Hi James, Tristan and ccp4 If the meeting is diffraction methods in structural biology then there would be plenty to discuss. My points are probably more relevant to other methods but cover areas raised in the discussion.
1. Electron cryo-tomography. My understanding is that if you are trying to image protein molecules within cells one can collect more particles even though the cells themselves are different. In principle, the signal from each protein molecule is not affected by the surrounding material as each element adds coherently. For tomography one fractionates the dose (Hoppe, W. & Hegerl, R. (1981). Ultramicroscopy. 6, 205-206) to get some 3D information. The signal from each protein for the same dose should be the same as for more normal single particle em. If one is interested in membrane protein the problem of alignment is reduced somewhat as 2 of the orientation parameters are at least partly determined. Admittedly one has to identify the protein. This is easier for larger proteins as shown by the work on the nuclear pore complex. My view is that these techniques will progress with time to smaller protein molecules once the technical challenges are overcome. 2. The complementary nature of electron microscopy and crystallography regarding floppy domains is a good point. For disorder on a scale less than a full domain the issue is what is realistic and representative. The crystal environment can force sidechains, loops, chain termini to have a particular position. I seem to remember James showing various structures of the same protein (lysozyme?) linked together to give a rather nice molecular dynamics type movie. The implication is that each structure selects one of the conformations which naturally occur in solution and one could use the movie to compare with molecular dynamics , though judging which of these is most realistic is another matter. Cheers Colin -----Original Message----- From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Tristan Croll Sent: 17 July 2019 09:19 To: ccp4bb <ccp4bb@jiscmail.ac.uk> Subject: Re: [ccp4bb] challenges in structural biology Hi Radu, Barring some truly spectacular advances, I think that crystallography is going to have a major role to play for a long time yet. Looking at single-particle cryoEM, for almost every target apart from the few ultra-rigid "rocks" the reconstruction will have a wide range of resolutions from (near)atomic in the rigid core to fuzzy blobs out on the floppy exterior. Yes, modern focused reconstruction techniques are improving upon this all the time, but there will be limits - and the problem is much more serious when it comes to electron cryo-tomography methods where you can't simply collect more particles! I think there's some lovely potential complementarity between cryo-EM and crystallography here: almost by definition, these peripheral mobile domains tend to be things that fold independently of the main body of the complex - so why not express them independently and see if they crystallise? That way you get the best of both worlds: the initial cryo-EM reconstruction allows some informed decision making on what construct(s) is/are likely to reliably crystallise, crystallisation of those domains gives you the atomic-resolution description you need, and modelling these back into the cryo-EM map allows you to study them in a more natural context. To bring things back to the original topic, I guess that's what I'd like to see more of: rather than seeing the methods as in fundamental competition, where are the *complementarities* between crystallography and newer techniques? Best regards, Tristan On 2019-07-17 08:43, r...@mrc-lmb.cam.ac.uk wrote: > Hi Both, > > I am not questioning the PDB stats, the issue was whether (crystal) > structures are sufficiently relevant to address biological questions > and justify the resources. Fragment screening is one example where > investment in protein crystallography can still be justified (for > now). But it doesn't really ask or answer biological questions... for > these, whether we like it or not, macromolecular crystallography (or > NMR, even in cell) cannot be the future. In my opinion :-) > > Best wishes, > > Radu > > >> Stating the crystallography is dead might be a bit premature, it is >> still king for depositions. >> >> >> >> In 2017 we had a large number of fragment screening experiments >> deposited. >> >> >> >> >> >> >> >> From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Nukri >> Sanishvili >> Sent: 15 July 2019 23:09 >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: Re: [ccp4bb] challenges in structural biology >> >> >> >> I know it is going to hijack the original topic but I could not >> help... >> >> >> >> “The reports of death of (macromolecular) crystallography are >> greatly exaggerated. >> >> If we believed the prognosticators, it has been dead since the 80s >> when some folks made the claim that the only relevant structures were >> those solved by NMR. >> >> I think we've done quite well since then... >> >> Best, >> >> Nukri >> >> >> >> On Mon, Jul 15, 2019 at 3:45 PM <r...