[3dem] [ccpem] Differences EM CP maps vs Xray ED Maps
Daniel Asarnow
asarnow at msg.ucsf.edu
Sat Oct 21 12:48:57 PDT 2023
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?
I would also appreciate a copy of that article, as you mention it is not
readily available online!
Best,
-da
On Sat, Oct 21, 2023 at 5:54 AM Benjamin Himes <himes.benjamin at gmail.com>
wrote:
> 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
> ZjQcmQRYFpfptBannerStart
> This Message Is From an Untrusted Sender
> You have not previously corresponded with this sender.
>
> ZjQcmQRYFpfptBannerEnd
>
> 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 filter, as Rasmus mentions. The latter leads to an even
> more convoluted discussion, and here, I will only refer to aperture
> contrast.
>
> 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 “*cryo-TEM simulations of amorphous radiation-sensitive samples
> using multislice wave propagation, Himes & Grigorieff 2021.” *A more
> accurate description of this contrast would almost certainly improve
> techniques that only require an accurate *forward model*, such as
> template matching.
>
> 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 *linear *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.
>
> 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 *“The Fourier Transform of an Electron Micrograph – First Order and
> Second Order theory of Image Formation”) *received little attention***. *In
> this paper, Harold shows nicely how second-order (quadratic) terms in the
> expansion give rise to aperture-based amplitude contrast***.
>
> I’m unsure precisely what Marin means by “*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*.” 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.
>
> 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:
>
> 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.
>
>
>
> *0 cents, eh?
>
> * ***I had to email H.P. Erickson years ago to get a copy. If would like
> a copy, feel free to email me.
>
> *****Quadratic terms are a minimum. In a full forward simulation, there
> is no approximation via power-series expansion and *all* terms are
> included in the calculations.
>
>
> cheers,
>
> ben
>
>
> On Wed, Oct 18, 2023 at 12:12 PM <3dem-request at ncmir.ucsd.edu> wrote:
>
>> Send 3dem mailing list submissions to
>> 3dem at ncmir.ucsd.edu
>>
>> To subscribe or unsubscribe via the World Wide Web, visit
>> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
>> <https://urldefense.com/v3/__https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem__;!!LQC6Cpwp!rgsqbgdMwR2bkbEX4SPCIOwBzw-xYdd2XtYVWF1t9JbG6idon1yxb5mbxQz_t8xhrYiOLnyC5Q8SKNInYo12SIxjfWw$>
>> or, via email, send a message with subject or body 'help' to
>> 3dem-request at ncmir.ucsd.edu
>>
>> You can reach the person managing the list at
>> 3dem-owner at ncmir.ucsd.edu
>>
>> When replying, please edit your Subject line so it is more specific
>> than "Re: Contents of 3dem digest..."
>>
>>
>> Today's Topics:
>>
>> 1. Re: [ccpem] Differences EM CP maps vs Xray ED Maps
>> (Schroeder, Rasmus)
>> 2. Re: [EXTERNAL] Re: [ccpem] Differences EM CP maps vs Xray ED
>> Maps (Frank, Joachim)
>>
>>
>> ----------------------------------------------------------------------
>>
>> Message: 1
>> Date: Wed, 18 Oct 2023 11:00:32 +0200
>> From: "Schroeder, Rasmus"
>> <rasmus.schroeder at bioquant.uni-heidelberg.de>
>> To: Marin van Heel <marin.vanheel at googlemail.com>
>> Cc: 3dem <3dem at ncmir.ucsd.edu>, CCPEM at jiscmail.ac.uk
>> Subject: Re: [3dem] [ccpem] Differences EM CP maps vs Xray ED Maps
>> Message-ID:
>> <FE068B80-64AC-40BC-AE88-2E2CB08E5B10 at bioquant.uni-heidelberg.de>
>> Content-Type: text/plain; charset="utf-8"
>>
>> Dear Marin - and all others here,
>>
>> 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.
>>
>> 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
>> (Angert et al., Ultramicroscopy 81 (2000) 203-222).
>> 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 ?
>> (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 ?)
>>
>> 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 ?.
>>
>> My 2 cent ?.
