Thanks @olibclarke for your input – by aligning calculated density, do you mean just manually superimposing in a molecular viewer? Or do you mean actually using the .mrc of my 75kDa protein in a processing job as a way of guiding things?
I tried your way of obtaining projections – I made an .mrc file from the crystal structure, imported it into CryoSPARC, but when I tried to create templates, there was an error when it tried to broadcast arrays between my volume’s cell and the one created by CryoSPARC:
[CPU: 211.0 MB] Traceback (most recent call last):
File "cryosparc_worker/cryosparc_compute/run.py", line 84, in cryosparc_compute.run.main
File "cryosparc_worker/cryosparc_compute/jobs/create_templates/run.py", line 47, in cryosparc_compute.jobs.create_templates.run.run
File "/home/nick/CryoSPARC/install/cryosparc_worker/cryosparc_compute/fourier.py", line 152, in zeropad
new_X[a:b,a:b,a:b] = X
ValueError: could not broadcast input array from shape (60,61,74) into shape (74,74,74)
Is there some way to tell molmap to create a cubic cell? I think CryoSPARC just takes the largest dimension and creates a cubic array from that.
Thank you @kyestachowski as well. I tried the following:
- Re-curating my exposures
- Re-picking particles (750)… anything that looked like continuous, isolated density. Big and small things, in the hopes that some would be ‘face-on’ orientations. Class averaging these, and using them as templates for the template picker which then picked 850,000 particles
- Class averaging the new particles with 80 classes, 50 iterations, ICUF of 2.0, Batchsize per class of 200, and Force max over poses/shifts set to False.
I obtained the following:
The ~50 classes below were basically all junk. I selected the first 17 classes and did an Ab initio reconstruction (I tried it twice, with 1 class and 3 classes to absorb junk). Both outputted similar maps. I then did a homogeneous refinement on the single class and got basically the same thing as before:
Green = calculated volume from crystal structure with cartoon alignment
Blue = my map with arbitrary alignment
My particle looks so different from the crystal structure, almost as though it is not big enough. But, it has central density that the crystal structure does not have.
Are these all artifacts from just not having enough orientations? Is maybe a tilt series the only way to go? You suggested I use different size masks, but I sadly don’t even see any ‘head-on’ smaller orientations like you describe 
Thanks again
PS. I am going to try the ‘Rebalance 2D Classes (BETA)’ job now, because I read in the docs if you observe smearing out along a particular axis due to preferential orientation, balancing the classes may help…
