[3dem] [ccpem] Relion 3.1 CtfRefine anisotropic magnification

[Marin van Heel]

Dear Omid,

Anisotropic magnification should be corrected first, even before any CTF
determination, since its presence messes up the defocus/astigmatism
assessment. The anisotropic magnification measurement is best/conveniently
performed on the movie data itself. It does not require any separate
calibration measurements since the water ring information is always present
in large cryo-EM datasets, and those contain all the necessary anisotropy
information. The Imagic procedures allow you to a posteriori correct
anistropic magnification even in "legacy" cryo-EM datasets. Depending on
how you perform the correction, the average pixel size may change, and
consistency is important. It is not a good idea to do this anisotropic
magnification correction as an "afterthought" at the late stages of
refinement since it may bias other intermediate processing steps like
particle picking.
We have described the procedure extensively in:

 Afanasyev P, Seer-Linnemayr C, Ravelli RBG, Matadeen R, De Carlo S,
Alewijnse B, Portugal RV, Pannu NS, Schatz M, van Heel M: *Single-particle
cryo-EM using alignment by classification (ABC): the structure of Lumbricus
terrestris hemoglobin*, *IUCrJ. **4* (2017) 678-694.


Hope this helps,


Marin van Heel

On Tue, Dec 24, 2019 at 7:30 PM Haji-Ghassemi, Omid <omidgh85 at mail.ubc.ca>
wrote:

> Dear All,
>
>
>
> I recently tried estimating the anisotropic magnification via CtfRefine in
> Relion 3.1 and I get the following warning once the run was finished:
>
> WARNING: Overall magnification of optics group #1 (opticsGroup1) differs
> from the nominal pixel size by 1.92044 %.
>
> WARNING: This overall difference changes the actual pixel size of the
> reconstruction!
>
>
>
> I also attached the resulting log pdf file.
>
> I then notice subsequent 3D refinement yielded strange 3D volume and
> really poor data quality. I have refined this dataset before and I obtain a
> resolution of ~-3.8-3.9 Ang. I also attempted to calibrate the pixel size
> on a region of the map where the resolution is the highest, and my pixel
> size seemed correct. The pixel size of the data is supposed to be around
> 1.08 or 1.09.
>
>
>
> So my questions are as follows:
>
> 1. Up to what resolutions can the anisotropic magnification be detected?
>
> 2. If indeed the data suffers from some degree of pixel error or
> magnification issues, do I need to adjust it manually before submitting the
> next refinement job? I assumed the correction is already applied after the
> CtfRefine job. If so where is this information recorded? The pixel size
> remained the same in the .star file of the header.
>
> 3. How do I analyze the log file data? It was not clear to me from after
> consulting the tutorial and the paper by Zivanov et al., 2019.
>
>
>
> Regards,
>
> Omid
>
> ---------------------------------------------------
>
> ---------------------------------------------------
>
> Omid Haji-Ghassemi, Ph.D.
>
> Postdoctoral Fellow
>
> Department of Biochemistry & Molecular Biology
>
> Faculty of Medicine
>
> University of British Columbia
>
> 2350 Health Sciences Mall
>
> Vancouver, BC, V6T 1Z3
>
> Canada
>
>
>
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