[3dem] Particle picking within Relion 1.3 using projection images

[mf2944 at columbia.edu]

Dear all,

first of all thanks for the suggestions. And sorry for the late reply,  
but I was testing a few options.

@Sjors:
I used now relion_project to create the projections (I was not aware
of this command, thanks). The result is exactly as before. However, in  
these attempts I used a map after post-processing. I now also tried to  
make reference projections from the map after the 3D refinement step  
and now there are a few particles picked with very low FOM values.  
Meaning the true particles have a FOM range of 0.01 to 0.1. But it is  
actually picking less efficient than using the classes from 2D  
classification missing my rare orientations (only ~100 particles  
compared to 300 particles using 2D classes).

Next time I will also used the proper list for the technical relion questions.

@Carlos Oscar:
No, I did not try to do particle picking in Xmipp yet. In principle I
can get particles picked using either arachnid or relion, but I was
hoping to pick more efficiently the less common orientations.

@Xiaochen:
The reference map was constructed from one of my previously refined
and post-processed reconstructions in relion. As stated above I used  
now also a reference projection from the 3D refinement step and the  
picking is better, but not good.

I will probably go ahead and do first a full 2D classification and use
the resulting 2D classes, which will also contain some less common
orientations, in a new round of particle picking.

Cheers
Marcus

Quoting Sjors Scheres <scheres at mrc-lmb.cam.ac.uk>:

> Dear Marcus,
> If you want to make your own references from projections of 3D maps,
> you should use relion_project to do so.
> It is very important that you use a 3D reconstruction that you made in
> RELION to make these projections.
> relion-project is not accessible from the GUI, type in the command
> without options to see the available options.
> HTH, S
> PS: These technical RELION_related questions are normally sent to the
> ccpem list.
>
>
>
> On 09/17/2014 04:22 PM, mf2944 at columbia.edu wrote:
>> Dear all,
>>
>> I recently tried using the particle picking within Relion and if I   
>>  use the recommended procedure doing first a small 2D    
>> classification, followed by autopick, the program works fine.
>> However, if I make a 2D classification from a few micrographs, the   
>>  2D classes are only representing some major orientations that I    
>> have in my sample. But for the more rare orientations (those I    
>> would especially like to pick more efficiently) I do not obtain a    
>> 2D class. These only come up if I make a complete 2D classification  
>>   using all micrographs.
>> Therefore I though to use 2D projections from an initial lower    
>> resolution volume I obtained using Relion.
>>
>> To create these projections i used xmipp
>> xmipp_image_convert -i xxx.mrc -o xxx.vol -t vol
>> xmipp_angular_project_library -i xxx.vol -o projection.mrc --sym x   
>>  --sampling_rate 22.5
>>
>> Since the pixel size of the input is twice as big as the pixel size  
>>   of the new dataset I am working is rescaled the projections
>> xmipp_image_resize -i projection.mrc -o projection_scaled.mrc --factor 2
>>
>> However, using these as input in the relion particle picking is not  
>>   successful.
>> My feeling is that the FOM for the true particles in the picking    
>> are very low.
>> As additional information I calculated the statistics of the    
>> reference images create in 2D classification vs xmipp projection
>>
>> projections
>> Total number of images/volumes: 13
>> min= -0.912874 max=  2.861991 avg=  0.022331 stddev=  0.633817
>>
>> 2D classes
>> Total number of images/volumes: 10
>> min= -0.481988 max=  0.763749 avg=  0.055558 stddev=  0.249007
>>
>> Could anybody help me out with what I could do to get this working?  
>>   Or am I missing something essential that this route will never  
>> work?
>>
>> Best regards
>> Marcus
>>
>>
>
> -- 
> 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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-- 
Marcus Fislage, PhD

Howard Hughes Medical Institute (HHMI)
Columbia University
Department of Biochemistry and Biophysics
Lab of Joachim Frank
New York, NY

Phone: 212.305.9524
Fax: 212.305.9500