<div dir="ltr"><div dir="ltr"><div>Dear Omid,</div><div><br></div><div>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.<br></div><div>We have described the procedure extensively in:
<p class="gmail-MsoListParagraph" style="margin:0cm 0cm 0.0001pt 8.5pt;text-align:justify;line-height:normal;font-size:11pt;font-family:"Calibri",sans-serif"><span style="font-family:"Times New Roman",serif"><span><span style="font:7pt "Times New Roman""> </span></span></span><span style="font-family:"Times New Roman",serif">Afanasyev P, Seer-Linnemayr C, Ravelli
RBG, Matadeen R, De Carlo S, Alewijnse B, Portugal RV, Pannu NS, Schatz M, van
Heel M: <b>Single-particle cryo-EM using
alignment by classification (ABC): the structure of Lumbricus terrestris
hemoglobin</b>, <i>IUCrJ. </i><b>4</b> (2017) 678-694.</span></p><p class="gmail-MsoListParagraph" style="margin:0cm 0cm 0.0001pt 8.5pt;text-align:justify;line-height:normal;font-size:11pt;font-family:"Calibri",sans-serif"><br></p><p class="gmail-MsoListParagraph" style="margin:0cm 0cm 0.0001pt 8.5pt;text-align:justify;line-height:normal;font-size:11pt;font-family:"Calibri",sans-serif">Hope this helps,</p><p class="gmail-MsoListParagraph" style="margin:0cm 0cm 0.0001pt 8.5pt;text-align:justify;line-height:normal;font-size:11pt;font-family:"Calibri",sans-serif"><br></p><p class="gmail-MsoListParagraph" style="margin:0cm 0cm 0.0001pt 8.5pt;text-align:justify;line-height:normal;font-size:11pt;font-family:"Calibri",sans-serif">Marin van Heel<br></p>
</div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Tue, Dec 24, 2019 at 7:30 PM Haji-Ghassemi, Omid <<a href="mailto:omidgh85@mail.ubc.ca">omidgh85@mail.ubc.ca</a>> wrote:<br></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex">
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<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">Dear All,<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif"><u></u> <u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">I recently tried estimating the anisotropic magnification via CtfRefine in Relion 3.1 and I get the following warning once the run was finished:<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">WARNING: Overall magnification of optics group #1 (opticsGroup1) differs from the nominal pixel size by 1.92044 %.<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">WARNING: This overall difference changes the actual pixel size of the reconstruction!<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif"><u></u> <u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">I also attached the resulting log pdf file.
<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">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.
<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif"><u></u> <u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">So my questions are as follows:<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">1. Up to what resolutions can the anisotropic magnification be detected?
<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">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.
<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">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.
<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif"><u></u> <u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">Regards,<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">Omid<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">---------------------------------------------------<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">---------------------------------------------------<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">Omid Haji-Ghassemi, Ph.D.<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">Postdoctoral Fellow<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">Department of Biochemistry & Molecular Biology<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">Faculty of Medicine<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">University of British Columbia<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">2350 Health Sciences Mall<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">Vancouver, BC, V6T 1Z3<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt;font-family:"Times New Roman",serif">Canada<u></u><u></u></span></p>
<p class="MsoNormal"><u></u> <u></u></p>
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