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

Sun Oct 15 13:22:42 PDT 2023

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.

https://mail.ncmir.ucsd.edu/pipermail/3dem/2014-July/003454.html

On Sun, Oct 15, 2023 at 10:01 AM Guillaume Gaullier <
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!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> 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
> hofkristallamt at gmail.com
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> -----------------------------------------------------------------
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