>>
>> Best,
>>
>> Rasmus
>>
>>
>> > On 15. Oct 2023, at 22:22, Marin van Heel <marin.vanheel at googlemail.com>
>> wrote:
>> >
>> >
>> > Dear All,
>> >
>> > 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
>> 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.
>> >
>> > Two more cents added!
>> >
>> > Marin
>> >
>> > 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.
>> >
>> > van Heel, M. (1978). On the imaging of relatively strong objects in
>> partially coherent illumination in optics and electron optics. Optik. 49,
>> 389?408.
>> >
>> > https://mail.ncmir.ucsd.edu/pipermail/3dem/2014-July/003454.html
>> <https://urldefense.com/v3/__https://mail.ncmir.ucsd.edu/pipermail/3dem/2014-July/003454.html__;!!LQC6Cpwp!rgsqbgdMwR2bkbEX4SPCIOwBzw-xYdd2XtYVWF1t9JbG6idon1yxb5mbxQz_t8xhrYiOLnyC5Q8SKNInYo12JnjKiZk$>
>> >
>> >
>> >
>> > On Sun, Oct 15, 2023 at 10:01?AM Guillaume Gaullier <
>> guillaume.gaullier at kemi.uu.se <mailto:guillaume.gaullier at kemi.uu.se>>
>> wrote:
>> >> Hello Bernhard,
>> >>
>> >> 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.
>> >>
>> >> 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.
>> >>
>> >> 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.
>> >> At least this is how I understand Tom Goddard?s explanation in this
>> discussion from last month on the chimerax-users list:
>> https://urldefense.com/v3/__https://mail.cgl.ucsf.edu/mailman/archives/list/chimerax-users@cgl.ucsf.edu/thread/3RNL6ODNP6QAL3BTHIPPQS2VE2B2YSVO/__;!!Mih3wA!F167aMQKY0npO0mDM2XnYUJMsI5KHiM2qByj0oICNWHtqYI1ub4ZRnTZOBwdK6UPjLGFDKL4R9ffbkAWKNY1BDEpbgUKIZYrTBIUZbKBW_r9pOE$
>> <
>> https://urldefense.com/v3/__https://mail.cgl.ucsf.edu/mailman/archives/list/chimerax-users@cgl.ucsf.edu/thread/3RNL6ODNP6QAL3BTHIPPQS2VE2B2YSVO/__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbMPdEMJ_w$
>> >
>> >> Maybe there are other reasons adding to this.
>> >>
>> >> I hope this helps.
>> >> Cheers,
>> >>
>> >> Guillaume
>> >>
>> >>
>> >>> On 15 Oct 2023, at 13:05, <Nonameavailable> <br at RUPPWEB.ORG <mailto:
>> br at RUPPWEB.ORG>> wrote:
>> >>>
>> >>> Dear EM Experts,
>> >>>
>> >>> 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:
>> >>>
>> >>> ?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??
>> >>>
>> >>> And here a potential partial answer:
>> >>>
>> >>> ?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.?
>> >>>
>> >>> What are we missing here?
>> >>>
>> >>> Thx, BR
>> >>>
>> >>> -----------------------------------------------------------------
>> >>> Bernhard Rupp (Hofkristallrat a. D)
>> >>> K.k. Hofkristallamt
>> >>> CA 92084 San Diego
>> >>> 001 (925) 209-7429
>> >>> +43 (676) 571-0536
>> >>> br at ruppweb.org <mailto:br at ruppweb.org>
>> >>> hofkristallamt at gmail.com <mailto:hofkristallamt at gmail.com>
>> >>>
>> https://urldefense.com/v3/__http://www.ruppweb.org/__;!!Mih3wA!F167aMQKY0npO0mDM2XnYUJMsI5KHiM2qByj0oICNWHtqYI1ub4ZRnTZOBwdK6UPjLGFDKL4R9ffbkAWKNY1BDEpbgUKIZYrTBIUZbKBEsdGQoE$
>> <
>> https://urldefense.com/v3/__http://www.ruppweb.org/__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbMv8ZhPSQ$>
>>
>> >>> -----------------------------------------------------------------
>> >>> All models are wrong but some are useful
>> >>> -----------------------------------------------------------------
>> >>>
>> >>>
>> >>>
>> >>> To unsubscribe from the CCPEM list, click the following link:
>> >>>
>> https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCPEM&A=1__;!!Mih3wA!F167aMQKY0npO0mDM2XnYUJMsI5KHiM2qByj0oICNWHtqYI1ub4ZRnTZOBwdK6UPjLGFDKL4R9ffbkAWKNY1BDEpbgUKIZYrTBIUZbKBbuni8so$
>> <
>> https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCPEM&A=1__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbPaA6gqVQ$
>> >
>> >>>
>> >>> VARNING: Klicka inte p? l?nkar och ?ppna inte bilagor om du inte
>> k?nner igen avs?ndaren och vet att inneh?llet ?r s?kert.
