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<p class="gmail-Default" style="margin:0cm 0cm 0.0001pt;font-size:12pt;font-family:"Times New Roman",serif;color:black"><span style="font-size:10pt;color:windowtext"><span> </span></span></p>
<p class="MsoNormal" style="margin:0cm 0cm 0.0001pt;line-height:normal;font-size:12pt;font-family:"Times New Roman",serif"><span>Hi Ed,<span></span></span></p>
<p class="MsoNormal" style="margin:0cm 0cm 0.0001pt;line-height:normal;font-size:12pt;font-family:"Times New Roman",serif"><span><span> </span></span></p>
<p class="MsoNormal" style="margin:0cm 0cm 0.0001pt;line-height:normal;font-size:12pt;font-family:"Times New Roman",serif"><span>Long time no talk! <br></span></p><p class="MsoNormal" style="margin:0cm 0cm 0.0001pt;line-height:normal;font-size:12pt;font-family:"Times New Roman",serif"><span>I started the #Why-o-Why series in Twitter to clarify some basic theoretical
cryo-EM principles – in digestible units – to both newcomers and established
senior scientists alike! <span></span></span></p>
<p class="MsoNormal" style="margin:0cm 0cm 0.0001pt;line-height:normal;font-size:12pt;font-family:"Times New Roman",serif"><span><span> </span></span></p>
<p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:12pt;font-family:"Times New Roman",serif"><span style="line-height:107%">In #Why-o-Why #13, for
example, I emphasise the fact that the FRC/FSC (and its thresholds) are not
tied to structural biology and also not tied to what you (want to) see in the
results. Judging the quality of a metric by whether you see protein side chains
is a methodological mistake! It is obviously inappropriate when you want
to use your intuitive side-chain metric to judge the “results-resolution”
achieved in an X-ray tomographic reconstruction of a human brain, or in the 3D tomographic
reconstruction of a computer chip. How would you react when a medic tells you
your 3D reconstruction of a ribosome is rubbish because he/she cannot even see the
2mm-sized kidney stones she/he likes to distinguish in the 3D reconstructions –
but in your ribosome map!<span></span></span></p>
<p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:12pt;font-family:"Times New Roman",serif"><span style="line-height:107%">All your FSC examples violate
this basic principle and thus do not represent appropriate, clean, scientific counter arguments for any FSC/FRC discussion!<span></span></span></p>
<p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:12pt;font-family:"Times New Roman",serif"><span style="line-height:107%">Moreover, issues like the
alignments of molecular images in the context single-particle cryo-EM analysis also
have ABSOLUTELY NOTHING to do with the FRC/FSC criterion for comparing of two
images or two 3D densities. Whatever reference bias or symmetry error you chose
to impose on the two images / volumes is your own responsibility. <span></span></span></p>
<p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:12pt;font-family:"Times New Roman",serif"><span style="line-height:107%">As I put it in an earlier internet
discussion: “If you overheat your living room, don’t blame your thermometer!”
