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Dear Sjors, I'd like to ask whether you would recommend to iteratively subtract unrelated densities ( as in "multi-body refinement") i.e. after 3D refine run on a protein domain of interest. As the reconstruction should improve, subtraction will become more precise. Another thing however is that probably subtraction should be done always from original particles in case that angles were not very good at the beginning and some signal was subtracted as well (e.g. for overlapping domain views)? I think this was planned for future Relion versions at some point but I don't have much info. Could you possibly comment on that? Another thing is what volume to use for subtraction. In the original gamma-secretase paper from Elife, 2015 you created a mask around full complex and then subtracted the "good domain", producing a masked volume that includes only "bad" domains but not the noise around the full complex (!). Then this volume was used for reprojecting and subtraction from the particles. This way you removed only "bad domains" but the noise stayed in the particles. I understand that the noise is important for Relion to properly estimate sigma. I was thinking whether this is a recommended way or it is better to also subtract the noise from the original particles: i.e. create an unmasked/unsharpened/unfiltered 3d volume using relion_reconstruct with original particles and then create a mask that includes not only "not interesting" domains but also noise around the full protein complex; then apply this mask to the reconstructed volume and reproject it to get the projections for subtraction. What do you think about it? Best regards, Grigory ------------------------------------------------------------ -------------------- Grigory Sharov, Ph.D. Institute of Genetics and Molecular and Cellular Biology Integrated Structural Biology Department (CBI) 1, rue Laurent Fries 67404 Illkirch, France tel. 03 69 48 51 00 e-mail: [log in to unmask]