>> >>> CAUTION: Do not click on links or open attachments unless you
>> recognise the sender and know the content is safe.
>> >>> <xray.png><EM.png>
>> >>
>> >>
>> >> <Screenshot 2023-10-15 at 14.22.31.png>
>> >>
>> >>
>> >>
>> >>
>> >>
>> >>
>> >>
>> >>
>> >> N?r du har kontakt med oss p? Uppsala universitet med e-post s?
>> inneb?r det att vi behandlar dina personuppgifter. F?r att l?sa mer om hur
>> vi g?r det kan du l?sa h?r:
>> https://urldefense.com/v3/__http://www.uu.se/om-uu/dataskydd-personuppgifter/__;!!Mih3wA!F167aMQKY0npO0mDM2XnYUJMsI5KHiM2qByj0oICNWHtqYI1ub4ZRnTZOBwdK6UPjLGFDKL4R9ffbkAWKNY1BDEpbgUKIZYrTBIUZbKBb2TM7vg$
>> <
>> https://urldefense.com/v3/__http://www.uu.se/om-uu/dataskydd-personuppgifter/__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbP9uI9duw$>
>>
>> >>
>> >> E-mailing Uppsala University means that we will process your personal
>> data. For more information on how this is performed, please read here:
>> https://urldefense.com/v3/__http://www.uu.se/en/about-uu/data-protection-policy__;!!Mih3wA!F167aMQKY0npO0mDM2XnYUJMsI5KHiM2qByj0oICNWHtqYI1ub4ZRnTZOBwdK6UPjLGFDKL4R9ffbkAWKNY1BDEpbgUKIZYrTBIUZbKB_zNAqb0$
>> <
>> https://urldefense.com/v3/__http://www.uu.se/en/about-uu/data-protection-policy__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbNcWzUBIQ$
>> >
>> >> To unsubscribe from the CCPEM list, click the following link:
>> >>
>> https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCPEM&A=1__;!!Mih3wA!F167aMQKY0npO0mDM2XnYUJMsI5KHiM2qByj0oICNWHtqYI1ub4ZRnTZOBwdK6UPjLGFDKL4R9ffbkAWKNY1BDEpbgUKIZYrTBIUZbKBbuni8so$
>> <
>> https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCPEM&A=1__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbPaA6gqVQ$
>> >_______________________________________________
>> > 3dem mailing list
>> > 3dem at ncmir.ucsd.edu
>> > https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
>> <https://urldefense.com/v3/__https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem__;!!LQC6Cpwp!rgsqbgdMwR2bkbEX4SPCIOwBzw-xYdd2XtYVWF1t9JbG6idon1yxb5mbxQz_t8xhrYiOLnyC5Q8SKNInYo12SIxjfWw$>
>>
>> --
>> ############################################################
>>
>> Rasmus R. Schroeder
>>
>> Cryo Electron Microscopy
>> Heidelberg University / Medical Faculty
>> BioQuant, Im Neuenheimer Feld 267
>> 69120 Heidelberg, Germany
>>
>> Tel. +49-(0)6221-5451350
>> e-mail rasmus.schroeder at bioquant.uni-heidelberg.de
>>
>> -------------- next part --------------
>> An HTML attachment was scrubbed...
