[3dem] [ccpem] Combining datasets with ctf parameters from ctffind & gctf

Penczek, Pawel A Pawel.A.Penczek at uth.tmc.edu
Tue May 30 08:39:20 PDT 2017


Dear Jin,

conversely, you can use CTER facility in SPHIRE package (http://sphire.mpg.de/wiki/doku.php).

It estimates error bounds of CTF parameters and it contains simple set of graphical tools that
allow one to reject/set to zero parameters that were found to be unreliable.  Therefore, instead of using
essentially random astigmatism angles that do no good it will set the astigmatism amplitudes
to zero based on significance thresholds set by the user.

Regards,
—
Pawel A. Penczek, Ph.D.
Professor
Structural Biology Imaging Center, Director
The University of Texas
phone: 713-500-5416
fax: 713-500-0652
https://med.uth.edu/bmb/faculty/pawel-a-penczek/



On May 30, 2017, at 9:46 AM, Jose Maria Carazo <carazo at cnb.csic.es<mailto:carazo at cnb.csic.es>> wrote:

Dear Jin,

You may want to check on a CTF Challenge we did a couple of years ago (Marabini et al. JSB 2015) for a more in depth analysis but, essentially, to assign a value to the astigmatism angle when your astigmatism is very small is not a very stable process.

Wbw..JM

On Tue, May 30, 2017 at 3:55 PM, Marin van Heel <marin.vanheel at googlemail.com<mailto:marin.vanheel at googlemail.com>> wrote:

Hi Sjors and Jin,

This type of anisotropy has a name: Astigmatism!
It may sound pedantic but anisotropy in EM normally refers to a magnification anisotropy, not to astigmatism.

Cheers,

Marin


On 29/05/2017 18:51, Sjors Scheres wrote:
Hi Jin,
In the case of anisotropy your Thon rings are elliptical instead of
circular because the defocus in one direction (defocusU) is not the same
as the defocus in the perpendicular direction (defocusV). DefocusAngle
describes the direction of this anisotropy. It is defined as the angle
between defocusU and the X-axis (if I remember correctly...). Both
ctffind4 and gctf estimate anisotropy for every micrograph, but if your
microscope was well aligned, then you may have hardly any anisotropy in
the data. This will result in very similar defocusU and defocusV values
for each micrograph. In that case, defocusAngle doesn't mean anything, and
can vary wildly between the different programs. I suspect this is what is
happening, but you can just check by plotting defocusU values against
defocusV values for the entire dataset. I suspect you can mix your
particles as you want without large effects on the reconstruction.
HTH,
Sjors


Hi All,

This may be very trivial thing. I am going to combine particles from two
dataset. And each dataset was processed with ctffind4 and gctf. I found
each program gives similar defocus values but different defocus angles per
image. What does defocus angle do in CTF and would it be okay to combine
the data from different softwares?

Thanks in advance,

Jin


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    Prof Dr Ir Marin van Heel

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--
Prof. Jose-Maria Carazo
Biocomputing Unit, Head, CNB-CSIC
Spanish National Center for Biotechnology
Darwin 3, Universidad Autonoma de Madrid
28049 Madrid, Spain

Cell: +34639197980
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