(But you may want to check the functioning of your thermostat!). This statement in its cryo-EM translation:
<span> </span>Don’t blame the FSC curve for your own Bias
Blunders.<span></span></span></p>
<p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:12pt;font-family:"Times New Roman",serif"><span style="line-height:107%">In your 2016 paper you
introduce the FSC as follows:<span></span></span></p>
<p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:12pt;font-family:"Times New Roman",serif"><b><i><span style="line-height:107%">“However, the definition of resolution in cryo-EM
is a much more nebulous entity than in X-ray crystallography, and the most
commonly used method of reporting resolution using a plot of the Fourier Shell
Correlation (FSC) is fraught with problems</span></i></b><span style="line-height:107%">”<span></span></span></p>
<p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:12pt;font-family:"Times New Roman",serif"><span style="line-height:107%">Hardly a clean neutral introduction
of a scientific topic and certainly a statement that is in flagrant contradiction with the facts (see:
#Why-o-Why #13). I know… I am not going to convince you after all these decades
of “some people” not willing to read - or listen to clean scientific arguments. I don’t
even expect a reaction from you! What I am more worried about is the effect of planting
wrong ideas in the minds of a new generation of cryo-EM scientists. <span></span></span></p>
<p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:12pt;font-family:"Times New Roman",serif"><span style="line-height:107%">So you cryo-EM newcomers out there, do take the time to read
some of the basics; and for those not on Twitter I added #Why-o-Why #13, below
(@Marin_van_Heel).<span></span></span></p>
<p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:12pt;font-family:"Times New Roman",serif"><span style="line-height:107%">Cheers,<span></span></span></p>
<p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:12pt;font-family:"Times New Roman",serif"><span style="line-height:107%">Marin</span></p><p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:12pt;font-family:"Times New Roman",serif"></p><div><img src="cid:ii_k6nzmu2n0" alt="image.png" width="389" height="494"><br><br></div><p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:12pt;font-family:"Times New Roman",serif"><br><span style="line-height:107%"><span></span></span></p>
</div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Thu, Feb 13, 2020 at 2:48 PM Egelman, Edward H (ehe2n) <<a href="mailto:egelman@virginia.edu">egelman@virginia.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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<p class="MsoNormal"><span style="font-size:14pt">I had studiously avoided getting involved in this thread which might lead to a long exchange with Marin about the history, significance and ontological status of the FSC, but I will suggest a paper that while
dated addresses some of these isssues:<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:14pt"><u></u> <u></u></span></p>
<p class="MsoNormal" style="margin-left:27pt"><span style="font-size:14pt;font-family:Helvetica;color:black">Subramaniam, S., Earl, L.A., Falconieri, V., Milne, J.L., and Egelman, E.H. (2016). Resolution advances in cryo-EM enable application
to drug discovery. Curr Opin Struct Biol<i> 41</i>, 194-202.<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:14pt">Abstract<u></u><u></u></span></p>
<p class="gmail-m_-1369160998631721172p1"><span style="font-size:14pt">The prospect that the structures of protein assemblies, small and large, can be determined using cryo-electron microscopy (cryo-EM) is beginning to transform the landscape of structural biology and cell biology.
Great progress is being made in determining 3D structures of biological assemblies ranging from icosahedral viruses and helical arrays to small membrane proteins and protein complexes. Here, we review recent advances in this field, focusing especially on the
emerging use of cryo-EM in mapping the binding of drugs and inhibitors to protein targets, an application that requires structure determination at the highest possible resolutions.
<b>We discuss methods used to evaluate the information contained in cryo-EM density maps and consider strengths and weaknesses of approaches currently used to measure map resolution.</b><u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:14pt"><u></u><u></u></span></p>
<p class="gmail-m_-1369160998631721172p1"><span style="font-size:14pt;font-family:"Calibri",sans-serif">We discuss that FSC is NOT a measure of resolution, it is a measure of reproducibility. We give examples for structures with symmetry where one can impose the wrong symmetry and get
