Icosahedral viruses

3D Reconstruction
1

Hello,
I am trying to process a Titan K3 data set of an Icosahedral virus. I am trying to get a decent ab initio model. I submit it for 3 ab initio model generation. From the results it appears that it is close but not quite there. I am wondering if I change the following parameters to improve the ab initio model generation.

Starting and target resolution. Currently set at 35 and 12 A. I am thinking of changing it to 40 and 15. Another one is to vary the number of initial models from 3 to say 5. If the number of models are increased, does that improve the chances of getting a correct initial model?

Additionally, should I also increase the class similarity parameter from 0.1 (default) to higher value.

Also, there are two other points that I am not really clear about. Initial and final iterations before annealing starts and ends and the other one being class similarity annealing.

Thanks

Best,

Abhishek

2

With high-symmetry particles you usually need finer sampling. I’d try setting the initial resolution to a higher resolution value.

3

Thank you Dan. The issue has been resolved. It is indeed now producing decent reconstructions. However, in terms of masking, it appears that cryoSPARC does not have any option of creating a disc shaped mask where one one can specify the inner and outer diameter. This often helps to mask out the rather poorly organized nucleocapsid part, thus improving the resolution largely of the glycoprotein shell, which is better defined. Is there any option like that in cryoSPARC?

Also, it does not seem to refine the magnification given the fact that the mag is nominal.

Thanks.

4

CryoSPARC doesn’t have that option, if it’s very important you might want to take your cryoSPARC angles out to cisTEM, then bring them back in again after classification/refinement with the inner mask.