[3dem] [ccp4bb] Review: Linearity and Resolution in X-Ray Crystallography and Electron Microscopy

Mon Oct 14 13:12:11 PDT 2024

Dear All,

To be honest, I already expected that my message on *Linearity and
Resolution* would not be well understood by especially the younger
generation of researchers in the X-ray crystallography (*XRC*) community. I
intend to make a special entry in my *Why-o-Why* series (moved from Twitter
to LinkedIn) to explain, using simple visual examples, why XRC is *not a
linear technique* and why such criteria like *Abbe's resolution metric* for
microscopes simply does not apply to *XRC*. In *XRC* there is no such thing
as an “*instrumental resolution*” that applies to *any sample* one mounts
in the diffractometer. Therefore the “*results resolution*” cannot be
directly coupled to *measured data*.

Much of what is covered in our review ( https://urldefense.com/v3/__https://osf.io/preprints/osf/5empt__;!!Mih3wA!FVNU4WysI0Eg7Ul5pEBJ8Z92lOWymfr05kCOZkOrOAKkWmdr13GZ-lkp2RkBzGFal7c-nFviBe_ziO1L67ZZPZow0w$ 
) was presented at a meeting in the “*Flat Iron Institute*” in New York in
2019 but the lecture got a bit lost because of the Pandemic. It is 5 years
old but little has changed and issues like “*instrumental resolution*”
and “*results
resolution*” are covered extensively:

https://urldefense.com/v3/__https://www.youtube.com/watch?v=ElWsgWfxp8I__;!!Mih3wA!FVNU4WysI0Eg7Ul5pEBJ8Z92lOWymfr05kCOZkOrOAKkWmdr13GZ-lkp2RkBzGFal7c-nFviBe_ziO1L67aX55KhcQ$ 

Why-O-why link:

https://urldefense.com/v3/__https://www.linkedin.com/posts/marin-van-heel-5845b422b_whyowhyarchive-activity-7149738255154946048-Oc93?utm_source=share&utm_medium=member_desktop__;!!Mih3wA!FVNU4WysI0Eg7Ul5pEBJ8Z92lOWymfr05kCOZkOrOAKkWmdr13GZ-lkp2RkBzGFal7c-nFviBe_ziO1L67aG-3lCZg$ 

Have fun,

Marin

*PS: I had an excellent teacher in understanding imaging:*

*(Sir William Bragg's book from my dad’s library I still use for teaching
and is sometimes still available in second-hand sites)*
[image: image.png]

On Mon, Oct 7, 2024 at 8:02 PM Marin van Heel <marin.vanheel at gmail.com>
wrote:

