e2ddd_external.py error with inputs

[Adam Miller]

Here is the e2version info:

EMAN 2.31 final (GITHUB: 2019-09-30 10:41 - commit: 194df95 )

Your EMAN2 is running on: Linux-5.4.0-53-generic-x86_64-with-debian-bullseye-sid 5.4.0-53-generic

Your Python version is: 2.7.14

I am getting these errors when trying to process a tilt series from a K3 with e2ddd_external.py (motioncor2 is installed and in my path). I have the raw tilts in a "movies" directory and I have the corresponding mdoc in the same directory. Here is my command:

e2ddd_external.py movies/ --program=ucsf_motioncor2 --device=gpu --mdoc=movies/t5.mdoc --apix=1.667 --gain=SuperRef_stack_00000.mrc --mc2_rotgain=0 --mc2_flipgain=0 --imod_rotflipgain=0 --device_num=0 --tomo --tiltseries_name=t5_50-1

Here is the error i get:

DOC: movies/t5.mdoc

Traceback (most recent call last):

  File "/home/apm4/EMAN2/bin/e2ddd_external.py", line 492, in <module>

    main()

  File "/home/apm4/EMAN2/bin/e2ddd_external.py", line 177, in main

    mdoc_params,tilt_source,raw_tilts,apix,options.mdoc = parse_mdoc(options.mdoc)

  File "/home/apm4/EMAN2/bin/e2ddd_external.py", line 486, in parse_mdoc

    filenames.append(i["SubFramePath"].split('\\')[-1])

KeyError: 'SubFramePath'

If I put the input as movies/t5.mrc I get a different error:

ERROR: When providing an mdoc in tomo mode, the input option must be a directory containing the raw files referenced in the mdoc.

Thanks for any help on this issue.

Best,

Adam

[Steve Ludtke]

Hi Adam,

we'd need to see the .mdoc file to figure out what's going on.

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[Adam Miller]

Here is a screen shot of the first part. The rest just follows the sequence for each tilt in the .mrc stack. Thank you.

[Steve Ludtke]

Hi Adam,

that doesn't really look like an mdoc file for a movie-mode tilt-series acquisition. ie - where each tilt is a movie. You'll note that there is a single filename at the beginning and the first 2 images in the file have different tilt angles. This looks like each tilt is a single exposure (or already averaged).

I'd also note some other oddities. It claims a DoseRate of 19 e-/pix/s, an exposure time of 0.35 s and a pixel spacing of 1.667 A/pix. If you do the math this comes out to 2.4 e-/A^2/s. With a 3 degree tilt step this is a total dose of ~100 e-/A^2. The fact that you're imaging at 1.667 A/pix would seem to imply you are targeting resolutions as high as ~5 A. While 100 e-/A is a fairly typical dose for a low resolution cellular tomogram, to achieve subnanometer resolution the dose is limited to ~20-30 e-/A^2, which in turn would mean you would only be able to use the first 8-12 of the 40 tilts in your tilt series.  While you may still get a little value out of the other tilts in terms of Z localization, you would need to be sure to place a tight limit on the tilts to include on any attempts at high resolution subtomogram averaging. If you aren't targeting high resolution, then you should probably be using a much lower magnification, and get a wider field of view, since you don't need the extra sampling....

 

On Nov 12, 2020, at 7:00 PM, Adam Miller <mille...@gmail.com> wrote:

Here is a screen shot of the first part. The rest just follows the sequence for each tilt in the .mrc stack. Thank you.

To view this discussion on the web visit https://groups.google.com/d/msgid/eman2/3fa7788f-0da1-4d8a-a066-6dcbc6d02edfn%40googlegroups.com.

[Adam Miller]

Thank you for the feedback Steve. This was my first collection, I am primarily trying to diagnose an air/water interface issue, so I am not trying to go to higher resolution. I will keep all of this in mind next time I am on the microscope and if I am trying for reconstructions.

Do you have any workflow advice on determining particles near the air/water interface? 

Thank you again.

Adam

[Steve Ludtke]

Hi Adam,

to perform the tomographic reconstruction in EMAN2 either you want your tilt series (which is different than a movie) to be in tilt order rather than collection order, in which case you can just specify a fixed tilt step, or you have a file in collection order, and you provide a .rawtlt file containing the tilt angle of each tilt in order. The .rawtlt file is extremely simple. It is just a text file with a single number (the tilt angle in degrees)  on each line.

If you are just trying to localize the particles in Z to see what's happening WRT the air-water interface this should be pretty simple. The layer should be reasonably thin, even with such a high A/pix value. Just make sure that the --clipz value you give to e2tomogram.py is thick enough (the number is in pixels, not Å) to accommodate the thickness of your ice. You will also likely want to select the --correctrot option which will try and put the strongest features in your tomogram in the same plane. In most cases this will make the ice layer nearly parallel to the X-Y plane, since often the physical tilt of the ice layer in the microscope isn't quite orthogonal to the beam when the tilt angle is 0. 

Once you have reconstructed the tomograms, simply open them in e2spt_boxer and you can look at slices along the 3 axes. If you drag the slider up and down through Z you should easily be able to tell if your particles are in a layer along the air/water interface.

To view this discussion on the web visit https://groups.google.com/d/msgid/eman2/44c1ef5e-6c49-4742-af60-61535e774485n%40googlegroups.com.