a better FSC than with the correct symmetry (this is also discussed in Egelman, E.H. 2014. Ambiguities in helical reconstruction. Elife<i> 3</i>.). While the map looks like garbage, the FSC does not care as the garbage is highly reproducible. As to actually
looking at the map, I would agree with previous people who have commented or implied that the gold standard in x-ray crystallography has always been the visual appearance and interpretability of the map, and not some single metric.<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:14pt">Regards,<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:14pt">Ed<u></u><u></u></span></p>
<div>
<div>
<p class="MsoNormal"><span style="font-size:10.5pt;color:black"><u></u> <u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:10.5pt;color:black"><u></u> <u></u></span></p>
</div>
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<p class="MsoNormal"><span style="font-size:10.5pt;color:black">Edward Egelman<u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:10.5pt;color:black">Harrison Distinguished Professor<u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:10.5pt;color:black">Department of Biochemistry and Molecular Genetics<u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:10.5pt;color:black">University of Virginia<u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:10.5pt;color:black">phone: 434-924-8210<u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:10.5pt;color:black">fax: 434-924-5069<u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:10.5pt;color:black"><a href="mailto:egelman@virginia.edu" target="_blank">egelman@virginia.edu</a><u></u><u></u></span></p>
</div>
</div>
<p class="MsoNormal"><span style="font-size:10.5pt;color:black"><a href="http://www.people.virginia.edu/~ehe2n" target="_blank"><span style="color:blue">www.people.virginia.edu/~ehe2n</span></a></span><span style="font-size:12pt"><u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:12pt"><u></u> <u></u></span></p>
<div style="border-color:rgb(181,196,223) currentcolor currentcolor;border-style:solid none none;border-width:1pt medium medium;padding:3pt 0in 0in">
<p class="MsoNormal"><b><span style="font-size:12pt;color:black">From: </span></b><span style="font-size:12pt;color:black">3dem <<a href="mailto:3dem-bounces@ncmir.ucsd.edu" target="_blank">3dem-bounces@ncmir.ucsd.edu</a>> on behalf of Pawel Penczek <<a href="mailto:Pawel.A.Penczek@uth.tmc.edu" target="_blank">Pawel.A.Penczek@uth.tmc.edu</a>><br>
<b>Date: </b>Thursday, February 13, 2020 at 8:42 AM<br>
<b>To: </b>3dem <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>><br>
<b>Subject: </b>[3dem] Which resolution?<u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
<div>
<p class="MsoNormal">Dear Teige, <u></u><u></u></p>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
<div>
<p class="MsoNormal">I am wondering whether you are familiar with<u></u><u></u></p>
</div>
<div>
<h2 style="margin-right:0in;margin-bottom:6pt;margin-left:0in;line-height:18pt;box-sizing:border-box">
<span style="font-size:15pt;font-family:"Lucida Grande",sans-serif"><u></u> <u></u></span></h2>
<h2 style="margin-right:0in;margin-bottom:6pt;margin-left:0in;line-height:18pt;box-sizing:border-box">
<span style="font-size:15pt;font-family:"Lucida Grande",sans-serif">Resolution measures in molecular electron microscopy.<u></u><u></u></span></h2>
<div id="gmail-m_-1369160998631721172cit_summary">
<div>
<p class="MsoNormal"><span style="font-size:10.5pt;font-family:"Arial",sans-serif;color:rgb(39,39,39)">Penczek PA. Methods Enzymol. 2010.<u></u><u></u></span></p>
</div>
</div>
<div id="gmail-m_-1369160998631721172cit_full">
<h3 style="margin-bottom:0.0001pt"><span style="font-size:10.5pt;font-family:"Lucida Grande",sans-serif;color:black">Citation<u></u><u></u></span></h3>
<p class="gmail-m_-1369160998631721172j" style="margin-right:0in;margin-bottom:1.5pt;margin-left:0in;box-sizing:border-box">
<span style="font-size:10.5pt;font-family:"Arial",sans-serif;color:rgb(39,39,39)">Methods Enzymol. 2010;482:73-100. doi: 10.1016/S0076-6879(10)82003-8.<u></u><u></u></span></p>
</div>
<div>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
<div>
<p class="MsoNormal">You will find there answers to all questions you asked and much more. <u></u><u></u></p>
</div>
<p class="MsoNormal"><u></u> <u></u></p>
<div>
<p class="MsoNormal">Regards, <u></u><u></u></p>
<div>
<p class="MsoNormal">Pawel Penczek<u></u><u></u></p>
</div>
</div>
</div>
</div>
<p class="MsoNormal" style="margin-bottom:12pt"><u></u> <u></u></p>
<div>
<p class="MsoNormal">Regards, <u></u><u></u></p>
<div>
<p class="MsoNormal">Pawel<u></u><u></u></p>
</div>
</div>
</div>
</div>
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