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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt">Hi Rasmus,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt">I’m in agreement with Marin about the necessity of using a correct element-specific spectral distribution of the amplitude contrast. But blaming a single paper is as you say unfair; the problem is rather
the uncritical adoption of an initial oversimplification by the users. There have been plenty of misjudgments like that.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt">Related to amplitude contrast, see my proof-of-concept paper on heavy/light atom discrimination using Peter Schiske’s method in Biophys. J. (1972), and a later paper with Pawel Penczek (Frank and Penczek,
Optik 1995) where we tried to use Schiske’s algorithm on the cryo-EM ribosome dataset collected in Heidelberg. The funny thing was, the L1 stalk lit up in the amplitude contrast image, so we thought something must have gone wrong since, to our knowledge then,
the L1 stalk was all protein and not, as we know now, part protein and part RNA.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt">my 1c,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt">--Joachim<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;color:black">Dr. Joachim Frank</span><span style="font-size:9.0pt;font-family:"Times New Roman",serif;color:black"><o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:9.0pt;color:black">Professor, Biochemistry and Molecular Biophysics & Biological Sciences, </span><span style="font-size:9.0pt;font-family:"Times New Roman",serif;color:black"><o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:9.0pt;color:black">Columbia University Irving Medical Center,
<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:9.0pt;color:black">Hammer Health Sciences Center, Room 616,
<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:9.0pt;color:black">701 West 168th Street, New York, NY 10032 -- </span><a href="mailto:jf2192@cumc.columbia.edu" title="mailto:jf2192@cumc.columbia.edu"><span style="font-size:9.0pt;color:#954F72">jf2192@cumc.columbia.edu</span></a><span style="font-size:9.0pt;color:black"><o:p></o:p></span></p>
<p class="MsoNormal"><i><span style="font-size:9.0pt;color:black">2017 Nobel Prize in Chemistry</span></i><span style="font-size:9.0pt;font-family:"Times New Roman",serif;color:black"><o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:9.0pt;color:black">Admin. Coordinator: Masgan Saidi 917 232 6545 </span><a href="mailto:ms4597@cumc.columbia.edu" title="mailto:ms4597@cumc.columbia.edu"><span style="font-size:9.0pt;color:#954F72">ms4597@cumc.columbia.edu</span></a><span style="font-size:9.0pt;font-family:"Times New Roman",serif;color:black"><o:p></o:p></span></p>
<p class="MsoNormal"><i><span style="font-size:9.0pt;color:black">Science: </span></i><a href="https://urldefense.proofpoint.com/v2/url?u=https-3A__joachimfranklab.org&d=DwMFAw&c=G2MiLlal7SXE3PeSnG8W6_JBU6FcdVjSsBSbw6gcR0U&r=WMEZpdnKefeJBKIRE4HFMD03dG6F5_6w7sRzzvkLMXQ&m=TYKF4If_gfUQerpbfW2d6Wtduj37ZOB7qemjTwlcxFU&s=imQWuSOK3f7uZgiFTjoAp76cDLVvhB8UwOJ3lnX6618&e=" title="https://urldefense.proofpoint.com/v2/url?u=https-3A__joachimfranklab.org&d=DwMFAw&c=G2MiLlal7SXE3PeSnG8W6_JBU6FcdVjSsBSbw6gcR0U&r=WMEZpdnKefeJBKIRE4HFMD03dG6F5_6w7sRzzvkLMXQ&m=TYKF4If_gfUQerpbfW2d6Wtduj37ZOB7qemjTwlcxFU&s=imQWuSOK3f7uZgiFTjoAp76cDLVvhB8Uw"><span style="font-size:9.0pt;color:#954F72">https://joachimfranklab.org</span></a><i><span style="font-size:9.0pt;color:black">
-- Fiction: franxfiction.com</span></i><span style="font-size:9.0pt;font-family:"Times New Roman",serif;color:black"><o:p></o:p></span></p>
<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal" style="margin-bottom:12.0pt"><b><span style="font-size:12.0pt;color:black">From:
</span></b><span style="font-size:12.0pt;color:black">3dem <3dem-bounces@ncmir.ucsd.edu> on behalf of Schroeder, Rasmus <rasmus.schroeder@bioquant.uni-heidelberg.de><br>
<b>Date: </b>Wednesday, October 18, 2023 at 5:02 AM<br>
<b>To: </b>Marin van Heel <marin.vanheel@googlemail.com><br>
<b>Cc: </b>3dem <3dem@ncmir.ucsd.edu>, CCPEM@jiscmail.ac.uk <CCPEM@jiscmail.ac.uk><br>
<b>Subject: </b>[EXTERNAL] Re: [3dem] [ccpem] Differences EM CP maps vs Xray ED Maps<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">Dear Marin - and all others here,<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">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.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">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 <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">(Angert et al., Ultramicroscopy 81 (2000) 203-222).<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">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 …<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">(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 …)<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">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 ….<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">My 2 cent ….<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">Best,<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">Rasmus<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<br>
<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">On 15. Oct 2023, at 22:22, Marin van Heel <marin.vanheel@googlemail.com> wrote:<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal" style="line-height:105%"><span lang="EN-IN" style="font-size:11.0pt;line-height:105%"> </span><span style="font-size:11.0pt;line-height:105%"><o:p></o:p></span></p>
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<p class="MsoNormal" style="margin-bottom:8.0pt;line-height:105%"><span lang="EN-IN" style="font-size:11.0pt;line-height:105%">Dear All,</span><span style="font-size:11.0pt;line-height:105%"><o:p></o:p></span></p>
<p class="MsoNormal" style="margin-bottom:8.0pt;line-height:105%"><span lang="EN-IN" style="font-size:11.0pt;line-height:105%">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. 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! That thus means that the <b><i>phase</i></b> at the origin must be
<b><i>zero</i></b>. (<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 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 to each other and will
also complicate any comparison with zero-average density maps in X-ray crystallography.</span><span style="font-size:11.0pt;line-height:105%"><o:p></o:p></span></p>
