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Manual Picking

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Marten edited this page Feb 7, 2023 · 13 revisions

Particle picking and deselection in 3D

PyTomGUI has a particle picking viewer that can be opened from the Particle Picking 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.

Go to: Particle Picking -> Manual Picking -> Select a tomogram.

After it opens, press the load button and save buttons (on top) to open and save particle lists. After loading a particle list you might need to adjust the Size Selection to get a good view of the subtomograms.

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. There are some methods for automated estimation of the thresholds (see the Template matching page). You can also use the subtomogram window opened by the viewer. When you click a subtomogram, the correlation score is shown in the window title. You can try to find a threshold where the results become visibly worse. This can then be entered as the minimal score.
  • You can apply a mask to the full 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 inside 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. More information in the Vesicle/organelle masking page.

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 `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.
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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