> Dear Marius Schmidt
>
> In my (our) *original FRC/FSC papers* (1982; 1986 ; 2000; 2004; 2017;
> 2020; 2024) the linearity of these correlation functions/metrics  have been
> extensively discussed. Historically, EM started at a low resolution
> "blobology" level whereas X-ray crystallography (XRC) at that time, already
> had reached atomic resolution. This led to the belief that the *XRC
> resolution metrics* ( like phase residuals and R-factors) were also
> appropriate as *resolution metrics for EM*. However, in XRC the
> measurables are *diffraction patterns* for which *amplitudes *corresponding
> *phases *had to be derived *iteratively*. In EM and in imagining in
> general, the measurables are the images themselves, that contain both the *amplitude
> information *and the *phase information*. To revert to the then already
> established *XRC resolution metrics* like phase residuals or R-factors,
> implied *discarding *the most important part of the available information
> (see the Why-O-Why ).
> (
> https://urldefense.com/v3/__https://www.linkedin.com/posts/marin-van-heel-5845b422b_whyowhyarchive-activity-7149738255154946048-Oc93/?utm_source=share&utm_medium=member_desktop__;!!Mih3wA!FVNU4WysI0Eg7Ul5pEBJ8Z92lOWymfr05kCOZkOrOAKkWmdr13GZ-lkp2RkBzGFal7c-nFviBe_ziO1L67Zz9YEvEw$ 
> ).
> That problem was realized soon and the mentioned *FRC and FSC metrics*
> were thus suggested which exploit all the available information. Thus, the *XRC
> atomic resolution* *technique *of the 1980s came with a *low-quality
> resolution metric* whereas the *Cryo-EM low-resolution blobology *approach
> of the 1980s came with a  *high-quality resolution metric*.
> Thus, in summary, *all resolution criteria in XRC* are *ad-hoc non-linear
> metrics* that have no general validity outside of *XRC*. Looking at only
> the amplitudes of a diffraction pattern is like finding the highest
> resolution spot in a diffraction pattern, where, even if the spot is
> clearly visible, that does not mean one would be able to find its phase. We
> need a more comprehensive metric that has a wide range of applicability.
> In other words, where a CC1-2 metric cannot be applied to assess the 3D
> brain scan of a brain-tumor patient, the FRC / FSC, and the newest FRI /
> FSI metrics can be applied in all cases
> where 2D and 3D data are dealt with!
>
> Hope this helps,
>
> Marin van Heel
>
> On Mon, Oct 7, 2024 at 3:04 PM Marius Schmidt <smarius at uwm.edu> wrote:
>
>> I think this is taken care of:
>> The CC1/2 and the CC1/2* are appropriate metrics for the resolution limit.
>> They are all spit out by newer data processing software.
>> The CC1/2 is directly comparable to the FSC. Many people use CC1/2 = 1/e
>> as
>> the resolution limit.
>> In many cases of data the CC1/2 = 1/e is equivalent to I/sigI of 1, which
>> is used sometimes as a metric for the resolution limit (some use I/sigI =
>> 2),
>> and in more cases the CC1/2 corresponds to Rmerge in the range of 40%.
>> For serial crystallography, the R-split goes through the roof at CC1/2 =
>> 1/e,
>> so the CC1/2 is the better metric.
>>
>> Best
>> Marius
>>
>>
>>
>>
>>
>> Marius Schmidt, Dr. rer. Nat. (habil.)
>> Professor
>> University of Wisconsin-Milwaukee
>> Kenwood Interdisciplinary Research Complex
>> Physics Department, Room 3087
>> 3135 North Maryland Avenue
>> Milwaukee, Wi 53211
>> phone (office): 1-414-229-4338
>> phone (lab): 414-229-3946
>> email: smarius at uwm.edu
>> https://urldefense.com/v3/__https://uwm.edu/physics/people/schmidt-marius/__;!!Mih3wA!FVNU4WysI0Eg7Ul5pEBJ8Z92lOWymfr05kCOZkOrOAKkWmdr13GZ-lkp2RkBzGFal7c-nFviBe_ziO1L67bNpWhOEw$ 
>> https://urldefense.com/v3/__https://sites.uwm.edu/smarius/__;!!Mih3wA!FVNU4WysI0Eg7Ul5pEBJ8Z92lOWymfr05kCOZkOrOAKkWmdr13GZ-lkp2RkBzGFal7c-nFviBe_ziO1L67ZOMxAY2Q$ 
>> https://urldefense.com/v3/__https://www.bioxfel.org/__;!!Mih3wA!FVNU4WysI0Eg7Ul5pEBJ8Z92lOWymfr05kCOZkOrOAKkWmdr13GZ-lkp2RkBzGFal7c-nFviBe_ziO1L67ZRzp61sQ$ 
>> Nature News and Views: https://urldefense.com/v3/__https://www.nature.com/articles/d41586-023-00504-4__;!!Mih3wA!FVNU4WysI0Eg7Ul5pEBJ8Z92lOWymfr05kCOZkOrOAKkWmdr13GZ-lkp2RkBzGFal7c-nFviBe_ziO1L67YIuM-jzg$ 
>>
>> ------------------------------
>> *From:* CCP4 bulletin board <CCP4BB at JISCMAIL.AC.UK> on behalf of Marin
>> van Heel <marin.vanheel at GMAIL.COM>
>> *Sent:* Monday, October 7, 2024 11:24 AM
>> *To:* CCP4BB at JISCMAIL.AC.UK <CCP4BB at JISCMAIL.AC.UK>
>> *Subject:* [ccp4bb] Review: Linearity and Resolution in X-Ray
>> Crystallography and Electron Microscopy
>>
>>
>> Dear All,
>>
>> Sayan Bhakta and I have recently posted the preprint of a review on
>> resolution and linearity which will appear in a book to be launched on the
>> 16th of October 2024.
>> ( https://urldefense.com/v3/__https://doi.org/10.1201/9781003326106__;!!Mih3wA!FVNU4WysI0Eg7Ul5pEBJ8Z92lOWymfr05kCOZkOrOAKkWmdr13GZ-lkp2RkBzGFal7c-nFviBe_ziO1L67aqgSDLLw$  ).
>> It is the first Cryo-EM review that I have been involved in for 25 years.
>> In our preparation, I was quite amazed about what other authors wrote (or
>> did not write) in their many reviews on these matters.
>> For example, I missed any serious discussion about resolution metrics in
>> X-ray crystallography, which technique is fundamentally non-linear.
>> Linearity is a prerequisite for defining the resolution of any
>> instrument. The iterative refinements applied in X-ray crystallography (and
>> sometimes Cryo-EM) makes that all Phase-residuals and R-factors or fixed
>> threshold values cannot be used to compare the results of independently
>> conducted experiments. What is an obvious consequence of the lack of
>> universality of such metrics like phase-residuals and R-factors, is that
>> they cannot be used outside of the immediate context in which they were
>> defined, like X-ray crystallography or structural biology.  In contrast,
>> the Fourier-Ring-Correlation (FRC); Fourier-Shell-Correlation (FSC) and
>> their recent successors: the Fourier-Ring-Information (FRI) and the
>> Fourier-Shell-Information (FSI), plus their integrated versions, are
>> universal metrics that are applicable to all fields of science where 2D and
>> 3D data are dealt with!
>>
>> https://urldefense.com/v3/__https://doi.org/10.31219/osf.io/5empt__;!!Mih3wA!FVNU4WysI0Eg7Ul5pEBJ8Z92lOWymfr05kCOZkOrOAKkWmdr13GZ-lkp2RkBzGFal7c-nFviBe_ziO1L67Yy7wSwrA$ 
>>
>> Have fun reading it!
>>
>> Marin
>>
>>
>>
>>
>>
>> ------------------------------
>>
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>>
>
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