Thanks for the helpful reply @mmclean!
I was not aware that cryosparc extraction filters out the particles at the micrograph edge and that explains why my particle stack decreased in size during an earlier extraction - thanks for clarifying 
In this case, however, this did not apply. I first extracted in the large box, refined and used that as input for the extraction into a small box so both stacks have the exact same 130675 particles. The only reason why I did this was so that the box size would be more computationally efficient for 3DVA - in hindsight I could have used the downsample particles job but in the end this was a fortunate ‘mistake’ since the small box gave me better alignment.
Regarding the precision, do higher values mean that the alignment is more accurate or is it just using finer shifts?
This is for the small box with higher resolution:
This is for the large box with poorer resolution:
The scale bar goes higher for the small box refinement.
Given that the protein is so flexible due to hinging and also motion of the large amount of glycans, any additional solvent noise is probably detrimental to the alignment.
I have tried the CTF tool from Takanori and just want to check if I understand it correctly.
I have no idea how to extract the CTF values from the .cs file so used pyem to convert it to a star file and used the following to calculate the average defocus U ($10), defocus V ($11) and defocus angle ($12) from the star file. I then used these values as input on the CTF calculator tool.
count=0; total=0; for i in $( awk '{ print $10; }' from_csparc_P7J208_ctfcalc.star ); do total=$(echo $total+$i | bc ); ((count++)); done; echo "scale=0; $total / $count" | bc
Using this, I got an average defocus U of 18602, average defocus V of 18393 and average defocus angle of 12.
I’m assuming astigmatism angle in this tool is the same as defocus angle?
My input for this tool was: defocus 18497A, astigmatism 104A, astigmatism angle 12, energy 300kV, Cs 2.7mm, phase shift 0, amplitude contrast 0.1.
For a box size of 280 at 1.06apix and ring at 3.7A:
For a box size of 576 at 1.06apix and ring at 3.7A:
Is it acceptable to use the 280 box??