[3dem] published B factors

[Alexis Rohou]

Hi Takanori,

Thank you for this clarification - my apologies if I missed this point in
your manuscript. I will correct my table accordingly.

Thanks also for the nice summary of why flattening spectra is likely
incorrect and leads to (slight?) oversharpening. I have not used REFMAC
myself, and I wonder whether you could describe a little more how you
arrived at the blurring factor of 20 Å^2 that you needed to apply in order
to ensure the inputs to REFMAC were as expected. Did you arrive at this
empirically? If so, what criterion were you using to judge whether you had
the correct value? Or perhaps REFMAC has a procedure to check that the
amplitude have the expected profile? My apologies if this is "in the
manual" somewhere!

Thanks again for sharing these details with us!

Cheers,
Alexis

On Sat, Aug 29, 2020 at 11:05 PM Takanori Nakane <tnakane at mrc-lmb.cam.ac.uk>
wrote:

> Hi Alexis,
>
> The B factor of 2.3 is not the value that made the spectrum flat.
> The value estimated by PostProcess was 22.3. However, this was blurred by
> 20
> before input into REFMAC. Thus, 2.3 in the "Refinement" table.
>
> The reason we need some blurring before model refinement is as follows.
>
> Wilson plot for crystallography is calculated after taking the resolution
> dependency of atomic scattering factors into account
> (see capital sigma in
> https://urldefense.com/v3/__https://smb.slac.stanford.edu/facilities/software/ccp4/html/pxmaths/bmg10.html__;!!Mih3wA!VbzHfREaHa5xs1lUjL_JGaOxMgD9UaTjmdYW60M0pH8wqO08R9mny2J8xHO6I1vSIA$
> ).
> This is not done in RELION's PostProcess. Thus, the value estimated by
> RELION "over-compensates"
> intrinsic resolution fall-off due to electron scattering factors, making
> them like
> normalized structure factors E in crystallography, which are not what
> REFMAC expects.
>
> Also note that some residues have lower flexibility than other residues.
> Sharpening them by a "global" B factor can make their ADPs negative.
>
> Best regards,
>
> Takanori Nakane
>
> On 2020/08/30 0:20, Alexis Rohou wrote:
> > Dear colleagues,
> >
> > Thanks to those who replied. Below are a few examples I gathered of
> structures for which both types of B factors were available.
> >
> > Cheers,
> > Alexis
> >
> >
> >
> >
> > B factor (Å^2 )
> >
> >
> >
> > Estimation method
> >
> >
> >
> > Reference
> >
> > In situ bacterial 70S ribosomal subunit at 3.7 Å
> >
> >
> >
> > 5
> >
> >
> >
> > Amplitude spectrum
> >
> >
> >
> > (Tegunov /et al./, 2020) and pers. comm. from D. Tegunov
> >
> > 86
> >
> >
> >
> > ResLog
> >
> > Mouse apoferritin at 1.22 Å
> >
> >
> >
> > 2.3
> >
> >
> >
> > Amplitude spectrum
> >
> >
> >
> > (Nakane /et al./, 2020)
> >
> > 32.5
> >
> >
> >
> > ResLog
> >
> > Urease at 1.98 Å
> >
> >
> >
> > 38
> >
> >
> >
> > Amplitude spectrum
> >
> >
> >
> > (Righetto /et al./, 2020) and pers. comm. from R. Righetto
> >
> > 64.1
> >
> >
> >
> > ResLog
> >
> > Apoferritin at 1.96 Å
> >
> >
> >
> > 50
> >
> >
> >
> > Amplitude spectrum
> >
> >
> >
> > Pers. comm. from R. Righetto
> >
> > 74
> >
> >
> >
> > ResLog
> >
> > Mycobacterial secretion system ESX-3 dimer at 3.8 Å
> >
> >
> >
> > 101
> >
> >
> >
> > Amplitude spectrum
> >
> >
> >
> > (Famelis /et al./, 2019) and pers. comm. from A. Rivera Calzada
> >
> > 144
> >
> >
> >
> > ResLog
> >
> >
> > Famelis, N. /et al./ (2019) ‘Architecture of the mycobacterial type VII
> secretion system.’, /Nature/, 576(7786), pp. 321–325. doi:
> > 10.1038/s41586-019-1633-1.
> >
> > Merk, A. /et al./ (2020) ‘1.8 Å resolution structure of β-galactosidase
> with a 200 kV CRYO ARM electron microscope’, /IUCrJ/. International Union
> of
> > Crystallography, 7(4), pp. 1–5. doi: 10.1107/s2052252520006855.
> >
> > Nakane, T. /et al./ (2020) ‘Single-particle cryo-EM at atomic
> resolution’.
> >
> > Righetto, R. D. /et al./ (2020) ‘High-resolution cryo-EM structure of
> urease from the pathogen Yersinia enterocolitica’, /bioRxiv/, p.
> > 2020.04.28.065599. doi: 10.1101/2020.04.28.065599.
> >
> > Tegunov, D. /et al./ (2020) ‘Multi-particle cryo-EM refinement with M
> visualizes ribosome-antibiotic complex at 3 . 7 Å inside cells’.
> >
> >
> >
> >
> > On Sun, Jul 5, 2020 at 11:49 AM Alexis Rohou <a.rohou at gmail.com <mailto:
> a.rohou at gmail.com>> wrote:
> >
> >     Dear colleagues,
> >
> >     If you know of a publication that includes, for the same dataset,
> both:
> >
> >     (1) an estimate of the "sharpening" B factor (i.e. based on the
> radial amplitude spectrum of a 3D reconstruction) as well as
> >     (2) the B factor estimated from a plot of resolution as a function
> of number of particles (also known as ResLog plot),
> >
> >     ... would you please point me to relevant citation(s)?
> >
> >     Thanks!
> >     Alexis
> >
> >     PS. Feel free to reply to me directly if you prefer - I can
> summarize findings to the list in a few days
> >
> >
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