<div dir="auto">Me again,<div dir="auto"><br></div><div dir="auto">As some of you might have already noticed, the link I sent was for the 1971 paper and not the 1973.</div><div dir="auto"><br></div><div dir="auto">So please, contact Ben 😉</div><div dir="auto"><br></div><div dir="auto">Cheers,</div><div dir="auto">Sylvain<br><br><div data-smartmail="gmail_signature" dir="auto">PhD<br>Research Fellow - Microscopy Australia<br><br>Monash University<br><br>Ramaciotti Centre for Cryo-EM<br>G92, 15 Innovation Walk, Clayton Campus<br>Victoria, 3800 Australia</div></div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Mon, 23 Oct 2023, 1:01 pm Sylvain Trepout, <<a href="mailto:sylvain.trepout@monash.edu">sylvain.trepout@monash.edu</a>> wrote:<br></div><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"><div dir="ltr">Hi there, <div><br></div><div>For those who want access to the article, it is available on Joachim's website:</div><div><br></div><div><div><a href="https://urldefense.com/v3/__https://joachimfranklab.org/Learning_Materials/Meeting_4_CTF/Papers/Erickson*2C*20Klug*20-*201971*20-*20Measurement*20and*20compensation*20of*20defocusing*20and*20aberrations*20by*20Fourier*20processing*20of*20electron*20micrographs.pdf__;JSUlJSUlJSUlJSUlJSUlJSUl!!Mih3wA!Dw68RByX_pnloZ6yxFGYqBPbonySfVLOUqoGIxZeGbk7r0EHDi8D9dy9rSsTIQCeCc-UDF9a7e1RxjknGMUo2R7g3phI65Lp$" target="_blank" rel="noreferrer">https://joachimfranklab.org/Learning_Materials/Meeting_4_CTF/Papers/Erickson%2C%20Klug%20-%201971%20-%20Measurement%20and%20compensation%20of%20defocusing%20and%20aberrations%20by%20Fourier%20processing%20of%20electron%20micrographs.pdf</a></div><div><br></div><div>Cheers,</div><div>Sylvain</div><div><div><div dir="ltr" class="gmail_signature" data-smartmail="gmail_signature"><div dir="ltr"><p style="color:rgb(34,34,34);margin-bottom:0px"><span style="color:rgb(80,80,80);font-family:sans-serif">PhD</span><br>Research Fellow - Microscopy Australia</p><p style="color:rgb(34,34,34);margin-bottom:0px"><b>Monash University</b><br></p><span style="color:rgb(34,34,34)">Ramaciotti Centre for Cryo-EM</span><br style="color:rgb(34,34,34)"><span style="color:rgb(34,34,34)">G</span><span style="color:rgb(34,34,34)">92, 15 Innovation Walk, Clayton Campus</span><br style="color:rgb(34,34,34)"><span style="color:rgb(34,34,34)">Victoria, 3800 Australia</span><p style="color:rgb(80,80,80);font-family:sans-serif;margin-bottom:0px">E: <a href="mailto:sylvain.trepout@monash.edu" style="color:rgb(17,85,204)" target="_blank" rel="noreferrer">sylvain.trepout@monash.edu</a></p></div></div></div><br></div></div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Sun, 22 Oct 2023 at 06:49, Daniel Asarnow <<a href="mailto:asarnow@msg.ucsf.edu" target="_blank" rel="noreferrer">asarnow@msg.ucsf.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"><div dir="ltr"><div>Thanks, Ben. Your point about the density distributions is clear. Even if the distributions are different, though, if we could calibrate (normalize to) a common physical unit then we should be able to calculate meaningful difference maps after resampling, no?<br></div><br><div>I would also appreciate a copy of that article, as you mention it is not readily available online!</div><div><br></div><div>Best,</div><div>-da<br></div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Sat, Oct 21, 2023 at 5:54 AM Benjamin Himes <<a href="mailto:himes.benjamin@gmail.com" target="_blank" rel="noreferrer">himes.benjamin@gmail.com</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>
<div style="display:none;font-size:1px;color:rgb(255,255,255);line-height:1px;height:0px;max-height:0px;opacity:0;overflow:hidden">
Well, if Joachim is adding only 1c, I guess I will perhaps add some crypto coin* One distinction to start with: the amplitude contrast from scattering outside the aperture is distinct from the amplitude contrast generated by using an energy
