Hi all,
Recently I’ve processed a dataset of an icosahedral virus, and reached an overall resolution of 3A with I symmetry applied for the capsid. Then I tried to go on processing with the portal reconstruction. I know the next thing is to define the coordinates of the 12 vertices then re-extract those sub-particles and do the localized classification to find the class showing portal density. Does anyone have any experience or detailed steps to do this in cryoSPARC?
Thanks.
Liz
Hi @liz,
Thanks for your patience, I had to spend some time exploring how to do this effectively within CS. I was able to make a workflow to find the portal on the Calicivirus VP2 dataset, EMPIAR-10193.
After Homogeneous Refinement of your virus you can do the following.
Symmetry expand the final particle stack with I symmetry.
Use ChimeraX to locate the vertex of which the particles and volume will be shifted to the center of the box. I am unsure if this will apply to your dataset, but for EMPIAR-10193 there were two possible vertices where the particle could be shifted too (red and yellow), of which the the red vertex was the correct one.
To find the coordinates of this position, place a marker on the surface, reposition the marker if needed using the “Move” mode in the “Markers” panel, and use measure center #{marker} to obtain coordinates in Å.
Back in cryosparc, connect the volume and symmetry expanded particles to a Volume Alignment Tools (VAT) job.
Use the 3D-coordinates output by the measure command in chimeraX as input for the VAT parameter “3D-coordinates of new center” and use ‘A’ for Angstroms. In my case, this was “366.67,419.19,559.45 A”.
Download your shifted volume, and find the right location for a mask using the same protocol from step 3.
Using the coordinates from step #6, I followed @olibclarke’s tutorial on creating cylindrical masks. I generated a mask around the area where the portal should be.
Import the mask into your workspace using the Import Volumes job.
Use a Volume Tools job to lowpass filter the mask to 15 Å and pad with 30 pixels. Although these are the values that worked for me, I didn’t test many – others will probably work well!
Next, re-extract the shifted particles using Extract From Micrographs.
This job then had one class which has the portal density clearly visible:
I hope this helps and please reach out if you have any further questions.
Best,
Kye
Hi @kstachowski,
Thank you so much for your very detailed protocol! I’ll give it a try on my own data set and let you know my progress.
Best regards,
Liz
Hi @kstachowski,
I’ve successfully screened out one class containing the portal density with your step-by-step instruction. After a further round of local refinement focused on the portal density, those structural details can be clearly distinguished. I’ll try to do further symmetry expansion and apply Cn symmetry to improve the resolution.
Thanks again!
Liz
Did you symmetry expand from icosahedral to C1? If so, re-applying higher symmetry orders is very dangerous as you will be reduplicating particles.
@liz I’m glad those instructions were able to get you to that result. As @rbs_sci mentioned, once you symmetry expand, you should not symmetry expand that particle set again. After getting the portal density, I used Volume alignment tools with Do symmetry alignment enabled and then I specified C3 since this portal was C3 symmetric. I then removed duplicates and performed a local refinement with a more specific mask for the portal and applied C3 symmetry within the local refinement job.
Best,
Kye
Thanks @kstachowski ! After using Volume alignment tools and removing duplicated particles, the additional local refinement gave me a high resolution map with portal density which has a C12 symmetry. And I found the mask to include the portal was deserved to try in the earlier step. Thanks again!
