<div dir="ltr"><div dir="ltr"><div>Dear Rasmus,</div><div><br></div><div>I am not really harsh on Harold Erickson. He is an open-minded scientist and that work was good for its time, When I called him a few years ago on the issue there was absolutely no problem in discussing the matter and he even remembered that we had spoken about it in 1977 during an EMBO EM school in Basel (when I was still writing my 1978 paper). The problem with such early papers is that science insights change and the approximations made by Erickson and Klug had their limitations. In my 1978 paper on partial coherence the REAL and the IMAGINARY parts of the complex object transmittance are treated fully independent. In fact the theory applies to light microscopy as well as electron microscopy and to other imaging systems. If any real criticism is appropriate here, that would apply to the later generations of researchers who just take over the old formulas without thinking about their meaning. <br></div></div><div><br></div><div>Adding two more cents</div><div><br></div><div>Marin<br></div><div><br></div><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Wed, Oct 18, 2023 at 6:01 AM Schroeder, Rasmus <<a href="mailto:rasmus.schroeder@bioquant.uni-heidelberg.de">rasmus.schroeder@bioquant.uni-heidelberg.de</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"><div>Dear Marin - and all others here,<div><br></div><div>I would not be so harsh with that paper from 1971, there is always a certain level of experimental data, and - as far as I understand it - at that time and resolution level obtainable it looked as if amplitude and phase potentials could be identical.</div><div><br></div><div>As you point out correctly, this is not the case, and I may add a study we did many, many years ago looking into the CTF for an energy filtered TEM </div><div>(Angert et al., Ultramicroscopy 81 (2000) 203-222).</div><div>Here a correct CTF needs to include yet another cos-term ….. and the zeros in the experimental and theoretical CTF only fit, if an additional cos-contrast (amplitude contrast produced by the energy filter) is added …</div><div>(And yes, I apologize for the quality of the data, but it was the best we managed to get with that very old hardware at that time …)</div><div><br></div><div>And I may add a bit of relief to all here: Do not get too excited that nobody uses this additional cos-term at present. The explanation is simple: Nobody cares for the extra bit of very low resolution data when fitting a CTF going out to better than a few Angstroms ….. But the problem with the density and the phase at “zero” remains ….</div><div><br></div><div>My 2 cent ….</div><div><br></div><div>Best,</div><div><br></div><div>Rasmus</div><div><br></div><div><div><div><br><blockquote type="cite"><div>On 15. Oct 2023, at 22:22, Marin van Heel <<a href="mailto:marin.vanheel@googlemail.com" target="_blank">marin.vanheel@googlemail.com</a>> wrote:</div><br><div><div dir="ltr"><div style="margin:0cm 0cm 8pt;line-height:107%;font-size:11pt;font-family:Calibri,sans-serif"><span lang="EN-IN"><span> </span></span><br></div><p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:11pt;font-family:"Calibri",sans-serif"><span lang="EN-IN">Dear All,<span></span></span></p><p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:11pt;font-family:"Calibri",sans-serif"><span lang="EN-IN">Unfortunately,
a fundamental<i> </i>mistake has been made in the much-cited paper by Erickson
and Klug [Erickson & Klug 1971] more than 50 years ago. <span> </span>They assumed that the EM <b><i>amplitude
contrast</i></b> of the (stained ) biological object to be <b><i>proportional</i></b>
to the <b><i>phase contrast</i></b> of the same object over all spatial
frequencies. If that were indeed the case, one single transfer function would
suffice to describe how the linear imaging device would generate an output
image. In reality, however, the <b>amplitude contrast</b> and <b>phase contrast</b>
two <b><i>separate properties</i></b> of the <b><i>complex transmission
function</i></b> of the object, and these are associated with different
physical properties [Van Heel 1978] . The problem is that that proportionality error
has crept into almost all popular CTF determination programs where, say, 10% or
15% amplitude contrast is suggested ab initio. Any percentage of amplitude
contrast erroneously causes the average density of the cryo-EM 3D reconstruction
to deviate from <b><i>zero</i></b>. Any phase-contrast image must yield a <b><i>zero
average</i></b> as it should be for any phase contrast image where what is
measured is the <b><i>difference</i></b> in <b><i>phase</i></b> between any
point in the back focal plane of the system with respect to the <b><i>phase</i></b>
