3DVA causes malfolmation of transmembrane helices

3D Variability Analysis
1

Hi, all

Currently I’m using 3DVA to resolve the potential continuous conformational changes of the ligand bound in the protein pocket. However, in almost all outputs of 3DVA display, malformation of the helices was observed. I post one of the example here and helices became a bubble.

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This phenomenon only appeared in some of the frames. And in the other frames where the helices are normal, the changes of the ligand could actually be observed.

I’m wondering if I could simply neglect those frames with malformation and only focus on the ones that seem to be normal? Or is there any other parameter that I should pay special attentian to?

2

How big is your mask? We have seen similar spurious density changes when the mask is too small

3

Hi Oli.

I created two types of mask, with one covering only the molecule, the other covering a quite large region enclosing the pocket. Both masks had dilation set to be 2.

For the larger mask, it seems no conformational changes exist according to 3DVA. I don’t know if I inteprete the information correct and here are the results.

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For the smaller mask, the difference seems to be more obvious, and it goes like this.

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But according to the 3DVA Display, it seems the difference between components was caused by the malformation of the transmembrane regions. But I did observe the density of my ligand shift upward and downward among different frames.

4

Yes my guess would be that this is artefactual due to the smaller mask. How much mass (kDa) are the two masks enclosing, approximately?

1 Like
5

About 10 kD for the larger mask. The smaller one only covers the molecule itself, which is about 500 Da.

6

right - that is way too small. Even the larger mask is borderline; the smaller one is far too small to yield sensible results in my experience.

7

@pywt901 Please can you run these commands (with appropriately replaced P99, J199 project and job IDs) to show the settings for the relevant 3D Variability Analysis and 3D Variability Display jobs:

cryosparcm cli "get_job('P99', 'J199', 'job_type', 'version',  'params_spec')"
8

Thank you! I’ll try larger mask to see if it work.

9

Sry I’m very new to the computer languages :cry:
I typed in this script right after logging in but it printed that “command not found” :joy:What should I do before running this command? Sry for the naive question…

10

The command would need to be run on the CryoSPARC master computer and under the Linux account user for CryoSPARC installation and operation.
Even if run on the correct computer under the correct account, the cryosparcm command may not be in your $PATH and may need to be run with the full path specified

/path/to/cryosparc_master/bin/cryosparcm cli "get_job('P99', 'J199', 'job_type', 'version',  'params_spec')"

where /path/to needs to be replaced with the actual directory path applicable to your specific CryoSPARC installation.