Poor CTF fit using patch CTF estimation in cryoSPARC v2.13.2

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#1

Hello everyone,

I have a question about the CTF fits that I have been getting using patch CTF estimation in cryoSPARC version 2.13.2. As shown in the screenshot below I am getting what appear to be acceptable fits until around 7Å. However, at higher resolution it does not appear to fit the CTF despite the presence of signal. I have collected a similar sample (same protein and grid type with similar ice) on another microscope without CTF issues. The sample was collected on C-flat grids on a Titan Krios with a Falcon III detector. I have double checked the pixel size, accelerating voltage, and Cs values.

Any suggestions on avoiding this situation in the future and/or improving the CTF fit for this dataset would be greatly appreciated.

Many thanks in advance,


#2

Hi @Andrew,

Was this data tilted, or was it phase plate data?
When you see the “3D” plot from patch CTF, does it show a large variation in the defocus landscape of the micrograph?

Can you try the following (this will force the job to assume the micrograph is flat):

  1. clone the patch CTF job
  2. set the “Number of movies to process” to 10 or so just for testing
  3. set the “Override Knots X” and “Override Knots Y” both to 1 (there are advanced params)

#3

Hi @apunjani,

Thanks for your reply. This collection is not from a tilted or phase plate data collection. Here is the 3D plot that goes with the patch ctf fit image that I posted previously:
image

I just tried the job you suggested where the micrograph is assumed to be flat. As you can see below I am still getting a similar issue.


Do you have any other suggestions?

Many thanks for you help.
Andrew