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Manual Picking

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Marten edited this page Feb 7, 2023 · 13 revisions

Manual particle picking in 3D

PyTomGUI has a particle picking viewer that can be opened from the Tomographic Reconstruction stage. After opening a tomogram, you can either start manual picking on the volume directly or open a particle list obtained by template matching. The latter is often used to clean template matching results. For detailed instructions on the viewer see the PyTomGUI tutorial on the wiki.

The viewer provides some options to assist in cleaning template matching results:

  • You can set a score threshold to remove particles with low correlation scores. You can determine the threshold by running template matching with an original vs mirrored template and plot the scores against each other (see the template matching page or the tutorial page on the wiki for further instructions). You can also visually inspect the subtomograms opened by the viewer. When you click one at the threshold where the results become visibly worse the viewer will display its correlation score on top. You can the fill it in to filter the results.
  • You can apply a binary mask to the tomogram to remove template matching results. For example, the mask can have the shape of a cell/organelle in the tomogram. This way you can filter out particles that are not in the cell/organelle. The mask should have higher values on the places of interest than elsewhere. Ideally, provide a binary mask, but if its not yet binary PyTomGUI will attempt to convert it. The mask should have the exact same dimensions as the tomogram.
    • Put information on structural mask generation here...

GENERAL NOTE ON FILTERING

When applying a mask or score threshold, PyTomGUI will reload the particle list and apply the filter to the original list. So, to not waste your precious energy only start manually deselecting particles after applying the filters!

When doing manual picking from scratch, PyTomGUI will save a coordinates.txt file (similar to EMAN2). This can be converted from the GUI ('Create Particle List' - tab in Tomographic Reconstruction) or from the command line (see below).

EMAN2 picking

The EMAN2 package provides a neat interface for interactive particle picking or 'boxing'. Hence, we support importing coordinates of subtomograms localized in EMAN2. This coordinate list is then transferred to the native PyTom format for dealing with subtomograms (particle list).

For a detailed tutorial on the usage of e2spt_boxer.py we refer to: EMAN2 single particle tomography tutorial.

Here, how we use it:

e2spt_boxer.py "MyTomogram.em" -­-­inmemory
There is also a argument --yshort, which you must not use (this option applies to somewhat weird coordinate systems like the one from IMOD).

In the e2spt_boxer.py you can conveniently mark the centers of the particles of your interest. Before leaving the GUI you save the coordinates in a file, which is then converted for usage in PyTom, e.g., for reference-free subtomogram alignment and averaging.

Convert particle coordinates to particle list using bin/coords2PL.py

The script bin/coords2PL.py conveniently converts the coordinates stored in the EMAN2 boxer tool into a PyTom particle list (XML file). In this script you can also specify the future names of the to-be-generated subtomograms as well as the respective missing wedges.
pytom "MyPyTomPath"/bin/coords2PL.py -p "particleList" -c "CoordinateFile" -s "MySubtomogramNames" -w "MyWedge"
In detail, the parameters are:
  • particleList: file-name for the XML file that is generated.
  • CoordinateFile: name of the ascii file containing the coordinates (i.e., the EMAN2 file).
  • MySubtomogramNames: Root for subtomogram files. Could be something like My/Subtomo/Directory/particle_ which would result in files named particle_1, particle_2, ... in the directory My/Subtomo/Directory/.
  • MyWedge: Missing wedge. Can be specified by one angle (symmetrical missing wedge) or by two numbers seperated by a comma, e.g. 30 or 30,35.3.
With the resulting particleList you can proceed to subtomogram reconstruction and analysis.

Particle Picking in Chimera

In UCSF Chimera positions can be marked in 3D. These coordinates can be stored in a CMM file (effectively an xml file containing coordinates. There is a little conversion script to create a particle list from such a cmm file in av3/tom: av3_cmm2particlelist.m.
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