Defocus estimation on images with hardly any thon rings

Submitted by chunfuxu on Tue, 2013-06-11 11:34

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ctffind & ctftilt

Dear Niko,

I have been trying to use ctffind3 to estimate the defocus of my cryoEM images. The parameters that i used are as follow:

CS[mm], HT[kV], AmpCnst, XMAG, DStep[um]
4.2, 200, 0.07, 106379.6, 15

Box, ResMin[A], ResMax[A], dFMin[A], dFMax[A], FStep[A], dAst[A]
256, 50, 4, 8000, 50000, 1000, 1000

and I get:
DFMID1 DFMID2 ANGAST CC

24050.82 21719.82 60.00 0.06457 Final Values

The right side of the output image showed hardly any thon rings(see attachment). I am wondering if the estimation is still reliable in this case.

Also when I change the box size to 512, the results I got was very different:
DFMID1 DFMID2 ANGAST CC

21413.42 22958.71 7.97 0.04508 Final Values

Is this normal? Could you please give me some advice? Really appreciate it.

-Chunfu

Axel Tue, 2013-06-11 11:49

Hi Chunfu,
As a first step, you could reduce the ResMax parameter, which is much too high in your case, try reducing it to 8, or 10 Angstroms. This should improve the reliability of your estimate (and is in general a good thing to do).

I would be a bit worried about using such an image, to be honest.

Axel

chunfuxu Tue, 2013-06-11 12:14

Thank you, Axel. I will try to reduce the ResMax to 10 and estimate again.

As of the image, what do you think could be the reason why it has such bad PSD profile? (forgive me if it is a stupid question) I was gonna upload the image and ask you to help me take a quick look at it. But I could not find where to attach it. Basically, it is a 4096*4096 image collected using Gatan CCD camera. I saw that people here use film and scanner more often. I am wondering if this could be why.

-Chunfu

Axel Tue, 2013-06-11 12:34

If you could upload a binned image, that could help us figure it out. Generally, low defocus images collected on CCD cameras which do not have either carbon or a lot of sample on them will not show a lot of Thon rings in CTFFIND3. If you see two zeros clearly though, that should be good enough, as long as the check images look good.

Axel