at the origin!<span> </span>That thus means that the <b><i>phase</i></b>
at the origin must be <b><i>zero</i></b>.<span>
</span>(<b><i>Zero</i></b> being the average density over the image around
which the phase information is modulated) . Adding a <b><i>cosine</i></b> component
to <span> </span>the CTF (a <b><i>sine</i></b>) will
shift the zeroes of the CTF and therewith shift the defocus values found in most
programs (no longer the real defocus!). That makes such results no longer comparable
<span> </span>to each other and will also complicate any
comparison with zero-average density maps in X-ray crystallography.<span></span></span></p><p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:11pt;font-family:"Calibri",sans-serif"><span lang="EN-IN">Two more
cents added!<sub><span></span></sub></span></p><p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:11pt;font-family:"Calibri",sans-serif"><span lang="EN-IN">Marin<span></span></span></p><p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:11pt;font-family:"Calibri",sans-serif">Erickson HP, Klug A (1971). Measurement and Compensation of
Defocusing and Aberrations by Fourier Processing of Electron Micrographs. Phil.
Trans. R. Soc. Lond. B. 261; 105-118.<span></span></p><p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:11pt;font-family:"Calibri",sans-serif">van Heel, M. (1978). On the imaging of relatively strong
objects in partially coherent illumination in optics and electron optics.
Optik. 49, 389–408.<span></span></p><p class="MsoNormal" style="margin:0cm 0cm 8pt;line-height:107%;font-size:11pt;font-family:"Calibri",sans-serif"><a href="https://mail.ncmir.ucsd.edu/pipermail/3dem/2014-July/003454.html" target="_blank">https://mail.ncmir.ucsd.edu/pipermail/3dem/2014-July/003454.html</a><span></span></p>
</div><div dir="ltr"><div><br></div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Sun, Oct 15, 2023 at 10:01 AM Guillaume Gaullier <<a href="mailto:guillaume.gaullier@kemi.uu.se" target="_blank">guillaume.gaullier@kemi.uu.se</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">
<div>
Hello Bernhard,
<div><br>
</div>
<div>According to PDB/EMDB validation reports, this spike in the voxel values histogram is caused by masking. One validation report I have says "A spike in this graph at zero usually indicates that the volume has been masked". You can probably also
find this note in validation reports of released PDB/EMDB entries.</div>
<div><br>
</div>
<div>Opening a map from 3D refinement and one of the two half-maps from the same job seems to confirm this. The map’s histogram shows this spike at zero, but the half-map’s histogram doesn’t. Half-maps are never filtered nor masked, whereas the main
map is masked (in cryoSPARC this is done by default, unless one turns off automatic masking and doesn’t provide any mask). See the attached histograms.</div>
<div><br>
</div>
<div>The fact that there is no absolute scale for contour level in cryoEM maps is indeed annoying (I would like to compare maps without worrying that maybe I chose inadequate contour levels). My understanding is that it is caused at least in part by
the fact that the size of the box enclosing the particle is arbitrary. Different amounts of low-value voxels between different maps give them different voxel value histograms, therefore choosing a contour level in terms of a certain number of standard deviations
above the mean produces different results with different maps. Electron density maps from crystallography don’t have this variability because the box always spans a full unit cell, without this variable padding around the region of high density.</div>
<div>At least this is how I understand Tom Goddard’s explanation in this discussion from last month on the chimerax-users list: <a href="https://urldefense.com/v3/__https://mail.cgl.ucsf.edu/mailman/archives/list/chimerax-users@cgl.ucsf.edu/thread/3RNL6ODNP6QAL3BTHIPPQS2VE2B2YSVO/__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbMPdEMJ_w$" target="_blank">https://mail.cgl.ucsf.edu/mailman/archives/list/chimerax-users@cgl.ucsf.edu/thread/3RNL6ODNP6QAL3BTHIPPQS2VE2B2YSVO/</a></div>
<div>Maybe there are other reasons adding to this.</div>
<div><br>
</div>
<div>I hope this helps.</div>
<div>Cheers,</div>
<div>
<div>
<div dir="auto" style="letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration:none">
<div dir="auto" style="letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration:none">
<div dir="auto" style="letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration:none">
<div dir="auto" style="letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration:none">
<div dir="auto" style="letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration:none">