>> URL: <
>> http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20231018/03b69f72/attachment-0001.html
>> <https://urldefense.com/v3/__http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20231018/03b69f72/attachment-0001.html__;!!LQC6Cpwp!rgsqbgdMwR2bkbEX4SPCIOwBzw-xYdd2XtYVWF1t9JbG6idon1yxb5mbxQz_t8xhrYiOLnyC5Q8SKNInYo12KOoVS8c$>
>> >
>>
>> ------------------------------
>>
>> Message: 2
>> Date: Wed, 18 Oct 2023 16:11:41 +0000
>> From: "Frank, Joachim" <jf2192 at cumc.columbia.edu>
>> To: "Schroeder, Rasmus" <rasmus.schroeder at bioquant.uni-heidelberg.de>,
>> Marin van Heel <marin.vanheel at googlemail.com>
>> Cc: 3dem <3dem at ncmir.ucsd.edu>, "CCPEM at jiscmail.ac.uk"
>> <CCPEM at jiscmail.ac.uk>
>> Subject: Re: [3dem] [EXTERNAL] Re: [ccpem] Differences EM CP maps vs
>> Xray ED Maps
>> Message-ID:
>> <
>> SA3PR02MB932735E3C16CCEED814C104780D5A at SA3PR02MB9327.namprd02.prod.outlook.com
>> >
>>
>> Content-Type: text/plain; charset="utf-8"
>>
>>
>> Hi Rasmus,
>>
>> 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.
>>
>> 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.
>>
>> my 1c,
>>
>> --Joachim
>>
>> Dr. Joachim Frank
>> Professor, Biochemistry and Molecular Biophysics & Biological Sciences,
>> Columbia University Irving Medical Center,
>> Hammer Health Sciences Center, Room 616,
>> 701 West 168th Street, New York, NY 10032 -- jf2192 at cumc.columbia.edu
>> <mailto:jf2192 at cumc.columbia.edu>
>> 2017 Nobel Prize in Chemistry
>> Admin. Coordinator: Masgan Saidi 917 232 6545 ms4597 at cumc.columbia.edu
>> <mailto:ms4597 at cumc.columbia.edu>
>> Science:
>> https://urldefense.com/v3/__https://joachimfranklab.org__;!!Mih3wA!Bg_Dt6-kIGGiCCVVKZc1MojKob6HpUPWS1D4XotzwDkNbu_UXbEJZDgQ8OD7NLiLt9z8iFlcSC1ells-0Xn8ptPLhg$
>> <
>> 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=>
>> -- Fiction: franxfiction.com
>> <https://urldefense.com/v3/__http://franxfiction.com__;!!Mih3wA!EvvpD3xjmy_w7Sc7XucWi3zEwyRIrPF390tkFzevMZ5YX13xqAJX1qB1BH4KFkccb63akwrGRoQsxWrPTjoQhPv0NGM$>
>>
>>
>>
>> From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Schroeder, Rasmus <
>> rasmus.schroeder at bioquant.uni-heidelberg.de>
>> Date: Wednesday, October 18, 2023 at 5:02 AM
>> To: Marin van Heel <marin.vanheel at googlemail.com>
>> Cc: 3dem <3dem at ncmir.ucsd.edu>, CCPEM at jiscmail.ac.uk <
>> CCPEM at jiscmail.ac.uk>
>> Subject: [EXTERNAL] Re: [3dem] [ccpem] Differences EM CP maps vs Xray ED
>> Maps
>> Dear Marin - and all others here,
>>
>> 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.
>>
>> 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
>> (Angert et al., Ultramicroscopy 81 (2000) 203-222).
>> 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 ?
>> (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 ?)
>>
>> 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 ?.
>>
>> My 2 cent ?.
>>
>> Best,
>>
>> Rasmus
>>
>>
>>
>> On 15. Oct 2023, at 22:22, Marin van Heel <marin.vanheel at googlemail.com>
>> wrote:
>>
>>
>> Dear All,
>> 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
>> 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.
>> Two more cents added!
>> Marin
>> 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.
>> van Heel, M. (1978). On the imaging of relatively strong objects in
>> partially coherent illumination in optics and electron optics. Optik. 49,
>> 389?408.
>> https://mail.ncmir.ucsd.edu/pipermail/3dem/2014-July/003454.html
>> <https://urldefense.com/v3/__https://mail.ncmir.ucsd.edu/pipermail/3dem/2014-July/003454.html__;!!LQC6Cpwp!rgsqbgdMwR2bkbEX4SPCIOwBzw-xYdd2XtYVWF1t9JbG6idon1yxb5mbxQz_t8xhrYiOLnyC5Q8SKNInYo12JnjKiZk$>
>> <
>> 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=
>> >
>>
>>
>> On Sun, Oct 15, 2023 at 10:01?AM Guillaume Gaullier <
>> guillaume.gaullier at kemi.uu.se<mailto:guillaume.gaullier at kemi.uu.se>>
>> wrote:
>> Hello Bernhard,
>>
>> 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.
