Hi All
I hope this is not too bone-headed of a question. I am new to using Relion (1.3), and I am doing 2D classification and I have been getting some results that look suspicious. I am using my own DD data set of yeast 80S particles. I followed the steps outlined in "Tutorial (The quickest way to learning RELION-1.3) ", for ctf-correction, autopicking, etc. - Now I am trying to do some particle pruning with the 2D classification. There are two things going on:
1) There are two highly populated classes (40% of ~60k particles) that look the same - they seem to be neither junk nor good classes.
2) There are two visible rings for each class - one is the soft mask, as I would expect, but the other seems to be related to the rotation of the particle during alignment - I've attached an image - it seems to exist on both good and bad classes - these are sorted by class distribution. I can't figure out what causes this, but I feel like its a good indicator I've picked some bad parameter. When I did 2D Classification on a very small manual picked set with a very slightly smaller mask (350 A vs 360 A) I did not see the same thing. My box size is 300 pix = 396 Ang.
Thanks for any help - please excuse such a possible inane question!
Liz Montabana
Here is the command:
mpirun -np 240 --bynode `which relion_refine_mpi` --o Class2D/autopick_sort2 --i particles_autopick_rd2.star --particle_diameter 360 --angpix 1.32 --ctf --iter 25 --tau2_fudge 2 --K 300 --flatten_solvent --oversampling 1 --psi_step 10 --offset_
range 5 --offset_step 2 --norm --scale --j 1 --memory_per_thread 8 > Class2D/autopick_sort2.out
and settings:
Class2D/autopick_sort2.gui_class2d.settings
is_continue == false
Output rootname: == Class2D/autopick_sort2
Continue from here: ==
Input images STAR file: == particles_autopick_rd2.star
Number of classes: == 300
Do CTF-correction? == Yes
Have data been phase-flipped? == No
Ignore CTFs until first peak? == No
Number of iterations: == 25
Regularisation parameter T: == 2
Mask individual particles with zeros? == No
Limit resolution E-step to (A): == -1
Perform image alignment? == Yes
In-plane angular sampling: == 5
Offset search range (pix): == 5
Offset search step (pix): == 1
Number of MPI procs: == 240
Number of threads: == 1
Available RAM (in Gb) per thread: == 8
Submit to queue? == Yes
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