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Hi Zhu, Did you try refining against a single model to high resolution (using auto-refine) and then perform 3D classification with local angular searches that are much finer than you would normally use in 3D classification? This is explained also at the end of the tutorial on the RELION Wiki. We often get good results with that in cases where the de novo classification fails. Hope that helps, Sjors > I have ran my ribosome data in relion-1.2. After 3d classification, I > could always get the state with three tRNA inside. And that must contain > two sub states at least. But no matter how many class numbers I set, I can > not separate them out. Could you give me some suggestions? Thanks! > -- 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