<p class="MsoNormal" style="margin-bottom:8.0pt;line-height:105%"><span lang="EN-IN" style="font-size:11.0pt;line-height:105%">Two more cents added!</span><span style="font-size:11.0pt;line-height:105%"><o:p></o:p></span></p>
<p class="MsoNormal" style="margin-bottom:8.0pt;line-height:105%"><span lang="EN-IN" style="font-size:11.0pt;line-height:105%">Marin</span><span style="font-size:11.0pt;line-height:105%"><o:p></o:p></span></p>
<p class="MsoNormal" style="margin-bottom:8.0pt;line-height:105%"><span style="font-size:11.0pt;line-height:105%">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.<o:p></o:p></span></p>
<p class="MsoNormal" style="margin-bottom:8.0pt;line-height:105%"><span style="font-size:11.0pt;line-height:105%">van Heel, M. (1978). On the imaging of relatively strong objects in partially coherent illumination in optics and electron optics. Optik. 49, 389–408.<o:p></o:p></span></p>
<p class="MsoNormal" style="margin-bottom:8.0pt;line-height:105%"><a href="https://urldefense.proofpoint.com/v2/url?u=https-3A__mail.ncmir.ucsd.edu_pipermail_3dem_2014-2DJuly_003454.html&d=DwMFaQ&c=009klHSCxuh5AI1vNQzSO0KGjl4nbi2Q0M1QLJX9BeE&r=wsoQjtpZ1rSBXeWc4Du-lO_6zaO7RA_HHVekUqQDowc&m=v_q3j9BRkOXQXVB_GGY6fjq8tAIBsPrdF8yYMFHB985rPAuC2c6lyIT_OU-2tl6i&s=0L2C6HNg3dI9xHXtUcSJXvrxQifPj0IQTR59uhdQd4w&e="><span style="font-size:11.0pt;line-height:105%">https://mail.ncmir.ucsd.edu/pipermail/3dem/2014-July/003454.html</span></a><span style="font-size:11.0pt;line-height:105%"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">On Sun, Oct 15, 2023 at 10:01 AM Guillaume Gaullier <</span><a href="mailto:guillaume.gaullier@kemi.uu.se" target="_blank"><span style="font-size:11.0pt">guillaume.gaullier@kemi.uu.se</span></a><span style="font-size:11.0pt">>
wrote:<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">Hello Bernhard, <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">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.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">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.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">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.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">At least this is how I understand Tom Goddard’s explanation in this discussion from last month on the chimerax-users list: </span><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"><span style="font-size:11.0pt">https://mail.cgl.ucsf.edu/mailman/archives/list/chimerax-users@cgl.ucsf.edu/thread/3RNL6ODNP6QAL3BTHIPPQS2VE2B2YSVO/</span></a><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">Maybe there are other reasons adding to this.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">I hope this helps.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">Cheers,<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:Helvetica"><br>
Guillaume<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:Helvetica"><o:p> </o:p></span></p>
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<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">On 15 Oct 2023, at 13:05, <Nonameavailable> <</span><a href="mailto:br@RUPPWEB.ORG" target="_blank"><span style="font-size:11.0pt">br@RUPPWEB.ORG</span></a><span style="font-size:11.0pt">> wrote:<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span lang="DE" style="font-size:11.0pt">Dear EM Experts,</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="DE" style="font-size:11.0pt"> </span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">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:<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"> <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">“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?”<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"> <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">And here a potential partial answer:<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"> <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">“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.”<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"> <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">What are we missing here?<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"> <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">Thx, BR<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"> <o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="DE" style="font-size:11.0pt">-----------------------------------------------------------------</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="DE" style="font-size:11.0pt">Bernhard Rupp (Hofkristallrat a. D)</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="ES" style="font-size:11.0pt">K.k. Hofkristallamt</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="ES" style="font-size:11.0pt">CA 92084 San Diego</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="ES" style="font-size:11.0pt">001 (925) 209-7429</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="ES" style="font-size:11.0pt">+43 (676) 571-0536</span><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><a href="mailto:br@ruppweb.org" target="_blank"><span lang="ES" style="font-size:11.0pt">br@ruppweb.org</span></a><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><a href="mailto:hofkristallamt@gmail.com" target="_blank"><span lang="ES" style="font-size:11.0pt">hofkristallamt@gmail.com</span></a><span style="font-size:11.0pt"><o:p></o:p></span></p>
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<p class="MsoNormal"><a href="https://urldefense.com/v3/__http:/www.ruppweb.org/__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbMv8ZhPSQ$" target="_blank"><span style="font-size:11.0pt">http://www.ruppweb.org/</span></a><span style="font-size:11.0pt">
<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">-----------------------------------------------------------------<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">All models are wrong but some are useful<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt">-----------------------------------------------------------------<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"> <o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><xray.png><EM.png><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><Screenshot 2023-10-15 at 14.22.31.png><o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:Helvetica;color:black">--<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 rasmus.schroeder@bioquant.uni-heidelberg.de<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"><o:p> </o:p></span></p>
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