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Hi Dominic, You cannot use the data.star as input STAR file for the references! Select a few nice class averages (by displaying the model.star from the last iteration of the initial 2D class averaging run; selecting the good classes by clicking (they get a red box) and save a new STAR file with the selected classes using the right-mouse popup menu) and use this STAR file for the references. Soon I hope to finish the 1.3 tutorial. This will describe the process in much detail. Until then I hope this will get you going. HTH, S On 07/01/2014 01:10 PM, Dominic Pollard wrote: > Dear Sjors, > > I have recently started using Relion-1.3 but am having some problems with the autopicking function. To test the function I followed instructions on the Relion-wiki pages: > > 1) I ran CTFFIND3 on several raw micrographs (mrc format) without any issues > 2) Created a new STAR file containing the two micrographs with highest and lowest defocus values from the relion_display program. This file was used as my INPUT for the autopicking > 3) My reference file was the data.star file output from a 2D-classification run from Relion1.3. This 2D-classification was run on particles picked and CTF corrected outside of Relion but otherwise preprocessed in Relion-1.2 > 4) Run relion_autopick in sequential mode with "Write FOM maps? - Yes" > > However, when I run relion_autopick from the GUI it does not generate FOM maps (no .spi files) or coordinate STAR files. Below is the terminal output. > > Executing: `which relion_autopick` --i NorR_Q304E_140618_Test.star --o autopick --particle_diameter 240 --angpix 2.05 --ref run3_it025_data.star --invert --ctf --ang 5 --lowpass 20 --write_fom_maps --threshold 0.25 --min_distance 40 & > Run autopicking on the following micrographs: > * NorR_Q304E_140618_0034.mrc > * NorR_Q304E_140618_0036.mrc > > When I open a micrograph and tick pick particles, ensuring the rootname is the same, there are no picked particles. > > Therefore, I have several questions: > > 1) Is there a problem that the original micrographs were mrc format? > 2) You mention that you can convert micrographs to STAR using relion_star_all_micrographs but this program isn't available in the Relion-1.3 directory we have downloaded, have I missed something in how to use this function? > 3) Could the problem be that my 2D classification reference file was created in relion-1.2 or that the particles for this reference file were CTF corrected outside of Relion > 4) Have I chosen the correct reference file? - data.star > 5) Or am I doing something wrong in the Relion-1.3 GUI? > > Thankyou very much in advance, > > Kind regards, > > Dominic Pollard > PhD student Imperial College London > Center for Structural Biology -- 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