How to obtain the portal vertices coordinates of an icosahedral virus

Cryo-EM Data Processing
1

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

2

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.

  1. Symmetry expand the final particle stack with I symmetry.

  2. 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.

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  1. Back in cryosparc, connect the volume and symmetry expanded particles to a Volume Alignment Tools (VAT) job.

  2. 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”.

  3. Download your shifted volume, and find the right location for a mask using the same protocol from step 3.

  4. 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.

    • I did not gaussian filter the volume, I lowpass filtered it in CS.

  5. Import the mask into your workspace using the Import Volumes job.

  6. 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!

  7. Next, re-extract the shifted particles using Extract From Micrographs.

    • For the dataset that I tested this workflow on, I re-extracted at a box size of 384 px and Fourier cropped to 300 px.
    • I then did a Homogeneous Reconstruction to ensure the putative viral portal was in the center of the box and that the mask I created fit over that region as well.

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  1. Connect the particle output of the homogeneous reconstruction to a 3D-classification job and the cylindrical mask as the focus mask. For parameter choices, I set the following:
    1. Number of classes: 80
    2. Filter resolution: 6
    3. Initial structure lowpass: 15

This job then had one class which has the portal density clearly visible:

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I hope this helps and please reach out if you have any further questions.

Best,
Kye

5 Likes
3

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

1 Like
4

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

5

Did you symmetry expand from icosahedral to C1? If so, re-applying higher symmetry orders is very dangerous as you will be reduplicating particles.

6

@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