[3dem] published B factors
Takanori Nakane
tnakane at mrc-lmb.cam.ac.uk
Sun Aug 30 15:34:07 PDT 2020
Hi,
> 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!
This is purely empirical. As long as we blur enough, the exact value does not really matter.
It simply shifts all refined ADPs. Of course the map can become dull, but the information
content does not change.
Best regards,
Takanori Nakane
> 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 <mailto: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> <mailto: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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