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<div><div><p class="MsoNormal" style="color:rgb(49,49,49);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:1px;text-decoration:none;margin:0in 0in 8pt;line-height:15.6933px;font-size:0.916667rem;font-family:Calibri,sans-serif;height:auto">Well, if Joachim is adding only 1c, I guess I will perhaps add some crypto coin*<span></span></p><p class="MsoNormal" style="color:rgb(49,49,49);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:1px;text-decoration:none;margin:0in 0in 8pt;line-height:15.6933px;font-size:0.916667rem;font-family:Calibri,sans-serif;height:auto">One distinction to start with: the amplitude contrast from scattering outside the aperture is distinct from the amplitude contrast generated by using an energy filter, as Rasmus mentions. The latter leads to an even more convoluted discussion, and here, I will only refer to aperture contrast.<span></span></p><p class="MsoNormal" style="color:rgb(49,49,49);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:1px;text-decoration:none;margin:0in 0in 8pt;line-height:15.6933px;font-size:0.916667rem;font-family:Calibri,sans-serif;height:auto">I agree with Joachim that a per-element consideration for amplitude contrast is required to consider the topic seriously. We began to explore this in “<i style="font-size:0.916667rem">cryo-TEM simulations of amorphous radiation-sensitive samples using multislice wave propagation, Himes & Grigorieff 2021.”<span> </span></i>A more accurate description of this contrast would almost certainly improve techniques that only require an accurate<span> </span><i style="font-size:0.916667rem">forward model</i>, such as template matching.<span></span></p><p class="MsoNormal" style="color:rgb(49,49,49);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:1px;text-decoration:none;margin:0in 0in 8pt;line-height:15.6933px;font-size:0.916667rem;font-family:Calibri,sans-serif;height:auto">How exactly to handle such information is not so clear for techniques that require a solution to the inverse problem, e.g., cryoEM reconstructions. That is to say, I believe one reason the additional phase shift (or cosine term) for the CTF was adopted is it is the only way, ad hoc as it may be, to account for amplitude contrast in<span> </span><b style="font-size:0.916667rem">linear<span> </span></b>image formation. It would be fine if we found some way to include the quadratic terms in how we relate the Fourier Transform of the experimental image and the Fourier Transform of the object. This was not at all lost on the author’s Marin mentions.<span></span></p><p class="MsoNormal" style="color:rgb(49,49,49);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:1px;text-decoration:none;margin:0in 0in 8pt;line-height:15.6933px;font-size:0.916667rem;font-family:Calibri,sans-serif;height:auto">However, as is often the case, the neat biological paper (1971 paper Marin calls out) was prioritized in publication, while the nuanced theory paper (1973<span> </span><i style="font-size:0.916667rem">“The Fourier Transform of an Electron Micrograph – First Order<span> </span><b style="font-size:0.916667rem">and Second Order</b><span> </span>theory of Image Formation”)<span> </span></i>received little attention<i style="font-size:0.916667rem">**.<span> </span></i>In this paper, Harold shows nicely how second-order (quadratic) terms in the expansion give rise to aperture-based amplitude contrast***.