>>
>> 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.
>>
>> 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.
>> At least this is how I understand Tom Goddard?s explanation in this
>> discussion from last month on the chimerax-users list:
>> 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$
>> <
>> https://urldefense.com/v3/__https:/mail.cgl.ucsf.edu/mailman/archives/list/chimerax-users@cgl.ucsf.edu/thread/3RNL6ODNP6QAL3BTHIPPQS2VE2B2YSVO/__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbMPdEMJ_w$
>> >
>> Maybe there are other reasons adding to this.
>>
>> I hope this helps.
>> Cheers,
>>
>> Guillaume
>>
>>
>>
>> On 15 Oct 2023, at 13:05, <Nonameavailable> <br at RUPPWEB.ORG<mailto:
>> br at RUPPWEB.ORG>> wrote:
>>
>> Dear EM Experts,
>>
>> 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:
>>
>> ?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??
>>
>> And here a potential partial answer:
>>
>> ?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.?
>>
>> What are we missing here?
>>
>> Thx, BR
>>
>> -----------------------------------------------------------------
>> Bernhard Rupp (Hofkristallrat a. D)
>> K.k. Hofkristallamt
>> CA 92084 San Diego
>> 001 (925) 209-7429
>> +43 (676) 571-0536
>> br at ruppweb.org<mailto:br at ruppweb.org>
>> hofkristallamt at gmail.com<mailto:hofkristallamt at gmail.com>
>>
>> https://urldefense.com/v3/__http://www.ruppweb.org/__;!!Mih3wA!Bg_Dt6-kIGGiCCVVKZc1MojKob6HpUPWS1D4XotzwDkNbu_UXbEJZDgQ8OD7NLiLt9z8iFlcSC1ells-0XmQcgWZfQ$
>> <
>> https://urldefense.com/v3/__http:/www.ruppweb.org/__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbMv8ZhPSQ$
>> >
>> -----------------------------------------------------------------
>> All models are wrong but some are useful
>> -----------------------------------------------------------------
>>
>>
>>
>> ________________________________
>>
>> To unsubscribe from the CCPEM list, click the following link:
>>
>> https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCPEM&A=1__;!!Mih3wA!Bg_Dt6-kIGGiCCVVKZc1MojKob6HpUPWS1D4XotzwDkNbu_UXbEJZDgQ8OD7NLiLt9z8iFlcSC1ells-0XkwAbHvVQ$
>> <
>> https://urldefense.com/v3/__https:/www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCPEM&A=1__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbPaA6gqVQ$
>> >
>>
>>
>> VARNING: Klicka inte p? l?nkar och ?ppna inte bilagor om du inte k?nner
>> igen avs?ndaren och vet att inneh?llet ?r s?kert.
>> CAUTION: Do not click on links or open attachments unless you recognise
>> the sender and know the content is safe.
