I combined 3 particle stacks from 3 datasets. Before I did that, I made sure that I extracted the particles from each dataset’s micrographs at the same box size of 540 px. Before that for refinement of each stack, I cropped two of the sets from 540 to 256, and in one dataset (my first) I cropped from 512 to 256. After I picked the desired particles from each of these datasets, I extracted each stack from the micrographs at 540 px, and combined using exposure group utilities (median) to average the CTF values.
I ended up with a group of 67k particles that I ran through homogenous refinement, and got a reconstruction @ 2.58 angstroms. I want to classify these particles using 3DVA, but the job fails as the size is too large for my current disk space. I want to downsample these particles to 128 px, but when I try to run the downsample job, I got an error “particles must all have same alignment pixel size”.
I do not understand why the alignment pixel size is different when I extracted each stack at the same size of 540 px. How can I solve this problem?
Now my particle stacks have been exported from different disk locations by creating a soft link in the current processing disk, so I feel that it will be too complicated to reextract the particles at the full size after running the cluster mode of 3DVA, since I won’t know which dataset the particles came from…so does this mean I will have to create soft links for the micrographs too, and use all of them as an input for the extract job??? these will be around 30 k micrographs…!!! So I am not sure how to navigate this. What could be the simplest way to run 3DVA on these combined particles?
Hi @Noha_Elshamy, I’d love to help figure this out for ya.
First, could you verify that I understand correctly what you’ve done:
You collected 3 datasets with the same pixel size
You extracted particles from all two datasets with the same box size (540 px) and one with a different box size (512 px)
You cropped all three datasets to 256 pixels. Now you have 3 datasets with the same box size and the same pixel size
You refined all three datasets together
You re-extracted the particles from all three datasets, this time all with a box size of 540 pixels. There are 67k particles in this new particle stack.
You tried to use Downsample Particles to take the stack from step 5 from 540 px → 128 px. This job failed with the error “particles must all have same alignment pixel size”
All is correct, except that I refined the datasets independently as they were not taken at the same time. I usually process at the 256 pixel size until I reach the resolution limit in my refinement. At that point, I would re-extract the particles from the micrographs at 540 px. What I mean is that I ended up with three constructions from my refinements, each at 540 px. I then exported the particles from each of the three constructions, imported them in a new workspace for processing the three sets, combined them (I did that by running them through a new ab-initio job, 1 class) followed by refinement.
I want to note also that because the defocus range for the 1st dataset was a little different from the subsequent two sets, I had to run the particles through an “Exposure group utilities” job in which I used a combine strategy of “median”.
You extracted particles from all two datasets with the same box size (540 px) and one with a different box size (512 px)
You cropped all three datasets to 256 pixels. Now you have 3 datasets with the same box size and the same pixel size
You refined all three datasets independently
You re-extracted particles with a box size of 540 px from all three datasets. You did not downsample or crop the images
You exported the particles from step 5 and imported them to a new workspace
You performed an Ab initio Reconstruction and Homogeneous or Non-Uniform Refinement with the particles from step 6. This refinement did not encounter any errors, and the map looked good.
You tried to perform 3D Variability Analysis, but this failed.
You tried to use Downsample Particles on the particles from step 7 (after the refinement), but this failed with the error “particles must all have same alignment pixel size”
If anything there is incorrect, could you let me know? Also, could you paste (as text) the error message you saw when 3DVA failed in step 8?
Yes this is correct. But as I noted, homogenous refinement with the option of “CTF refinement” “On” failed before I did the combination of the particles stacks using the exposure group utilities and using a combine strategy as “median”