the resolution from 3.8Å for the latter to 3.6 Å for the former. Further analysis showed that indeed there are subtle differences between those two reference volumes. That could explain the limited improvement in resolution with a big increase in particle numbers. Thanks again.

7

Some structures - or even certain classes - will tend to converge to a certain handedness for idiosyncratic reasons. Since you were able to run a consensus refinement, you could now restart classification at a somewhat higher resolution, perhaps even using both references aligned and in the physically correct hand. If the starting resolution is 15A, say, the hand should be stable. Using a higher initial resolution is fine if your refinement is already significantly beyond.