[scipion-users] Cryo-EM tomography using Scipion

[范宏成 <187...@16...>]

Dear all,
I am new to the cryo-EM tomography. I have some questions  when using some softwares.
1. I watched  subtomogram-averaging videos using relion3 on the scipion3 website. I noticed that adding extra restraints like --sigma_tilt 3, --sigma_psi 3 in relion-3d classification maybe in order to improve the alignment accuracy. I wanna know whether these values (--sigma_tilt 3, --sigma_psi 3)  are generally applicable. For subtomogram-averaging analysis, what is the ordinary range of these values when using the relion-3d classification? I recently tried the relion4 too. I found that the relion4 tomo-processing strategy (pseudo-subtomograms) showed much difference from the relion3. I'm not sure whether I need to add these extra retraints in relion4-3d classification or not. 


2. Does relion4 can be used in the Scipion3 now？I noticed the tilt handedness parameter in the relion_tomo_import_tomograms protocol.  The default values is -1. I wanna know the influence of this parameter for subtomo-averaging and which condition to choose the value of 1 . I found that I could also got same chirality and roughly same resolution 3D structure when I chose the 1 for  the tilt handedness parameter.


3. How to  manually judge the alignment accuracy of particles when doing the sub-tomogram analysis in Scipion. There existed some mis-oriented particles after relion-refinement. Can I put these aligned particles to original tomograms to check  which particles are mis-oriented and thus I can manually changed  their relevant euler angles to make them better fit the geometric constraint in tomograms or just deleted them.


4.I recently tried the Aretomo (v1.1.0) for my project(FIB-milled). I found better tomography reconstruction in aretomo compared to using imod patch-tracking in my project.  I am wondering whether aretomo results can be better than imod even using the sub-tilt refinement in relion4 ? Dose anyone test the performance of aretomo for subtomo-averaing?


5. I am doing the tomogram segmentation. My tomogram have some microstructures like  mitochondrion, ribosome, microtubule, lipid droplets, endoplasmic reticulum. Are there some softwares recommended or  worth a try ?


Looking forward to your response. Thank you！




Hongcheng Fan