Dear John, I really like your lecture notes! My Imperial/Leiden lecture notes – currently being updated – look a lot like yours. The earlier version:
(https://www.single-particles.org/methodology/MvH_Phase_Contrast.pdf) And you are fully correct: “resolution of cryoEM imaging varies locally”! That is exactly the point! The existing cryo-EM dogmas, however, PREVENT you from doing things right, which seriously hampers progress (see our BioRxiv paper: https://www.biorxiv.org/content/10.1101/224402v1 ). Cheers, Marin On Tue, Feb 18, 2020 at 4:36 AM John R Helliwell <[email protected]> wrote: > Hi Colin, > Yes I agree, see eg page 7 of > https://www2.physics.ox.ac.uk/sites/default/files/2011-06-08/optics_notes_and_slides_part_5_pdf_63907.pdf > (and > there maybe better weblinks). > > The resolution of cryoEM imaging varies locally and so the “local scaling > in a complex way” is what we have to get into in practice...... > > Greetings, > John > > Emeritus Professor John R Helliwell DSc > > > > On 17 Feb 2020, at 21:57, Nave, Colin (DLSLtd,RAL,LSCI) < > [email protected]> wrote: > > > > Hi John > > I agree that neutrons have a role to increase the contrast for certain > atoms. The “water window” for x-ray imaging also fulfils a similar role. > The “locally scaled in a complex way” is a bit beyond me. > > > > The relationship between “ diffraction” errors and “imaging” errors is > based on Parseval’s theorem applied to the errors for electron densities > and structure factors. See for example > https://www-structmed.cimr.cam.ac.uk/Course/Fourier/Fourier.html and > scroll down to Parseval’s theorem. Admittedly not a primary reference but I > think Randy (and Parseval, not to be confused with Wagner’s opera), are > unlikely to have got it wrong. > > > > Imaging (with both electrons and x-rays) can be lensless (as in MX, CDI > and variants) or with an objective lens (electron microscopes have nice > objective lenses). The physical processes are the same up to any lens but > MX, CDI etc. use a computer to replace the lens. The computer algorithm > might be imperfect resulting in visible termination errors. With a decent > lens, one can also see diffraction ripples (round bright stars in a > telescope image) due to the restricted lens aperture. > > > > Good debate though. > > > > Colin > > *From:* John R Helliwell <[email protected]> > *Sent:* 17 February 2020 16:36 > *To:* Nave, Colin (DLSLtd,RAL,LSCI) <[email protected]> > *Cc:* [email protected] > *Subject:* Re: [ccp4bb] FW: [ccp4bb] [3dem] Which resolution? > > > > Hi Colin, > > Neutrons are applied to the uranyl hydrides so as to make their scattering > lengths much more equal than with X-rays, and so side step ripple effects > of the uranium in the Xray case, which obscures those nearby hydrogens. > > In terms of feature resolvability the email exchange (and there may be > better ones):- > http://www.phenix-online.org/pipermail/phenixbb/2017-March/023326.html > > refers to “locally scaled in a complex way”. So, is the physics of the > visibility of features really comparable between the two methods of cryoEM > and crystal structure analysis? > > Greetings, > > John > > Emeritus Professor John R Helliwell DSc > > > > > > > > On 17 Feb 2020, at 13:59, Nave, Colin (DLSLtd,RAL,LSCI) < > [email protected]> wrote: > > > > Hi John > > I agree that if I truncate the data at a high information content > threshold (e.g. 2 bits) series termination errors might hide the lighter > atoms (e.g. the hydrogens in uranium hydride crystal structures). However, > I think this is purely a limitation of producing electron density maps via > Fourier transforms (i.e. not the physics). A variety of techniques are > available for handling series termination including ones which are > maximally non-committal with respect to the missing data. The issue is > still there in some fields (see > https://onlinelibrary.wiley.com/iucr/itc/Ha/ch4o8v0001/ ). For protein > crystallography perhaps series termination errors have become less > important as people are discouraged from applying some I/sigI type cut off. > > > > Cheers > > Colin > > > > > > > > *From:* John R Helliwell <[email protected]> > *Sent:* 17 February 2020 12:09 > *To:* Nave, Colin (DLSLtd,RAL,LSCI) <[email protected]> > *Subject:* Re: [ccp4bb] FW: [ccp4bb] [3dem] Which resolution? > > > > Hi Colin, > > I