Then I use the new 2D class to run template picker again but this time the extracted micrographs are less accurate than the old 2D that is generated from my manual pick. I’m expecting the 2D class will be improved each iteration.
My question is how to accurately select apoferritin and generate high resolution 2D class, thanks a lot!
What thresholds are you using to set those picks? How many picks are made straight from the blob picker? If that is the raw output with no thresholding, something else is wrong.
Here’s what one of my micrographs looks like with apoF circled:
Hi @rbs_sci , yours looks amazing! And thanks a lot for your help!
I set Lowpass filter to templates and to micrographs as 5 Å and the picking seems much better. It start to pick particles.
But still some miss picking, is there any other parameters can help this?
“What thresholds are you using to set those picks?”
Which thresholds do you mean, the power histogram from inspect picks?
I used 330-360 for power score and 0.13 for NCC Score for the picture below, but whatever I used the circle is not picking the particles.
I tried to measure the size as below, they are slight larger than 120 A (138, 132 and 146)
Can I ask why pixel size will cause off picking? Lots of particles pick in my template picker are at empty region.
OK, then check with the microscope facility that the magnification the data were collected with is correct. Because that currently looks like ~1.4Å/pixel…
Thank you so much! I know it is 130k magnification but couldnt find a chart to show how large is the pixel size. Do you know the pixel size of K3 under 130k?
That will depend on the microscope and whether there is an energy filter, how the lenses are tuned, and so forth - it’s better to check with the facility.
Is it possible that you set F-crop to 1/2 during motion correction? The calculated pixel size based on your measurements is 1.65Å, which is exactly 2x your nominal pixel size of 0.824.
What I meant re a pixel size error is that if your pixel size is off from the true value by a significant amount, then a ring template based on the “true” size of apoferritin will no longer correlate well with your particles.
So in your case, if you are measuring the particles at a nominal outer diameter of 140 Å, you might want to try a ring with a min diameter of 110-120 and a max diameter of 140-150 and see how that goes. You can always calibrate the pixel size later, after refinement.
I correct my raw pixel size to 0.66, the images were taken by krios under 130x, I found parameters in mdoc file.
I compared template picker and blob picker. Blob picker with ring blob and pick range 110-140, it does work better, starts to pick particle but mostly pick the edge of the particle
The template picker I select 1500 particles, the 2D was great but when I use it to template pick, the result is not good, still can’t pick the particles.
Your data should be perfectly fine for picking… not sure why it is not working. What particle diameter did you set for template picking? I would go a bit larger than the actual particle - maybe 140 or 150 Å
Because that currently looks like ~1.4Å/pixel…
