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Thanks for your suggestion Giulia. I tried 25A, and it seems to have worked as both the artifacts seem to be gone, and the resolution has improved somewhat. Gökhan -----Original Message----- From: Collaborative Computational Project in Electron cryo-Microscopy [mailto:[log in to unmask]] On Behalf Of Sjors Scheres Sent: Thursday, March 31, 2016 1:53 AM To: [log in to unmask] Subject: Re: [ccpem] Artifacts in the map (due to misaligned particles?) Hi, I think Giulia's suggestion is a good one. Sometimes at low-res the orientations aren't well defined and starting from a high-res reference may help. As always: just maintain your own critical judgement regarding overfitting and final resolution estimates. HTH, Sjors > Dear Gökhan, > > I had a similar issue. I didn't see any clear X-shaped artifacts but > the map clearly wasn't right. The way I noticed was by running 3D > classification from the refined particles, without allowing for > alignments, and I got classes that were clearly the same molecule > rotated. You might want to try this to see if this really is your problem. > > The way I solved it was by increasing the low-pass filter for the 3d > refinements to 20A. Even a 40A filtered map was too "pseudo-2-fold" > for the aligments to work, whereas the 20A-filtered one worked beautifully. > You might need to find the coarser possible filter that eliminates the > error - this will depend on the size and shape of your object. > > As long as the final resolution goes significantly beyond your initial > filter I think you'll be fine - although I'd be very interested in > hearing people's comments regarding this. > > Giulia > > On 28/03/2016 23:55, Gökhan Tolun wrote: >> >> Hi, >> >> I am working on two projects with no symmetry, and in the maps >> generated by the refinement process I see artifacts that look as if >> some of the particles were misaligned 180 degrees in one axis, >> creating a pattern in the 3D reconstruction slices that looks like >> two densities crossing each other (with a general X or + appearance >> at the points where the two densities meet). Also, if I move through >> the slices of the reconstructed 3d volume (e.g., using 3DMOD), the >> density continuity moves in two different axes that are 90 degrees to >> each other (best-effort explanation), so I am quite sure that there >> is something wrong. >> >> I do not see any relevant parameters I can play with in the GUI, and >> when I looked at the relion_refine parameters from the command line, >> nothing obvious caught my eye that is listed as may be related to >> such an issue. >> >> So, is there a way to avoid misalignment of particles during a C1 >> refinement? May any of the “expert” or other command-line parameters >> be fiddled-with to fix such a faulty behavior during a 3D refinement, >> and if so, which ones and how? >> >> Thanks. >> >> Gökhan >> > > -- > Giulia Zanetti > ISMB, Birkbeck College > Malet St. London > WC1E 7HX > 02076316898 > > -- 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