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Hi Ed, Long time no talk! I started the #Why-o-Why series in Twitter to clarify some basic theoretical cryo-EM principles – in digestible units – to both newcomers and established senior scientists alike! In #Why-o-Why #13, for example, I emphasise the fact that the FRC/FSC (and its thresholds) are not tied to structural biology and also not tied to what you (want to) see in the results. Judging the quality of a metric by whether you see protein side chains is a methodological mistake! It is obviously inappropriate when you want to use your intuitive side-chain metric to judge the “results-resolution” achieved in an X-ray tomographic reconstruction of a human brain, or in the 3D tomographic reconstruction of a computer chip. How would you react when a medic tells you your 3D reconstruction of a ribosome is rubbish because he/she cannot even see the 2mm-sized kidney stones she/he likes to distinguish in the 3D reconstructions – but in your ribosome map! All your FSC examples violate this basic principle and thus do not represent appropriate, clean, scientific counter arguments for any FSC/FRC discussion! Moreover, issues like the alignments of molecular images in the context single-particle cryo-EM analysis also have ABSOLUTELY NOTHING to do with the FRC/FSC criterion for comparing of two images or two 3D densities. Whatever reference bias or symmetry error you chose to impose on the two images / volumes is your own responsibility. As I put it in an earlier internet discussion: “If you overheat your living room, don’t blame your thermometer!” (But you may want to check the functioning of your thermostat!). This statement in its cryo-EM translation: Don’t blame the FSC curve for your own Bias Blunders. In your 2016 paper you introduce the FSC as follows: *“However, the definition of resolution in cryo-EM is a much more nebulous entity than in X-ray crystallography, and the most commonly used method of reporting resolution using a plot of the Fourier Shell Correlation (FSC) is fraught with problems*” Hardly a clean neutral introduction of a scientific topic and certainly a statement that is in flagrant contradiction with the facts (see: #Why-o-Why #13). I know… I am not going to convince you after all these decades of “some people” not willing to read - or listen to clean scientific arguments. I don’t even expect a reaction from you! What I am more worried about is the effect of planting wrong ideas in the minds of a new generation of cryo-EM scientists. So you cryo-EM newcomers out there, do take the time to read some of the basics; and for those not on Twitter I added #Why-o-Why #13, below (@Marin_van_Heel). Cheers, Marin [image: image.png] On Thu, Feb 13, 2020 at 2:48 PM Egelman, Edward H (ehe2n) < [log in to unmask]> wrote: > I had studiously avoided getting involved in this thread which might lead > to a long exchange with Marin about the history, significance and > ontological status of the FSC, but I will suggest a paper that while dated > addresses some of these isssues: > > > > Subramaniam, S., Earl, L.A., Falconieri, V., Milne, J.L., and Egelman, > E.H. (2016). Resolution advances in cryo-EM enable application to drug > discovery. Curr Opin Struct Biol* 41*, 194-202. > > Abstract > > The prospect that the structures of protein assemblies, small and large, > can be determined using cryo-electron microscopy (cryo-EM) is beginning to > transform the landscape of structural biology and cell biology. Great > progress is being made in determining 3D structures of biological > assemblies ranging from icosahedral viruses and helical arrays to small > membrane proteins and protein complexes. Here, we review recent advances in > this field, focusing especially on the emerging use of cryo-EM in mapping > the binding of drugs and inhibitors to protein targets, an application that > requires structure determination at the highest possible resolutions. *We > discuss methods used to evaluate the information contained in cryo-EM > density maps and consider strengths and weaknesses of approaches currently > used to measure map resolution.* > > We discuss that FSC is NOT a measure of resolution, it is a measure of > reproducibility. We give examples for structures with symmetry where one > can impose the wrong symmetry and get a better FSC than with the correct > symmetry (this is also discussed in Egelman, E.H. 2014. Ambiguities in > helical reconstruction. Elife* 3*.). While the map looks like garbage, > the FSC does not care as the garbage is highly reproducible. As to actually > looking at the map, I would agree with previous people who have commented > or implied that the gold standard in x-ray crystallography has always been > the visual appearance and interpretability of the map, and not some single > metric. > > Regards, > > Ed > > > > > > Edward Egelman > > Harrison Distinguished Professor > > Department of Biochemistry and Molecular Genetics > > University of Virginia > > phone: 434-924-8210 > > fax: 434-924-5069 > > [log in to unmask] > > www.people.virginia.edu/~ehe2n > > > > *From: *3dem <[log in to unmask]> on behalf of Pawel Penczek < > [log in to unmask]> > *Date: *Thursday, February 13, 2020 at 8:42 AM > *To: *3dem <[log in to unmask]> > *Subject: *[3dem] Which resolution? > > > > 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 > [log in to unmask] > 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