<div style="letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px">
<div style="letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px">
<div style="font-family:Helvetica;font-size:12px;font-style:normal;font-variant-caps:normal;font-weight:normal;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px">
<div>
<div style="font-family:Helvetica;font-size:12px;font-style:normal;font-variant-caps:normal;font-weight:normal;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px">
<br>
Guillaume</div>
<div style="font-family:Helvetica;font-size:12px;font-style:normal;font-variant-caps:normal;font-weight:normal;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px">
<br>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
<div><br>
<blockquote type="cite">
<div>On 15 Oct 2023, at 13:05, <Nonameavailable> <<a href="mailto:br@RUPPWEB.ORG" target="_blank">br@RUPPWEB.ORG</a>> wrote:</div>
<br>
<div>
<div style="font-family:Helvetica;font-size:12px;font-style:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration:none">
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<span lang="DE">Dear EM Experts,<u></u><u></u></span></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<span lang="DE"><u></u> <u></u></span></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
Some of my crystallographer colleagues and I wonder about the fundamentally different appearance of density histograms in EM Coulomb potential maps vs the X-ray Electron density maps. Here is the question:<u></u><u></u></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
“What I've noticed is that they are on different scales. That is understandable, a e/A^3 is different than V. But there are papers showing that these values are somewhat proportional to each other for lower resolutions. But the other thing that I have noticed
is that electron density maps have close to normally distributed value distributions, whereas cryoEM maps have a sharp spike and a very long tail. As a result, an electron density blob in an X-ray map looks nice somewhere around 3sigma, whereas for cryoEM
it's sometime 10sigma, 17sigma, 20sigma, all over the place. I'm thinking of using thresholds based on percentiles rather than sigmas, but my main question is: shouldn't the values on cryoEM maps be also approximately normally distributed? what is the cause
of this non-normality? sharpening? the raw experimental data themselves?”<u></u><u></u></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
And here a potential partial answer:<u></u><u></u></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
“In cryo-EM, there is no absolute scaling, which means that density values can vary significantly. This variability can explain why you consistently encounter different sigma values. I can confirm that the density value distribution behaves as you described,
and I have also observed this. However, I cannot provide a definitive answer as to why this occurs. My best guess is that it may be related to B factor weighting in motion correction, but I cannot provide a conclusive explanation, I'm afraid.”<u></u><u></u></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
What are we missing here?<u></u><u></u></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
Thx, BR<u></u><u></u></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<span lang="DE">-----------------------------------------------------------------<u></u><u></u></span></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<span lang="DE">Bernhard Rupp (Hofkristallrat a. D)<u></u><u></u></span></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<span lang="ES">K.k. Hofkristallamt<u></u><u></u></span></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<span lang="ES">CA 92084 San Diego<u></u><u></u></span></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<span lang="ES">001 (925) 209-7429<u></u><u></u></span></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<span lang="ES">+43 (676) 571-0536<u></u><u></u></span></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<span lang="ES"><a href="mailto:br@ruppweb.org" target="_blank">br@ruppweb.org</a><u></u><u></u></span></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<span lang="ES"><a href="mailto:hofkristallamt@gmail.com" target="_blank">hofkristallamt@gmail.com</a><u></u><u></u></span></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<span><a href="https://urldefense.com/v3/__http://www.ruppweb.org/__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbMv8ZhPSQ$" target="_blank">http://www.ruppweb.org/</a> <u></u><u></u></span></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<span>-----------------------------------------------------------------<u></u><u></u></span></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<span>All models are wrong but some are useful<u></u><u></u></span></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<span>-----------------------------------------------------------------<u></u><u></u></span></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<span><u></u> <u></u></span></div>
