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Hi all, I have a question regarding helical reconstruction in Relion 2.1. I am processing a filament data with a rise of 44A. For a box size of 300 pix (1 pix=1.169A), when I am choosing the number of asymmetric units in 3D auto-refine equal to 4, I am getting an error message: helix.cpp::chechParametersFor3DHelicalReconstruction(): Number of asymmetrical units exceeds its upper bound! The central z length is set to 60%. I am confused as to why I am getting this error because as I understand: Number of asu should be less than (Box size (A) * central z-length) / (Helical rise) Therefore according to my calculations: ( 300 * 1.169 * 0.6 )/ 44 = 4.78 A which should accommodate 4 asu. What I find surprising is that the program runs smoothly if I choose the number of asu= 3 or if I reduce the central z length to 40% without changing any other parameter. Any help in this regard would be highly appreciated. Also, since I am new to the field, I must apologize in advance if there is a lack of understanding or oversight on my part. For reference, I have listed below all the relevant parameters from Particle Extraction and 3D auto-refine below that return the error message: Particle Extraction parameters: Particle box size = 300 pix Rescale particles = No Extract helical segments = Yes Tube diameter = 100 A Co-ordinates are start-to-end only = Yes Cut helical tubes into segments = Yes Number of asu = 1 Helical rise = 44 A Refinement parameters: Mask diameter = 260 A Helical reconstruction = Yes Inner tube diameter = 10 A Outer tube diameter = 100 A Apply helical symmetry= Yes Number of asu = 4 Initial twist = 80 deg Helical rise = 44 A Central Z-length = 60% Thank and best wishes, Sadeem