2D classes not centered

[Annelie Olofsson]

Hi,

I am trying to do 2D classifications on negative stained particles and my 2D classes does not get centered in one of my data sets (see attachment). It works fine with other similar particles using the same procedure (using auto-center when picking particles manually and centeracf when generating 2D classes). The pixel size is however smaller on the particle set that I have troubles with (6,57Å compared to 10,73Å) and I do not have that many particles either (1330). It would be great if you could help me understand what could be wrong and if I can avoid it to get centered 2D classifications. Thank you! /Annelie

e2version.py:

EMAN 2.1 (CVS 2014/12/09 16:26:21)
Your EMAN2 is running on: Mac OS 10.7.5x86_64
Your Python version is:  2.7.1

Command:

e2refine2d.py --input=sets/my_stack_ptcls.lst --ncls=100 --normproj --fastseed --iter=10 --nbasisfp=12 --naliref=3 --parallel="thread:8" --centeracf --simalign=rotate_translate_flip --simaligncmp=ccc --simraligncmp=dot --simcmp=ccc --classkeep=0.85 --classiter=5 --classalign=rotate_translate_flip --classaligncmp=ccc --classraligncmp=ccc --classaverager=mean --classcmp=ccc --classnormproc=normalize.edgemean

SPR - Box size: 84

Microscope: 80 kV

GUI: e2projectmanager.py

[Steven Ludtke]

Hi Annelie,

We made a number of improvements to the centering problem, specifically focused on negative stain data, over the last 3 months.  I'd like to encourage you to download the nightly snapshot version and give it another try with exactly the same options. While the new version may still not be perfect in all cases, I suspect for data like you show that it will be much much better.

If there are still centering issues with the new version, another thing to try is to apply a modest high-pass filter to your data to help get rid of some of the very low resolution artifacts (gradients, etc.) in the raw images which can bias the alignment of the particles. I think there are some discussions of this in the google group over the last couple of months you can refer to.

Please feel free to followup after testing.

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<MS P13 MSP2 200x 20classes.png><MS P13 MSP2 200x 100classes.png>

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Steven Ludtke, Ph.D.
Professor, Dept. of Biochemistry and Mol. Biol.                Those who do

Co-Director National Center For Macromolecular Imaging            ARE

Baylor College of Medicine                                     The converse
slu...@bcm.edu  -or-  ste...@alumni.caltech.edu               also applies
http://ncmi.bcm.edu/~stevel

[Annelie Olofsson]

Hi Steven, 

Thank you for your help! 

After installing the nightly snapshot version, centering of the 2D classes worked fine.

Best regards,

Annelie

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[Haixin Sui]

Hi Steve,

Is the e2spt_boxer.py still under development or ready to use?

I tried the "e2spt_boxer.py" program of the daily distribution version EMAN 2.1 (CVS 2015/03/25 09:00:01). With this programs, I could extract map boxes by clicking objects in the tomogram, and I can save out the coordinates of the boxes.  In addition, I could delete unwanted map boxes during the process of marking objects.  However, if I save the marker coordinates out after deleting some map boxes, the coordinates of the deleted boxes still present in the coordinate file.

Any advice on how to delete or correct markers during marking objects using e2spt_boxer.py?

Thanks

Haixin

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Haixin Sui
Wadsworth Center

[Steven Ludtke]

Hi Haixin,

Yes, while I could envision making SPT a bit more integrated, like SPA is, e2spt_boxer should be fully functional. I just ran a test:

- opened a tomogram which was previously boxed

- read in the ".box" file associated with it

- deleted several particles (by holding down shift when clicking on a particle)

- wrote a new ".box" file

- compared the old and new .box files and the particles I had deleted were, indeed, missing from the second file...

If this is the process you followed, we need to try and figure out what is going wrong, and where. You might check the console for error messages...

----------------------------------------------------------------------------

Steven Ludtke, Ph.D.

Professor, Dept of Biochemistry and Mol. Biol.         (www.bcm.edu/biochem)

Co-Director National Center For Macromolecular Imaging        (ncmi.bcm.edu)

Co-Director CIBR Center                          (www.bcm.edu/research/cibr)

Baylor College of Medicine                             

slu...@bcm.edu

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[Haixin Sui]

Hi Steve,

The program works fine when I follow the instruction in your email. The marker coordinates were deleted if I click on the unwanted boxes while holding the shift key.

Before I read your email, I tried to delete the boxes with the following method:
In the particle list window, we clicked the center button of the mouse. It brought out a new window showing options such as "App" "Del" "Darg" "Sets"... We activated the "Del" and then clicked on the unwanted boxes.  The image boxes disappeared as we expected. We then saved the marker coordinates out in a new file. The marker coordinates of the deleted boxes still presented in the new file.  I guess this is not the right way to delete image boxes.

Thank you for the response. We will use the method in your email.

Haixin

Haixin Sui
Wadsworth Center

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Subject: Re: [EMAN2] e2spt_boxer.py
From: slud...@gmail.com
Date: Tue, 31 Mar 2015 00:16:06 -0500
To: em...@googlegroups.com

[Steven Ludtke]

Yes, I understand the confusion.  The "del" mode in the image widget control panel deletes particle images from that widget. If you then saved the 2-D images visible in the tiled display to a file, the ones you deleted from that window would be absent. However, this sort of deletion doesn't tell the overall boxing program that you wanted to delete particles.

Again, I understand why you did it the way you did, and we should probably do something about this (disabling the "del" button in the widget or something).

----------------------------------------------------------------------------
Steven Ludtke, Ph.D.

Professor, Dept. of Biochemistry and Mol. Biol.                Those who do