>> <xray.png><EM.png>
>>
>> <Screenshot 2023-10-15 at 14.22.31.png>
>>
>>
>>
>>
>>
>>
>>
>>
>> N?r du har kontakt med oss p? Uppsala universitet med e-post s? inneb?r
>> det att vi behandlar dina personuppgifter. F?r att l?sa mer om hur vi g?r
>> det kan du l?sa h?r:
>> https://urldefense.com/v3/__http://www.uu.se/om-uu/dataskydd-personuppgifter/__;!!Mih3wA!Bg_Dt6-kIGGiCCVVKZc1MojKob6HpUPWS1D4XotzwDkNbu_UXbEJZDgQ8OD7NLiLt9z8iFlcSC1ells-0Xm4ssdvyA$
>> <
>> https://urldefense.com/v3/__http:/www.uu.se/om-uu/dataskydd-personuppgifter/__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbP9uI9duw$
>> >
>>
>> E-mailing Uppsala University means that we will process your personal
>> data. For more information on how this is performed, please read here:
>> https://urldefense.com/v3/__http://www.uu.se/en/about-uu/data-protection-policy__;!!Mih3wA!Bg_Dt6-kIGGiCCVVKZc1MojKob6HpUPWS1D4XotzwDkNbu_UXbEJZDgQ8OD7NLiLt9z8iFlcSC1ells-0Xl9jvn8Ww$
>> <
>> https://urldefense.com/v3/__http:/www.uu.se/en/about-uu/data-protection-policy__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbNcWzUBIQ$
>> >
>>
>> ________________________________
>>
>> To unsubscribe from the CCPEM list, click the following link:
>>
>> https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCPEM&A=1__;!!Mih3wA!Bg_Dt6-kIGGiCCVVKZc1MojKob6HpUPWS1D4XotzwDkNbu_UXbEJZDgQ8OD7NLiLt9z8iFlcSC1ells-0XkwAbHvVQ$
>> <
>> https://urldefense.com/v3/__https:/www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCPEM&A=1__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbPaA6gqVQ$
>> >
>> _______________________________________________
>> 3dem mailing list
>> 3dem at ncmir.ucsd.edu
>> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
>> <https://urldefense.com/v3/__https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem__;!!LQC6Cpwp!rgsqbgdMwR2bkbEX4SPCIOwBzw-xYdd2XtYVWF1t9JbG6idon1yxb5mbxQz_t8xhrYiOLnyC5Q8SKNInYo12SIxjfWw$>
>> <
>> https://urldefense.proofpoint.com/v2/url?u=https-3A__mail.ncmir.ucsd.edu_mailman_listinfo_3dem&d=DwQFaQ&c=009klHSCxuh5AI1vNQzSO0KGjl4nbi2Q0M1QLJX9BeE&r=wsoQjtpZ1rSBXeWc4Du-lO_6zaO7RA_HHVekUqQDowc&m=v_q3j9BRkOXQXVB_GGY6fjq8tAIBsPrdF8yYMFHB985rPAuC2c6lyIT_OU-2tl6i&s=8bwVfajFx72MhgQgncx08ORiLKAKTeNH2DFBwpAJo9E&e=
>> >
>>
>> --
>> ############################################################
>>
>> Rasmus R. Schroeder
>>
>> Cryo Electron Microscopy
>> Heidelberg University / Medical Faculty
>> BioQuant, Im Neuenheimer Feld 267
>> 69120 Heidelberg, Germany
>>
>> Tel. +49-(0)6221-5451350
>> e-mail rasmus.schroeder at bioquant.uni-heidelberg.de
>>
>> -------------- next part --------------
>> An HTML attachment was scrubbed...
>> URL: <
>> http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20231018/0c3eddf4/attachment.html
>> <https://urldefense.com/v3/__http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20231018/0c3eddf4/attachment.html__;!!LQC6Cpwp!rgsqbgdMwR2bkbEX4SPCIOwBzw-xYdd2XtYVWF1t9JbG6idon1yxb5mbxQz_t8xhrYiOLnyC5Q8SKNInYo12AJUpfWY$>
>> >
>>
>> ------------------------------
>>
>> Subject: Digest Footer
>>
>> _______________________________________________
>> 3dem mailing list
>> 3dem at ncmir.ucsd.edu
>> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
>> <https://urldefense.com/v3/__https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem__;!!LQC6Cpwp!rgsqbgdMwR2bkbEX4SPCIOwBzw-xYdd2XtYVWF1t9JbG6idon1yxb5mbxQz_t8xhrYiOLnyC5Q8SKNInYo12SIxjfWw$>
>>
>>
>> ------------------------------
>>
>> End of 3dem Digest, Vol 194, Issue 20
>> *************************************
>>
> _______________________________________________
> 3dem mailing list
> 3dem at ncmir.ucsd.edu
>
> https://urldefense.com/v3/__https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem__;!!LQC6Cpwp!rgsqbgdMwR2bkbEX4SPCIOwBzw-xYdd2XtYVWF1t9JbG6idon1yxb5mbxQz_t8xhrYiOLnyC5Q8SKNInYo12SIxjfWw$
>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20231021/b6fef80c/attachment-0001.html>
More information about the 3dem
mailing list