<span> </span><span></span></p><p class="MsoNormal" style="color:rgb(49,49,49);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:1px;text-decoration:none;margin:0in 0in 8pt;line-height:15.6933px;font-size:0.916667rem;font-family:Calibri,sans-serif;height:auto">I’m unsure precisely what Marin means by “<i style="font-size:0.916667rem">In reality, however, the amplitude contrast and phase contrast two separate properties of the complex transmission function of the object, and these are associated with different physical properties</i>.” There is no special magic here; the amplitude contrast (from the aperture losses) is produced by the same phase-object via elastic scattering as the phase contrast. As with many confusing theoretical issues, it is not nature that is confused; it is our math.<span></span></p><p class="MsoNormal" style="color:rgb(49,49,49);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:1px;text-decoration:none;margin:0in 0in 8pt;line-height:15.6933px;font-size:0.916667rem;font-family:Calibri,sans-serif;height:auto">While the discussion on the proper application of amplitude contrast is quite interesting, I’m not sure it gets to the heart of Bernhard’s original question. To paraphrase, “Should one be able to use standard deviations of voxel values to compare different maps?” I’m not sure it should. Consider just the ability to compare two cryoEM maps at a standard isosurface threshold:<span></span></p><p class="MsoNormal" style="color:rgb(49,49,49);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:1px;text-decoration:none;margin:0in 0in 8pt;line-height:15.6933px;font-size:0.916667rem;font-family:Calibri,sans-serif;height:auto">Let’s imagine we’ve sorted out CTF issues and other errors in the inverse image reconstruction problem, so we’ve got a “perfect” reconstructed 3D. What do you expect if you now imagine gradually decreasing the voxel sampling (larger pixel size)? The value in each voxel will tend toward the average value of the map, and you will have an approximately uniform distribution of voxel values. What about the opposite case with finer sampling up until we have on average one atom/voxel? Would the distribution then be Gaussian? Probably not. Would it be comparable between, say a Ribosome (RNA + protein) and Apoferritin (protein)? Almost certainly not as the distribution of atom types; hence, the potential well “seen” by the imaging electrons would not be similar. So, variable sampling rate is one (of several) things that may confound direct comparison of maps of even the same molecule. Even normalizing for this, there is no apparent reason I can see why the distribution should be the same between different molecules, or Gaussian for any.<span></span></p><p class="MsoNormal" style="color:rgb(49,49,49);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:1px;text-decoration:none;margin:0in 0in 8pt;line-height:15.6933px;font-size:11pt;font-family:Calibri,sans-serif;height:auto"><span> </span></p><p class="MsoNormal" style="color:rgb(49,49,49);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:1px;text-decoration:none;margin:0in 0in 8pt;line-height:15.6933px;font-size:0.916667rem;font-family:Calibri,sans-serif;height:auto">*0 cents, eh?<span></span></p><p class="MsoNormal" style="color:rgb(49,49,49);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:1px;text-decoration:none;margin:0in 0in 8pt;line-height:15.6933px;font-size:0.916667rem;font-family:Calibri,sans-serif;height:auto"><i style="font-size:0.916667rem"><span> </span>**</i>I had to email H.P. Erickson years ago to get a copy. If would like a copy, feel free to email me.<i><span></span></i></p><p class="MsoNormal" style="color:rgb(49,49,49);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:1px;text-decoration:none;margin:0in 0in 8pt;line-height:15.6933px;font-size:0.916667rem;font-family:Calibri,sans-serif;height:auto"><i style="font-size:0.916667rem">***</i>Quadratic terms are a minimum. In a full forward simulation, there is no approximation via power-series expansion and<span> </span><i style="font-size:0.916667rem">all</i><span> </span>terms are included in the calculations.