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Dear colleagues, I am trying to use relion for subtomogram averaging. Unlike the data in protocol, my sample was from FIB milling cells, as a result, there is a 13 degree pre-tilt in my tomograms. I found the tilt direction of the 3D subtomogram ctf file is mirrored compared with the power spectrum calculated from the subtomograms (see attachment, left: ctf, right: power spectrum of the same subtomogram ). Does this really mean that the ctf files are applied a wrong sign or just display difference in different software packages. I reconstructed my tomogram with IMOD, then everything was done in relion just following the protocol. Best Qiang -- Qiang (John) Guo Department of Molecular Structural Biology Max Planck Institute of Biochemistry Am Klopferspitz 18 82152 Martinsried Germany Email: [log in to unmask]