Issues with Patch CTF after Patch Motion Correction

Troubleshooting
1

I am used to preprocessing my cryo-EM data with MotionCor2, but wanted to give Patch Motion Correction a try in CryoSPARC. I had previously imported my MotionCor2-corrected micrographs into CryoSPARC and ran Patch CTF for downstream template picking, which produced good results (graphs looked normal, CTF estimates were in expected 3-5A range). Then in a separate workspace, I imported my raw .tif images with a gain.mrc reference file without issue, ran patch motion correction job without noticing any issue, but then in my patch CTF job the graphs look extremely weird and CTF estimates are way off (right above my minimum 30A resolution specified at 30.7A). What could be causing this and how could I fix it?

2

Hi @amcc,

Thanks for trying out Patch Motion Correction in cryoSPARC! By any chance did you turn on the “Flip gain ref in Y?” parameter in the Import Movies job? Can you take a screenshot of the overview tab of the Import Movies job where one of the images is plotted with the gain reference applied?

5

Hi,
could this issue be sorted? I am having similar issue with my data. Patch motion/CTF jobs did really well with some of my image sets. I wanted to try it with one of the old image set. With this particular data, I did not notice any peculiarity with the patch motion job but the patch CTF estimations are way off. I repeated the full frame motion and Gctf estimation with the same data and they behave as expect. Please see the sceenshots attached (one each for single ctf fit jobs

Screen Shot 2020-05-26 at 3.18.47 PM
Screen Shot 2020-05-26 at 3.18.47 PM2068×668 495 KB
Screen Shot 2020-05-26 at 3.07.50 PM
Screen Shot 2020-05-26 at 3.07.50 PM2124×1004 502 KB
and an overall CTF plot
Screen Shot 2020-05-26 at 3.09.03 PM
Screen Shot 2020-05-26 at 3.09.03 PM1296×554 97.7 KB
Screen Shot 2020-05-26 at 3.08.28 PM
Screen Shot 2020-05-26 at 3.08.28 PM1260×558 125 KB
in (curate micrographs job) done using full frame motion/gctf estimation or patch motion/patch ctf respectively. )

6

Hi,

My issue was that I didn’t use the super-resolution pixel size when importing my raw date movies, and I needed to do that instead of using my physical pixel size. must be a different issue for you, but good luck!

7

Thanks for the update. @stephan can you please look into the issue i am having?

8

Hi @kplkmrgoutam,

I’m sorry for replying to this months later, but it’s perhaps worth mentioning that cryoSPARC v3, released yesterday, includes a few fixes to patchCTF that make it perform better on difficult data sets. You may consider trying these data again with version 3.

If you’re still having trouble, you could try changing the minimum and maximum search defocus parameters to narrow the search range around the actual values.

Harris