</p><p class="MsoNormal" style="color:rgb(49,49,49);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:1px;text-decoration:none;margin:0in 0in 8pt;line-height:15.6933px;font-size:0.916667rem;font-family:Calibri,sans-serif;height:auto"><br></p><p class="MsoNormal" style="color:rgb(49,49,49);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:1px;text-decoration:none;margin:0in 0in 8pt;line-height:15.6933px;font-size:0.916667rem;font-family:Calibri,sans-serif;height:auto" dir="auto">cheers, </p><p class="MsoNormal" style="color:rgb(49,49,49);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:1px;text-decoration:none;margin:0in 0in 8pt;line-height:15.6933px;font-size:0.916667rem;font-family:Calibri,sans-serif;height:auto" dir="auto">ben</p></div><br></div><div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Wed, Oct 18, 2023 at 12:12 PM <<a href="mailto:3dem-request@ncmir.ucsd.edu" target="_blank" rel="noreferrer">3dem-request@ncmir.ucsd.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">Send 3dem mailing list submissions to<br>
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Today's Topics:<br>
<br>
1. Re: [ccpem] Differences EM CP maps vs Xray ED Maps<br>
(Schroeder, Rasmus)<br>
2. Re: [EXTERNAL] Re: [ccpem] Differences EM CP maps vs Xray ED<br>
Maps (Frank, Joachim)<br>
<br>
<br>
----------------------------------------------------------------------<br>
<br>
Message: 1<br>
Date: Wed, 18 Oct 2023 11:00:32 +0200<br>
From: "Schroeder, Rasmus"<br>
<<a href="mailto:rasmus.schroeder@bioquant.uni-heidelberg.de" target="_blank" rel="noreferrer">rasmus.schroeder@bioquant.uni-heidelberg.de</a>><br>
To: Marin van Heel <<a href="mailto:marin.vanheel@googlemail.com" target="_blank" rel="noreferrer">marin.vanheel@googlemail.com</a>><br>
Cc: 3dem <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank" rel="noreferrer">3dem@ncmir.ucsd.edu</a>>, <a href="mailto:CCPEM@jiscmail.ac.uk" target="_blank" rel="noreferrer">CCPEM@jiscmail.ac.uk</a><br>
Subject: Re: [3dem] [ccpem] Differences EM CP maps vs Xray ED Maps<br>
Message-ID:<br>
<<a href="mailto:FE068B80-64AC-40BC-AE88-2E2CB08E5B10@bioquant.uni-heidelberg.de" target="_blank" rel="noreferrer">FE068B80-64AC-40BC-AE88-2E2CB08E5B10@bioquant.uni-heidelberg.de</a>><br>
Content-Type: text/plain; charset="utf-8"<br>
<br>
Dear Marin - and all others here,<br>
<br>
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.<br>
<br>
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 <br>
(Angert et al., Ultramicroscopy 81 (2000) 203-222).<br>
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 ?<br>
(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 ?)<br>
<br>
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 ?.<br>
<br>
My 2 cent ?.<br>
<br>
Best,<br>
<br>
Rasmus<br>
<br>
<br>
> On 15. Oct 2023, at 22:22, Marin van Heel <<a href="mailto:marin.vanheel@googlemail.com" target="_blank" rel="noreferrer">marin.vanheel@googlemail.com</a>> wrote:<br>
> <br>
> <br>
> Dear All,<br>
> <br>
> Unfortunately, a fundamental 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 amplitude contrast of the (stained ) biological object to be proportional to the phase contrast 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 amplitude contrast and phase contrast two separate properties of the complex transmission function 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 zero. Any phase-con<br>
trast image must yield a zero average as it should be for any phase contrast image where what is measured is the difference in phase between any point in the back focal plane of the system with respect to the phase at the origin! That thus means that the phase at the origin must be zero. (Zero being the average density over the image around which the phase information is modulated) . Adding a cosine component to the CTF (a sine) 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.<br>
