Tutorial?

[Mtsaha]

Is there a tutorial (or testcase with example) for running Auto3dEM?

I am having difficulty in following:

http://cryoem.ucsd.edu/programs/auto3dem_usersguide_v4.04.htm

 

[Che-Yen Wang]

Hi Mitul,

You can download it from here: http://cryoem.ucsd.edu/programs/code/P22demo_v2.tar.gz

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[Mtsaha]

Thank you so much Joseph! Let me check this ...

[Mtsaha]

Hi there,

I am trying to go through this tutorial.

The first thing i notice is that the ctf estimation is done from the whole micrograph than from boxed particles.

Am i right?

If yes then how can i do ctf estimation from boxed particles instead? As I heard that the latter gives better ctf estimation.

Thanks,

Mitul

On Tuesday, June 3, 2014 9:57:08 PM UTC-5, Joseph Wang wrote:

[Giovanni Cardone]

Hi Mitul,

you are correct: the defocus is estimated from the whole micrograph. For the estimation Auto3DEM relies on CTFFIND3, developed in Niko Grigorieff lab and used in many groups, and the program works only with micrographs. Working on micrographs rather than on boxed particles has some advantages, such as:
- you can do better periodogram averaging, which increases the quality of the spectrum;
- by doing the estimation on tiles extracted from the micrographs you are automatically including all the particles (including those ones that you would not pick for the reconstruction) and possible portions of carbon present in the field of view, maximizing the signal useful for the estimation (CTFFIND3 also excludes those tiles that are void of signal).
The only disadvantage that I can think is that with the 'micrograph approach' you can not get an estimate of the SNR of the particles (like done in EMAN), but this is unrelated to the quality of the defocus estimation.
Regarding the possibility to estimate the defocus from a stack of particles, the only way to do it in Auto3DEM is using the graphical interface of RobEM, which means that the procedure is completely manual.

I hope it helps.
Giovanni

-- 
Giovanni Cardone
Baker's Laboratory
University of California, San Diego
Department of Chemistry and Biochemistry
9500 Gilman Drive, NSB 4107, MC-0378
La Jolla, CA, 92093-0378
(858)534-5852

[Mtsaha]

Hi Giovanni,

Thanks! Also when I run autoapp to do decus estimation, I need to enter "Amplitude contrast percentage".
Can you suggest how I find this parameter for my dataset?

Thanks,
Mitul

[Che-Yen Wang]

Hi Mitul,

I think you could use either 0.07 or 0.05 for regular cryo-EM. However, if you use energy filter or phase plate to collect the data, this value may be changed.

Kind regards,

Joe

[Mtsaha]

Thanks Joe!

[David Michael Belnap]

Mitul,

You could analyze the particle files separately in CTFFIND3, and then edit the output files to put your data back into the AUTO3DEM stream.

David

[Mtsaha]

Thanks! I will try this!

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