[3dem] [ccpem] Differences EM CP maps vs Xray ED Maps
Benjamin Himes
himes.benjamin at gmail.com
Mon Oct 23 08:04:13 PDT 2023
Hi Daniel,
I think you are asking a slightly different question. As you mention a
difference map, am I correct to assume it is calculated between two maps of
nominally identical molecules? Perhaps with a different ligand or
conformation?
In that case, yes, this should be just fine. To help account for variable
image processing and data quality artifacts, you might try a program
distributed with cisTEM called “scale_with_mask.” This will normalize the
average intensity in each Fourier shell from one map to the average
intensity in each Fourier shell of another, prior to making a difference
map. Just be sure to normalize to the lower quality map as it sets your
bounds on interpretation : )
Cheers,
Ben
------------------------
Benjamin A. Himes, PhD
Managing Member
Stochastic Analytics, LLC
------------------------
On Sat, Oct 21, 2023 at 3:49 PM 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
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>> 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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>>>
>>> >>> -----------------------------------------------------------------
>>> >>> 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
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>>> 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$
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>>> >
>>>
>>>
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>>> <Screenshot 2023-10-15 at 14.22.31.png>
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>>>
>>>
>>>
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>>>
>>>
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>>> https://urldefense.com/v3/__http:/www.uu.se/om-uu/dataskydd-personuppgifter/__;!!Mih3wA!BGIjNxSHKX_ICKU_6CFanyPV1Mdi8YchWr8HV5aQnLt4dvOJSmWTZyuazSRj4rRmTlL8O2gx5mA3rFLt4AbnqbP9uI9duw$
>>> >
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>>> https://urldefense.com/v3/__http://www.uu.se/en/about-uu/data-protection-policy__;!!Mih3wA!Bg_Dt6-kIGGiCCVVKZc1MojKob6HpUPWS1D4XotzwDkNbu_UXbEJZDgQ8OD7NLiLt9z8iFlcSC1ells-0Xl9jvn8Ww$
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>>> >
>>>
>>> ________________________________
>>>
>>> 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
>>>
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>>> ------------------------------
>>>
>>> Subject: Digest Footer
>>>
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>>>
>>> End of 3dem Digest, Vol 194, Issue 20
>>> *************************************
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