<div dir="ltr"><div>Hi David,</div><div>That's what I thought you meant - as a practical matter, this issue is resolved by the patch approach. How cryoSPARC averages the patch fit quality has not been described, but tilted images can be filtered very well in my experience. The local defocus patch estimates are also generally very accurate - it's common for CTFFIND to fit a highly tilted image to 6-8Å, but patch fits of the same image extend to 3-4Å (the programs use the same definition of the fit resolution), and I've only single-particle estimates improve on the patch values for large particles like ribosomes or the 20S proteasome. These notes are all from a single-particle perspective.<br></div><div><br></div><div>Best,</div><div>-da<br></div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Tue, Mar 1, 2022 at 10:50 AM Morgan, David Gene <<a href="mailto:dagmorga@indiana.edu">dagmorga@indiana.edu</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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Daniel,</div>
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Programs such as ctffind give some sort of measure of the quality of the CTF estimate by comparing the real data with calculated CTF parameters. Unless the programs know how to account for tilt when doing this comparision, the quality of the fit will be artificially
bad because of Thon ring incoherence due to the tilt.</div>
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Since one of the first ways to eliminate bad images acquired with automated single particle data collection methods is to "filter out" the bad images based on things like the CTF results, this above-mentioned measure of the quality of the fit needs to be robust
to tilting the grid.</div>
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I remember that there once was at least one program that could do this, but I haven't been able to find it. I have been told (since yesterday) that this is implemented in some alpha releases of cisTEM, for example, and I thought that the Grigorieff group had
released a stand-alone version of that software. But I haven't been able to find a recent version. I will hunt through my archives...<br>
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<div id="gmail-m_5007608132264124042divRplyFwdMsg" dir="ltr"><font style="font-size:11pt" face="Calibri, sans-serif" color="#000000"><b>From:</b> Daniel Asarnow <<a href="mailto:asarnow@msg.ucsf.edu" target="_blank">asarnow@msg.ucsf.edu</a>><br>
<b>Sent:</b> Tuesday, March 1, 2022 2:49 AM<br>
<b>To:</b> Philip Köck <<a href="mailto:koeck@kth.se" target="_blank">koeck@kth.se</a>>; Benjamin Himes <<a href="mailto:himes.benjamin@gmail.com" target="_blank">himes.benjamin@gmail.com</a>><br>
<b>Cc:</b> Morgan, David Gene <<a href="mailto:dagmorga@indiana.edu" target="_blank">dagmorga@indiana.edu</a>>; <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a> <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>><br>
<b>Subject:</b> Re: [3dem] [External] Re: tilted transfer functions?</font>
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<div>Just to clarify, the patch methods don't require particle locations. The gCTF method requires particle locations as input but there's no need for them to be real particles; it too just estimates the CTF on the patches.</div>
<div><br>
</div>
<div>David, what do you mean by the goodness of fit estimates being incorrect? In these methods a goodness of fit measure is used to pick the optimal defocus values in the patch, and these values do give superior reconstruction resolution and appear to accurately
define the real tilt axis.</div>
<div><br>
</div>
<div>Best,</div>
<div>-da<br>
</div>
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<div>
<div dir="ltr">On Mon, Feb 28, 2022 at 10:51 PM Philip Köck <<a href="mailto:koeck@kth.se" target="_blank">koeck@kth.se</a>> wrote:<br>
</div>
<blockquote style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex">
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<div id="gmail-m_5007608132264124042x_gmail-m_-1457521378416033752gmail-m_-8199452785360907602divtagdefaultwrapper" dir="ltr" style="font-size:12pt;color:rgb(0,0,0);font-family:Calibri,Helvetica,sans-serif">
<p>Hi.</p>
<p><br>
</p>
<p>There are two separate problems here.</p>
<p>The easier one is to simply determine the defocus in various positions of the specimen.</p>
<p>If you only want to work with small cut-outs (such as single particles) then you just use the normal CTF-correction with the local defocus. Others pointed to software that does that.</p>
<p><br>
</p>
<p>If you want to work with the image as a whole (for example for 2D crystals) the problem is more difficult. Essentially you don't have a CTF in that case (it's not a transfer function).</p>
<p>Look at the paper by Ansgar Philippsen (<span>Ultramicroscopy 107 (2007) 202–212) if that's what you're after. Also check software for 2D crystallography, but I don't what is actually implemented.</span></p>
<p><span><br>
</span></p>
<p><span>All the best,</span></p>
<p><span><br>
</span></p>
<p><span>Philip</span></p>
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<font style="font-size:11pt" face="Calibri, sans-serif" color="#000000"><b>Från:</b> 3dem <<a href="mailto:3dem-bounces@ncmir.ucsd.edu" target="_blank">3dem-bounces@ncmir.ucsd.edu</a>> för Morgan, David Gene <<a href="mailto:dagmorga@indiana.edu" target="_blank">dagmorga@indiana.edu</a>><br>
<b>Skickat:</b> den 28 februari 2022 23:50:32<br>
<b>Till:</b> Daniel Asarnow<br>
<b>Kopia:</b> <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a><br>
<b>Ämne:</b> Re: [3dem] [External] Re: tilted transfer functions?</font>
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Daniel,</div>
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The problem is that the tilt causes the higher resolution Thon rings to become out-of-phase, and so the standard programs can't give a decent estimate of the goodness of-fit. I vaguely remember that someone had a program that took the tilt into account a number
of years ago, but a quick hunt for it didn't turn up anything.<br>
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<font style="font-size:11pt" face="Calibri, sans-serif" color="#000000"><b>From:</b> Daniel Asarnow <<a href="mailto:asarnow@msg.ucsf.edu" target="_blank">asarnow@msg.ucsf.edu</a>><br>
<b>Sent:</b> Monday, February 28, 2022 5:45 PM<br>
<b>To:</b> Morgan, David Gene <<a href="mailto:dagmorga@indiana.edu" target="_blank">dagmorga@indiana.edu</a>><br>
<b>Cc:</b> <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a> <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>><br>
<b>Subject:</b> [External] Re: [3dem] tilted transfer functions?</font>
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<div>I think any patch-based CTF program, like the one in cryosparc or gCTF's local mode (with evenly spaced coordinates instead of real particle locations) would work well. With cryoSPARC there's an easy method to plot the tilt axis; you can also choose a
specific number X and Y divisions for the patches.</div>
<div><br>
</div>
<div>Best,</div>
<div>-da<br>
</div>
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<div>
<div dir="ltr">On Mon, Feb 28, 2022 at 2:41 PM Morgan, David Gene <<a href="mailto:dagmorga@indiana.edu" target="_blank">dagmorga@indiana.edu</a>> wrote:<br>
</div>
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<div style="font-family:Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)">
Hi,</div>
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<br>
</div>
<div style="font-family:Calibri,Helvetica,sans-serif;font-size:12pt;color:rgb(0,0,0)">
What's the best way to evaluate the CTF of an image that has a significant amount of tilt?<br>
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