[3dem] [ccpem] Differences EM CP maps vs Xray ED Maps
Sylvain Trepout
sylvain.trepout at monash.edu
Mon Oct 23 02:10:27 PDT 2023
Me again,
As some of you might have already noticed, the link I sent was for the 1971
paper and not the 1973.
So please, contact Ben 😉
Cheers,
Sylvain
PhD
Research Fellow - Microscopy Australia
Monash University
Ramaciotti Centre for Cryo-EM
G92, 15 Innovation Walk, Clayton Campus
Victoria, 3800 Australia
On Mon, 23 Oct 2023, 1:01 pm Sylvain Trepout, <sylvain.trepout at monash.edu>
wrote:
> Hi there,
>
> For those who want access to the article, it is available on Joachim's
> website:
>
>
> 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$
>
> Cheers,
> Sylvain
>
> PhD
> Research Fellow - Microscopy Australia
>
> *Monash University*
> Ramaciotti Centre for Cryo-EM
> G92, 15 Innovation Walk, Clayton Campus
> Victoria, 3800 Australia
>
> E: sylvain.trepout at monash.edu
>
>
> On Sun, 22 Oct 2023 at 06:49, Daniel Asarnow <asarnow at msg.ucsf.edu> wrote:
>
>> 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:
>>>
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>>>> 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>
>>>> >>>
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>>>> <
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>>>>
>>>> >>> -----------------------------------------------------------------
>>>> >>> All models are wrong but some are useful
>>>> >>> -----------------------------------------------------------------
>>>> >>>
>>>> >>>
>>>> >>>
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>>>> >_______________________________________________
>>>> > 3dem mailing list
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>>>>
>>>> --
>>>> ############################################################
>>>>
>>>> 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
>>>>
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>>>> >
>>>>
>>>> ------------------------------
>>>>
>>>> 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
>>>> -----------------------------------------------------------------
>>>>
>>>>
>>>>
>>>> ________________________________
>>>>
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>>>>
>>>> <Screenshot 2023-10-15 at 14.22.31.png>
>>>>
>>>>
>>>>
>>>>
>>>>
>>>>
>>>>
>>>>
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>>>> >
>>>>
>>>> --
>>>> ############################################################
>>>>
>>>> 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
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