[3dem] [EXTERNAL] Re: [ccpem] Differences EM CP maps vs Xray ED Maps

Wed Oct 18 09:11:41 PDT 2023

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

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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-contrast 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.
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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

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Rasmus R. Schroeder

Cryo Electron Microscopy
Heidelberg University / Medical Faculty
BioQuant, Im Neuenheimer Feld 267
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Tel. +49-(0)6221-5451350
e-mail  rasmus.schroeder at bioquant.uni-heidelberg.de

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