> <br>
> Two more cents added!<br>
> <br>
> Marin<br>
> <br>
> 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.<br>
> <br>
> van Heel, M. (1978). On the imaging of relatively strong objects in partially coherent illumination in optics and electron optics. Optik. 49, 389?408.<br>
> <br>
> <a href="https://urldefense.com/v3/__https://mail.ncmir.ucsd.edu/pipermail/3dem/2014-July/003454.html__;!!LQC6Cpwp!rgsqbgdMwR2bkbEX4SPCIOwBzw-xYdd2XtYVWF1t9JbG6idon1yxb5mbxQz_t8xhrYiOLnyC5Q8SKNInYo12JnjKiZk$" rel="noreferrer noreferrer" target="_blank">https://mail.ncmir.ucsd.edu/pipermail/3dem/2014-July/003454.html</a><br>
> <br>
> <br>
> <br>
> On Sun, Oct 15, 2023 at 10:01?AM Guillaume Gaullier <<a href="mailto:guillaume.gaullier@kemi.uu.se" target="_blank" rel="noreferrer">guillaume.gaullier@kemi.uu.se</a> <mailto:<a href="mailto:guillaume.gaullier@kemi.uu.se" target="_blank" rel="noreferrer">guillaume.gaullier@kemi.uu.se</a>>> wrote:<br>
>> Hello Bernhard,<br>
>> <br>
>> 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.<br>
>> <br>
>> 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.<br>
>> <br>
>> 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.<br>
>> 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!F167aMQKY0npO0mDM2XnYUJMsI5KHiM2qByj0oICNWHtqYI1ub4ZRnTZOBwdK6UPjLGFDKL4R9ffbkAWKNY1BDEpbgUKIZYrTBIUZbKBW_r9pOE$" rel="noreferrer noreferrer" target="_blank">https://urldefense.com/v3/__https://mail.cgl.ucsf.edu/mailman/archives/list/chimerax-users@cgl.ucsf.edu/thread/3RNL6ODNP6QAL3BTHIPPQS2VE2B2YSVO/__;!!Mih3wA!F167aMQKY0npO0mDM2XnYUJMsI5KHiM2qByj0oICNWHtqYI1ub4ZRnTZOBwdK6UPjLGFDKL4R9ffbkAWKNY1BDEpbgUKIZYrTBIUZbKBW_r9pOE$</a> <<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$" rel="noreferrer noreferrer" target="_blank">https://urldefense.com/v3/__https://mail.cgl.ucsf.edu/mailman/archives/list/chimerax-users@cgl.ucsf.edu/thread/3RNL6ODNP6QAL3BTHIPPQS2VE2B2YSVO/__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbMPdEMJ_w$</a>><br>
>> Maybe there are other reasons adding to this.<br>
>> <br>
>> I hope this helps.<br>
>> Cheers,<br>
>> <br>
>> Guillaume<br>
>> <br>
>> <br>
>>> On 15 Oct 2023, at 13:05, <Nonameavailable> <<a href="mailto:br@RUPPWEB.ORG" target="_blank" rel="noreferrer">br@RUPPWEB.ORG</a> <mailto:<a href="mailto:br@RUPPWEB.ORG" target="_blank" rel="noreferrer">br@RUPPWEB.ORG</a>>> wrote:<br>
>>> <br>
>>> Dear EM Experts,<br>
>>> <br>
>>> 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:<br>
>>> <br>
>>> ?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??<br>
>>> <br>
>>> And here a potential partial answer:<br>
>>> <br>
>>> ?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.?<br>
>>> <br>
>>> What are we missing here?<br>
>>> <br>
>>> Thx, BR<br>
>>> <br>
>>> -----------------------------------------------------------------<br>
>>> Bernhard Rupp (Hofkristallrat a. D)<br>
>>> K.k. Hofkristallamt<br>
>>> CA 92084 San Diego<br>
>>> 001 (925) 209-7429<br>
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>>> All models are wrong but some are useful<br>
>>> -----------------------------------------------------------------<br>
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--<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" rel="noreferrer">rasmus.schroeder@bioquant.uni-heidelberg.de</a><br>
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------------------------------<br>
<br>
Message: 2<br>
Date: Wed, 18 Oct 2023 16:11:41 +0000<br>
From: "Frank, Joachim" <<a href="mailto:jf2192@cumc.columbia.edu" target="_blank" rel="noreferrer">jf2192@cumc.columbia.edu</a>><br>
To: "Schroeder, Rasmus" <<a href="mailto:rasmus.schroeder@bioquant.uni-heidelberg.de" target="_blank" rel="noreferrer">rasmus.schroeder@bioquant.uni-heidelberg.de</a>>,<br>
