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CCPEM  July 2014

CCPEM July 2014

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Subject:

Re: Relion-1.3 autopicking and relion_star_all_micrographs

From:

Sjors Scheres <[log in to unmask]>

Reply-To:

Sjors Scheres <[log in to unmask]>

Date:

Tue, 1 Jul 2014 14:56:07 +0100

Content-Type:

text/plain

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Parts/Attachments

text/plain (56 lines)

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

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