Help with merging particles from 2 datasets

Dear cryoSPARC community,

I am wondering if I am doing something fundamentally wrong when I am merging particles from 2 datasets.

I have two datasets (dataset A ~109K and B 280K particles) that were collected at the same pixel size. I’ve processed the data individually and I got maps at ~4.5Å and ~3.5Å for each dataset.

I re-extracted particles at the same box size and tried to combine them by simply dragging particles from both data sets as input to 2D classification.

Based on the output (2D class representative picture attached) it doesn’t look like the particles are being merged – The contrast is very different. Similar results was observed when I run an ab initio with 2 classes.

Any thoughts, ideas, on the matter would be much appreciated

Hi @gptomaleri,

Were the two datasets collected during different collection sessions? If you haven’t already, you might want to turn on Min over scale after first iteration in 2D Classification – this will re-estimate the contrast (i.e. multiplicative scale factor) of each particle, which can help when the sample and ice characteristics vary between the datasets. Let me know if that helps!

Best,
Michael

2 Likes

Hi @mmclean,

Thanks for getting back to me so quickly.
Yeah, we have collected them 3 months apart at the same microscope. That means that our dataset A was processed in an older version of the CryoSPARC, I doubt that this is a problem, right? The only difference between the datasets was the spot size…

Now, I’ve turned on Min over scale after first iteration in 2D Classification but there is an error message:

I tried to turn it on for datasets A and B individually and the same problem occurred.

Any thoughts?
Best,
Gio

Hi @gptomaleri,

Thanks for the additional info. Do you know the version of cryoSPARC that dataset A was initially processed with? I assume this means that motion correction, CTF estimation, and particle picking/extraction was all done with the older version?

Regarding the final error, are you currently on Patch 210615 or later? We resolved a bug in that patch that may be responsible for the ValueError – do let us know if updating to patch 210615 (or the following patch 210622) doesn’t resolve it.

Best,
Michael

1 Like

Hi @mmclean,

Yeah, we use V2.15.0 for motion correction, CTF estimation, and particle picking of dataset A. Do you think that could be the problem?

We recently update (06/28/2021) to V3.2.0 and still got the error.

Any help?
Best,
Gio

Hi,
I encounter a similar problem processing two datasets collected months apart on the same microscope with same parameters. In my case one of the samples had substantially thinner ice.