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Hi Sjors and all others, In order to perform the signal subtraction, we have to do 3D auto-refine for C1 using expanded_particles.star with ONLY local angular searches (e.g., 1.8 degrees). Is my understanding correct? Thanks, Dong-Hua On Thu, Mar 7, 2019 at 11:56 AM Sjors Scheres <[log in to unmask]> wrote: > Dear Julian, > > More classes just gives it more space to separate out, as you do not align > anyway, the increase in number of parameters is not large. > The higher T fudges for the observation that the signal now lives in a > small part of the box, while it is measured as its power from the whole > box. > > You say refinement has failed. You could try to select your class from the > expanded classification, and then perform a auto-refinement where you only > perform local angular searches: that is, use a start sampling that is just > as large as the one from where you do local searches, for example both at > 1.8 degree. The input map for that refinement could be the class you had > identified. Doing only local searches will prevent particles 'loosing > their way again' if you start from a low-resolution initial model. This > whole procedure also works with signal subtraction, but make sure to > revert back to the non-subtracted particles before the 3D refinement (i.e. > only use subtracted images in the classification). > > HTH, > Sjors > > > > > Dear Sjors and all the others, > > > > thank you very much for the very helpful input! > > > > We now expanded the star file and made a masked classification which > > resulted in one class nicely showing the expected conformational > > change. However, refinement of this class has led to a bad result. > > > > We will now do the particle substraction as you suggested and try again. > > > > For my understanding, could you clarify the need of many classes and > > the higher-than usual T-factor? > > > > Best, > > Julian > > > > > > Zitat von Sjors Scheres <[log in to unmask]>: > > > >> Dear Julian, > >> It is as Bayly says: > >> > >> relion_particle_symmetry_expand --i refine3D/jobXXX/run_data.star > >> --sym C3 --o expanded_particles.star > >> > >> Then there are multiple options. I would suggest making a mask > >> around 1 monomer and another mask around the other 2 monomers. Then > >> subtract 2 monomers from all expanded particles, and feed the > >> resulting images into a 3D classification (without re-alignment) > >> that uses the single-monomer mask. Use many classes (10?) and a > >> higher-than usual T-factor, say 10-20. > >> > >> The original reference is : > >> S.H.W. Scheres (2016) "Processing of Structurally Heterogeneous > >> Cryo-EM Data in RELION" Meth. Enzym. 579, 125-157 (section 4.7) > >> > >> HTH, > >> Sjors > >> > >> > >> Charles Bayly-Jones wrote: > >>> Yes, the relion_particle_symmetry_expand function will allow you to > >>> accomplish this. Then perform masked 3D classification as you > >>> described. It is very useful indeed :) > >>> > >>> You could also use the localised reconstruction method from Ilca et > >>> al. (2015) doi:10.1038/ncomms9843 > >>> < > http://www.nature.com/ncomms/2015/151104/ncomms9843/full/ncomms9843.html> > >>> Best of luck, > >>> Charlie > >>> _________________* > >>> * > >>> > >>> *Charles Bayly-Jones* > >>> BSc(ScSchProg)(Hons) > >>> > >>> *PhD Candidate* > >>> *Teaching Associate* > >>> * > >>> * > >>> *Monash Biomedicine Discovery Institute* > >>> *Department of Biochemistry and Molecular Biology* > >>> Level 2, Building 77 > >>> 23 Innovation Walk > >>> Clayton VIC 3800 > >>> Australia > >>> > >>> > >>> On Tue, 5 Mar 2019 at 21:01, Julian Reitz <[log in to unmask] > >>> <mailto:[log in to unmask]>> wrote: > >>> > >>> Dear all, > >>> > >>> we are working on a dataset from a homo-trimeric protein. Until > >>> now we have very successfully refined the structure to good > >>> resolution applying C3 symmetry in RELION. > >>> > >>> However, we also have a sample from this protein that contains a > >>> low affinity binding substrate and we would like to do a > >>> classification to see if there are some subunits showing the > >>> expected conformational change due to substrate binding. The point > >>> is, that we do not expect the ligand to bind to all three monomers > >>> of one particle but that the conformational change might only > >>> happen in one of the monomers of a particle. Therefore, normal > >>> 3D-classification seems unrewarding and a masked classification > >>> would suffer from the aspect, that we always only look on one of > >>> the monomers ignoring two-thirds of the data. > >>> > >>> We were thinking that it might be helpful if we somehow triplicate > >>> the particles, each rotated by 120° to get all of the three > >>> monomers from one particle inside the mask. Is there any clever > >>> way to do this in RELION or does anybody else has a good idea how > >>> we could proceed? > >>> > >>> Best, > >>> Julian > >>> > >>> > ######################################################################## > >>> > >>> To unsubscribe from the CCPEM list, click the following link: > >>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCPEM&A=1 > >>> > >>> > >>> > ------------------------------------------------------------------------ > >>> > >>> To unsubscribe from the CCPEM list, click the following link: > >>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCPEM&A=1 > >>> > >> > >> -- > >> Sjors Scheres > >> MRC Laboratory of Molecular Biology > >> Francis Crick Avenue, Cambridge Biomedical Campus > >> Cambridge CB2 0QH, U.K. > >> tel: +44 (0)1223 267061 > >> http://www2.mrc-lmb.cam.ac.uk/groups/scheres > >> > >> ######################################################################## > >> > >> To unsubscribe from the CCPEM list, click the following link: > >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCPEM&A=1 > > > > > > -- > > Julian Reitz, Dipl.-Biochem. > > J.W. Goethe Universität Frankfurt am Main > > BMLS - Buchmann Institut for Molecular Life Sciences > > Frangakis Group > > Max-von-Laue-Str. 15 Raum 1.658, D-60438 > > Tel: +49 (0)69 798-46429 > > e-mail: [log in to unmask] > > http://fcem.uni-frankfurt.de/em > > > > ######################################################################## > > > > To unsubscribe from the CCPEM list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCPEM&A=1 > > > > > -- > Sjors Scheres > MRC Laboratory of Molecular Biology > Francis Crick Avenue, Cambridge Biomedical Campus > Cambridge CB2 0QH, U.K. > tel: +44 (0)1223 267061 > http://www2.mrc-lmb.cam.ac.uk/groups/scheres > > ######################################################################## > > To unsubscribe from the CCPEM list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCPEM&A=1 > ######################################################################## To unsubscribe from the CCPEM list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCPEM&A=1