think the physics of the imaging and the crystal structure analysis, > respectively without and with Fourier termination ripples, are different. > For the MX re Fourier series for two types of difference map see our > contribution:- > > > > http://scripts.iucr.org/cgi-bin/paper?S0907444903004219 > > > > Greetings, > > John > > > > Emeritus Professor John R Helliwell DSc > > > https://www.crcpress.com/The-Whats-of-a-Scientific-Life/Helliwell/p/book/9780367233020 > > > > > > > > > On 17 Feb 2020, at 11:26, "[email protected]" < > [email protected] wrote: > > > > > > Dear all. > > Would it help to separate out the issue of the FSC from the value of the > threshold? My understanding is that the FSC addresses the spatial > frequency at which there is a reliable information content in the image. > This concept should apply to a wide variety of types of image. The issue is > then what value of the threshold to use. For interpretation of protein > structures (whether by x-ray or electron microscopy), a half bit threshold > appears to be appropriate. However, for imaging the human brain (one of > Marin’s examples) a higher threshold might be adopted as a range of > contrasts might be present (axons for example have a similar density to the > surroundings). For crystallography, if one wants to see lighter atoms > (hydrogens in the presence of uranium or in proteins) a higher threshold > might also be appropriate. I am not sure about this to be honest as a 2 bit > threshold (for example) would mean that there is information to higher > resolution at a threshold of a half bit (unless one is at a diffraction or > instrument limited resolution). > > > > Most CCP4BBers will understand that a single number is not good enough. > However, many users of the protein structure databases will simply search > for the structure with the highest named resolution. It might be difficult > to send these users to re-education camps. > > > > Regards > > Colin > > > > *From:* CCP4 bulletin board <[email protected]> *On Behalf Of *Petrus > Zwart > *Sent:* 16 February 2020 21:50 > *To:* [email protected] > *Subject:* Re: [ccp4bb] [3dem] Which resolution? > > > > Hi All, > > > > How is the 'correct' resolution estimation related to the estimated error > on some observed hydrogen bond length of interest, or an error on the > estimated occupancy of a ligand or conformation or anything else that has > structural significance? > > > > In crystallography, it isn't really (only in some very approximate > fashion), and I doubt that in EM there is something to that effect. If you > want to use the resolution to get a gut feeling on how your maps look and > how your data behaves, it doesn't really matter what standard you use, as > long as you are consistent in the use of the metric you use. If you want to > use this estimate to get to uncertainties of model parameters, you better > try something else. > > > > Regards > > Peter Zwart > > > > > > > > On Sun, Feb 16, 2020 at 8:38 AM Marin van Heel < > [email protected]> wrote: > > Dear Pawel and All others .... > > This 2010 review is - unfortunately - largely based on the flawed > statistics I mentioned before, namely on the a priori assumption that the > inner product of a signal vector and a noise vector are ZERO (an > orthogonality assumption). The (Frank & Al-Ali 1975) paper we have refuted > on a number of occasions (for example in 2005, and most recently in our > BioRxiv paper) but you still take that as the correct relation between SNR > and FRC (and you never cite the criticism...). > > Sorry > > Marin > > > > On Thu, Feb 13, 2020 at 10:42 AM Penczek, Pawel A < > [email protected]> wrote: > > Dear Teige, > > > > I am wondering whether you are familiar with > Resolution measures in molecular electron microscopy. > > Penczek PA. Methods Enzymol. 2010. > Citation > > Methods Enzymol. 2010;482:73-100. doi: 10.1016/S0076-6879(10)82003-8. > > > > You will find there answers to all questions you asked and much more. > > > > Regards, > > Pawel Penczek > > > > Regards, > > Pawel > > _______________________________________________ > 3dem mailing list > [email protected] > https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > > > > -- > > ------------------------------------------------------------------------ > P.H. 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