<div style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif">
<span lang="DE"><u></u> <u></u></span></div>
</div>
<br style="font-family:Helvetica;font-size:12px;font-style:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration:none">
<hr style="font-family:Helvetica;font-size:12px;font-style:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration:none"><p align="center" style="font-family:Helvetica;font-size:12px;font-style:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration:none">
To unsubscribe from the CCPEM list, click the following link:<br>
<a href="https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCPEM&A=1__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbPaA6gqVQ$" target="_blank">https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCPEM&A=1</a></p><p style="font-family:Helvetica;font-size:12px;font-style:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration:none">
<br>
</p>
<div style="font-style:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration:none;background-color:rgb(255,255,255);width:846px;border:1pt solid rgb(0,0,0);padding:2pt;font-size:10pt;line-height:12pt;font-family:Calibri,serif,EmojiFont;text-align:left">
<span>VARNING:</span><span> </span>Klicka inte på länkar och öppna inte bilagor om du inte känner igen avsändaren och vet att innehållet är säkert.<br>
<span>CAUTION:</span><span> </span>Do not click on links or open attachments unless you recognise the sender and know the content is safe.</div>
<span id="m_4380002884934045966m_-5234759440403458115m_-3628768259685584288cid:82E1FF61-85B5-4FA3-897E-AF854060A7AF"><xray.png></span><span id="m_4380002884934045966m_-5234759440403458115m_-3628768259685584288cid:1756B97C-763C-4A76-BBB7-2FC698B2E724"><EM.png></span></div>
</blockquote>
</div>
<div><br>
</div>
<span id="m_4380002884934045966cid:18b34b2023d814147c71"><Screenshot 2023-10-15 at 14.22.31.png></span></div>
<u></u>
<br>
<br>
<br>
<br>
<br>
<br>
<br>
<br>
När du har kontakt med oss på Uppsala universitet med e-post så innebär det att vi behandlar dina personuppgifter. För att läsa mer om hur vi gör det kan du läsa här: <a href="https://urldefense.com/v3/__http://www.uu.se/om-uu/dataskydd-personuppgifter/__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbP9uI9duw$" target="_blank">http://www.uu.se/om-uu/dataskydd-personuppgifter/</a>
<br>
<br>
E-mailing Uppsala University means that we will process your personal data. For more information on how this is performed, please read here: <a href="https://urldefense.com/v3/__http://www.uu.se/en/about-uu/data-protection-policy__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbNcWzUBIQ$" target="_blank">http://www.uu.se/en/about-uu/data-protection-policy</a>
</div>
<br>
<hr><p align="center">To unsubscribe from the CCPEM list, click the following link:<br>
<a href="https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCPEM&A=1__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbPaA6gqVQ$" target="_blank">https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCPEM&A=1</a>
</p>
</blockquote></div>
_______________________________________________<br>3dem mailing list<br><a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a><br><a href="https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem" target="_blank">https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem</a><br></div></blockquote></div><br><div>
<div style="color:rgb(0,0,0);font-family:Helvetica;font-size:12px;font-style:normal;font-variant-caps:normal;font-weight:normal;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration:none">--<br>############################################################<br><br>Rasmus R. Schroeder<br><br>Cryo Electron Microscopy<br>Heidelberg University / Medical Faculty<br>BioQuant, Im Neuenheimer Feld 267<br>69120 Heidelberg, Germany<br><br>Tel. +49-(0)6221-5451350<br>e-mail <a href="mailto:rasmus.schroeder@bioquant.uni-heidelberg.de" target="_blank">rasmus.schroeder@bioquant.uni-heidelberg.de</a></div>
</div>
<br></div></div></div>_______________________________________________<br>
3dem mailing list<br>
<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a><br>
<a href="https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem" rel="noreferrer" target="_blank">https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem</a><br>
</blockquote></div></div>