@mrc-lmb.cam.ac.uk >> <mailto:r...@mrc-lmb.cam.ac.uk> > wrote: >> >> Hi Tassos, Tim, >> >> I wonder why would you or anyone on this list worry whether >> biological questions that can be asked and answered with structures >> are relevant to justify the resources? I think there is abundant >> evidence that this is the case. Unless your point is that >> crystallography is now dead for all practical purposes... then yes, I >> fully agree :-) It would however be wrong to erase its historical >> contribution to understanding biology. >> >> Best wishes, >> >> Radu >> >> >>> I would wonder more if the biological questions you can *ask* with a >>> (crystal) >>> structure are sufficiently relevant to justify the resources. >>> >>> Sent from my iPhone >>> >>>> On 15 Jul 2019, at 22:08, Tim Grüne <tim.gru...@univie.ac.at >>>> <mailto:tim.gru...@univie.ac.at> > wrote: >>>> >>>> Dear James, >>>> >>>> 10) are the biological questions that you can answer with a >>>> (crystal) >>>> structure sufficiently relevant to justify the resources? >>>> >>>> Best, >>>> Tim >>>> >>>> >>>> >>>> Am 15.07.2019 21:44, schrieb Holton, James M: >>>>> Hello folks, >>>>> I have the distinct honor of chairing the next Gordon Research >>>>> Conference on Diffraction Methods in Structural Biology (July >>>>> 26-31 2020). This meeting will focus on the biggest challenges >>>>> currently faced by structural biologists, and I mean actual >>>>> real-world challenges. As much as possible, these challenges will >>>>> take the form of friendly competitions with defined parameters, >>>>> data, a scoring system, and "winners", to be established along >>>>> with other unpublished results only at the meeting, as is >>>>> tradition at GRCs. >>>>> But what are the principle challenges in biological structure >>>>> determination today? I of course have my own ideas, but I feel >>>>> like I'm forgetting something. Obvious choices are: >>>>> 1) getting crystals to diffract better >>>>> 2) building models into low-resolution maps (after failing at #1) >>>>> 3) telling if a ligand is really there or not >>>>> 4) the phase problem (dealing with weak signal, twinning and >>>>> pseudotranslation) >>>>> 5) what does "resolution" really mean? >>>>> 6) why are macromolecular R factors so much higher than >>>>> small-molecule ones? >>>>> 7) what is the best way to process serial crystallography data? >>>>> 8) how should one deal with non-isomorphism in multi-crystal >>>>> methods? >>>>> 9) what is the "structure" of something that won't sit still? >>>>> What am I missing? Is industry facing different problems than >>>>> academics? Are there specific challenges facing electron-based >>>>> techniques? If so, could the combined strength of all the world's >>>>> methods developers solve them? I'm interested in hearing the >>>>> voice of this community. On or off-list is fine. >>>>> -James Holton >>>>> MAD Scientist >>>>> ################################################################## >>>>> ###### To unsubscribe from the CCP4BB list, click the following >>>>> link: >>>>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB >>>>> <https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1> >>>>> &A=1 >>>> >>>> -- >>>> -- >>>> Tim Gruene >>>> Head of the Centre for X-ray Structure Analysis Faculty of >>>> Chemistry University of Vienna >>>> >>>> Phone: +43-1-4277-70202 >>>> >>>> GPG Key ID = A46BEE1A >>>> >>>> ################################################################### >>>> ##### >>>> >>>> To unsubscribe from the CCP4BB list, click the following link: >>>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB >>>> <https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1> >>>> &A=1 >>> >>> #################################################################### >>> #### >>> >>> To unsubscribe from the CCP4BB list, click the following link: >>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB >>> <https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1> &A=1 >>> >> >> >> -- >> Radu Aricescu >> MRC Laboratory of Molecular Biology >> Francis Crick Avenue >> Cambridge Biomedical Campus >> Cambridge CB2 0QH, U.K. >> tel: +44-(0)1223-267049 >> fax: +44-(0)1223-268305 >> www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu-aricescu >> >> ##################################################################### >> ### >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB >> <https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1> &A=1 >> >> >> >> _____ >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB >> <https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1> &A=1 >> >> >> ##################################################################### >> ### >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 >> ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. 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