Marin van Heel <<a href="mailto:marin.vanheel@googlemail.com" target="_blank" rel="noreferrer">marin.vanheel@googlemail.com</a>><br>
Cc: 3dem <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank" rel="noreferrer">3dem@ncmir.ucsd.edu</a>>, "<a href="mailto:CCPEM@jiscmail.ac.uk" target="_blank" rel="noreferrer">CCPEM@jiscmail.ac.uk</a>"<br>
<<a href="mailto:CCPEM@jiscmail.ac.uk" target="_blank" rel="noreferrer">CCPEM@jiscmail.ac.uk</a>><br>
Subject: Re: [3dem] [EXTERNAL] Re: [ccpem] Differences EM CP maps vs<br>
Xray ED Maps<br>
Message-ID:<br>
<<a href="mailto:SA3PR02MB932735E3C16CCEED814C104780D5A@SA3PR02MB9327.namprd02.prod.outlook.com" target="_blank" rel="noreferrer">SA3PR02MB932735E3C16CCEED814C104780D5A@SA3PR02MB9327.namprd02.prod.outlook.com</a>><br>
<br>
Content-Type: text/plain; charset="utf-8"<br>
<br>
<br>
Hi Rasmus,<br>
<br>
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.<br>
<br>
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.<br>
<br>
my 1c,<br>
<br>
--Joachim<br>
<br>
Dr. Joachim Frank<br>
Professor, Biochemistry and Molecular Biophysics & Biological Sciences,<br>
Columbia University Irving Medical Center,<br>
Hammer Health Sciences Center, Room 616,<br>
701 West 168th Street, New York, NY 10032 -- <a href="mailto:jf2192@cumc.columbia.edu" target="_blank" rel="noreferrer">jf2192@cumc.columbia.edu</a><mailto:<a href="mailto:jf2192@cumc.columbia.edu" target="_blank" rel="noreferrer">jf2192@cumc.columbia.edu</a>><br>
2017 Nobel Prize in Chemistry<br>
Admin. Coordinator: Masgan Saidi 917 232 6545 <a href="mailto:ms4597@cumc.columbia.edu" target="_blank" rel="noreferrer">ms4597@cumc.columbia.edu</a><mailto:<a href="mailto:ms4597@cumc.columbia.edu" target="_blank" rel="noreferrer">ms4597@cumc.columbia.edu</a>><br>
Science: <a href="https://urldefense.com/v3/__https://joachimfranklab.org__;!!Mih3wA!Bg_Dt6-kIGGiCCVVKZc1MojKob6HpUPWS1D4XotzwDkNbu_UXbEJZDgQ8OD7NLiLt9z8iFlcSC1ells-0Xn8ptPLhg$" rel="noreferrer noreferrer" target="_blank">https://urldefense.com/v3/__https://joachimfranklab.org__;!!Mih3wA!Bg_Dt6-kIGGiCCVVKZc1MojKob6HpUPWS1D4XotzwDkNbu_UXbEJZDgQ8OD7NLiLt9z8iFlcSC1ells-0Xn8ptPLhg$</a> <<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=" rel="noreferrer noreferrer" target="_blank">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=</a>> -- Fiction: <a href="https://urldefense.com/v3/__http://franxfiction.com__;!!Mih3wA!EvvpD3xjmy_w7Sc7XucWi3zEwyRIrPF390tkFzevMZ5YX13xqAJX1qB1BH4KFkccb63akwrGRoQsxWrPTjoQhPv0NGM$" rel="noreferrer noreferrer" target="_blank">franxfiction.com</a><br>
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<br>
<br>
From: 3dem <<a href="mailto:3dem-bounces@ncmir.ucsd.edu" target="_blank" rel="noreferrer">3dem-bounces@ncmir.ucsd.edu</a>> on behalf of Schroeder, Rasmus <<a href="mailto:rasmus.schroeder@bioquant.uni-heidelberg.de" target="_blank" rel="noreferrer">rasmus.schroeder@bioquant.uni-heidelberg.de</a>><br>
Date: Wednesday, October 18, 2023 at 5:02 AM<br>
To: Marin van Heel <<a href="mailto:marin.vanheel@googlemail.com" target="_blank" rel="noreferrer">marin.vanheel@googlemail.com</a>><br>
Cc: 3dem <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank" rel="noreferrer">3dem@ncmir.ucsd.edu</a>>, <a href="mailto:CCPEM@jiscmail.ac.uk" target="_blank" rel="noreferrer">CCPEM@jiscmail.ac.uk</a> <<a href="mailto:CCPEM@jiscmail.ac.uk" target="_blank" rel="noreferrer">CCPEM@jiscmail.ac.uk</a>><br>
Subject: [EXTERNAL] Re: [3dem] [ccpem] Differences EM CP maps vs Xray ED Maps<br>
Dear Marin - and all others here,<br>
<br>
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.<br>
<br>
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<br>
(Angert et al., Ultramicroscopy 81 (2000) 203-222).<br>
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 ?<br>
(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 ?)<br>
<br>
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 ?.<br>
<br>
My 2 cent ?.<br>
<br>
Best,<br>
<br>
Rasmus<br>
<br>
<br>
<br>
On 15. Oct 2023, at 22:22, Marin van Heel <<a href="mailto:marin.vanheel@googlemail.com" target="_blank" rel="noreferrer">marin.vanheel@googlemail.com</a>> wrote:<br>
<br>
<br>
Dear All,<br>
Unfortunately, a fundamental 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 amplitude contrast of the (stained ) biological object to be proportional to the phase contrast 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 amplitude contrast and phase contrast two separate properties of the complex transmission function 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 zero. Any phase-contr<br>
ast image must yield a zero average as it should be for any phase contrast image where what is measured is the difference in phase between any point in the back focal plane of the system with respect to the phase at the origin! That thus means that the phase at the origin must be zero. (Zero being the average density over the image around which the phase information is modulated) . Adding a cosine component to the CTF (a sine) 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.<br>
Two more cents added!<br>
Marin<br>
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.<br>
van Heel, M. (1978). On the imaging of relatively strong objects in partially coherent illumination in optics and electron optics. Optik. 49, 389?408.<br>
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On Sun, Oct 15, 2023 at 10:01?AM Guillaume Gaullier <<a href="mailto:guillaume.gaullier@kemi.uu.se" target="_blank" rel="noreferrer">guillaume.gaullier@kemi.uu.se</a><mailto:<a href="mailto:guillaume.gaullier@kemi.uu.se" target="_blank" rel="noreferrer">guillaume.gaullier@kemi.uu.se</a>>> wrote:<br>
Hello Bernhard,<br>
<br>
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.<br>
<br>
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.<br>
<br>
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.<br>
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!Bg_Dt6-kIGGiCCVVKZc1MojKob6HpUPWS1D4XotzwDkNbu_UXbEJZDgQ8OD7NLiLt9z8iFlcSC1ells-0Xm3pfu--w$" rel="noreferrer noreferrer" target="_blank">https://urldefense.com/v3/__https://mail.cgl.ucsf.edu/mailman/archives/list/chimerax-users@cgl.ucsf.edu/thread/3RNL6ODNP6QAL3BTHIPPQS2VE2B2YSVO/__;!!Mih3wA!Bg_Dt6-kIGGiCCVVKZc1MojKob6HpUPWS1D4XotzwDkNbu_UXbEJZDgQ8OD7NLiLt9z8iFlcSC1ells-0Xm3pfu--w$</a> <<a href="https://urldefense.com/v3/__https:/mail.cgl.ucsf.edu/mailman/archives/list/chimerax-users@cgl.ucsf.edu/thread/3RNL6ODNP6QAL3BTHIPPQS2VE2B2YSVO/__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbMPdEMJ_w$" rel="noreferrer noreferrer" target="_blank">https://urldefense.com/v3/__https:/mail.cgl.ucsf.edu/mailman/archives/list/chimerax-users@cgl.ucsf.edu/thread/3RNL6ODNP6QAL3BTHIPPQS2VE2B2YSVO/__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbMPdEMJ_w$</a>><br>
Maybe there are other reasons adding to this.<br>
<br>
I hope this helps.<br>
Cheers,<br>
<br>
Guillaume<br>
<br>
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<br>
On 15 Oct 2023, at 13:05, <Nonameavailable> <<a href="mailto:br@RUPPWEB.ORG" target="_blank" rel="noreferrer">br@RUPPWEB.ORG</a><mailto:<a href="mailto:br@RUPPWEB.ORG" target="_blank" rel="noreferrer">br@RUPPWEB.ORG</a>>> wrote:<br>
<br>
Dear EM Experts,<br>
<br>
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:<br>
<br>
?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??<br>
<br>
And here a potential partial answer:<br>
<br>
?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.?<br>
<br>
What are we missing here?<br>
<br>
Thx, BR<br>
<br>
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Rasmus R. Schroeder<br>
<br>
Cryo Electron Microscopy<br>
Heidelberg University / Medical Faculty<br>
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