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Particle Picking in Chimera

In this section, we will pick spheres in UCSF Chimera using the Volume Tracer and Kun Qu’s Pick Particle plugin. We will then update these metadata on the tomolist and generate a STOPGAP motivelist of these particles. We will then visualize the particle positions in UCSF Chimera using Kun Qu’s Place Object plugin. The Pick Particle and Place Object plugins are not standard in Chimera and have been installed separately.

Opening a Tomogram in Chimera

  1. Open a new bash terminal (i.e. not in the TOMOMAN console), load and open Chimera.

    module load chimera
chimera

    NOTE: Given that chimera uses many different windows, we recommend you go to a new workspace to open chimera. You can move to the window on the right by using the keyboard shortcut Ctrl + Shift + Right Arrow.

  2. For particle picking, we recommend using the non-CTF-corrected tomogram from AreTomo saved in bin8_aretomo/TS_01_dose-filt_bin8.mrc. This is because non-CTF-corrected tomograms typically have higher contrast, which is useful for this type of visual analysis. Open it in Chimera with File > Open.

  3. Chimera may open the tomogram as an isosurface volume. If so, visualize it as planes (in the Volume Viewer window select Features > Planes, click "One," and use the slider) and set the appropriate levels on the histogram.

    You may want to play around in the Chimera viewer to get familiar with the functions of all three mouse buttons in panning, zooming, moving planes, etc.

  4. In the Volume Viewer window, open the Coordinates panel by going to Features > Coordinates. Set the Origin index to 0 and the Voxel size to 1. Press enter after changing each setting. You may need to recenter and reorient the view; there are buttons to help with that.

  5. If you want more contrast to better visualize the VLPs, you can apply a gaussian filter (Volume Viewer > Tools > Volume Filter). A gaussian with of 1 should be sufficient. Uncheck "Displayed subregion only" under Options before clicking Filter.

  6. Before picking VLPs, it may be useful to shift the camera to use orthographic projections (Main Window > Tools > Camera > Projection > Orthographic). Orthographic side view disables "scaling with distance" of objects far from viewing plane due to perspective effect.

Picking Spheres

In Chimera, we will pick VLP centers using Volume Tracer and set radii for each sphere using the Pick Particle tool.

Picking Centers

  1. In the Volume Viewer, open the Tools > Volume Tracer. Try setting the marker radius to 20, which makes a sphere smaller than the VLPs, but large enough to assess centering. Move through the planes and place a marker at the center of each VLP. We recommend taking only the complete VLPs. This tomogram has around 9 complete VLPs.

  2. When you have finished marking the VLPs, select Volume Tracer > File > "Save current marker set as..." and save the marker set as sphere.cmm in TS_01/metadata/sphere/. You will need to create the subdirectories inside TS_01. This naming convention is important for parsing the metadata files into the tomolist later.

Picking Particles

  1. In the main window, open the Pick Particles tool (Tools > Utilities > Pick Particle).

  2. Under Marker File, click browse and open the marker file we just saved. Ensure that Object Style is set to Sphere and press Display. A series of size sliders will a appear for each marker.

  3. Adjust the sliders to place the edge of each sphere at the edge of its VLP. Since the VLPs are not perfect spheres, adjust the radius to a point with the best compromise.

    NOTE: There are several concentric layers to each VLP; set the radius for each sphere so that the edge is on similar layers. For these VLPs, we suggest setting the radius to between the two outermost layers. If your diameters are not around 40-50 voxels, you may have forgotten to set your Voxel Size to 1. If so, you will need to re-pick the volume centers.

  4. When all radii are set, press Save. For now, you can click Reset to remove the spherical wireframes and close the Pick Particles window.

Leave Chimera open and go back to the TOMOMAN standalone console.

Generating Motivelist

After picking our spheres, we can append this data to the tomolist and use functions in the STOPGAP toolbox to generate a motivelist.

  1. Add the picked spheres to the tomolist using tm_metadata_add_new. The parameters for this function are root_dir, tomolist_name, and type. The function loads a tomolist, scans the metadata/ subfolder for each tilt series for a subfolder named type, and stores the names of all files in that folder. These types can then be used by other functions that parse the tomolist. In this case, type is sphere. If your TOMOMAN Console is still in tomo/ you can use this command:

    tm_metadata_add_new(pwd, 'tomolist.mat', 'sphere');

  2. To generate a motivelist, we will use the sg_motl_batch_sphere function. This function generates particle coordinates along the surface of the input spheres in a defined way. The parameters for this function are:

    • tomolist_name – Name of tomolist
    • output_name – Name of output motivelist (file extension should be .star)
    • metadata_type – Type of metadata; in this case ‘sphere’
    • binning – Binning of input files
    • p_dist – Distance between particles; set to half the true distance (e.g. 3.5) for oversampling. The distance between particles can be measured on the tomogram using 3dmod by opening your tomogram, selecting Special-Drawing Tools-Measure from the 3dmod menu, and middle clicking to measure the distance between two subunits.
    • rand_phi – Randomize starting phi angles; set to 1
    • padding – Removes particles within a given distance of the edge of the tomogram
    • subset_list – Optional file listing a subset of tomograms to process

    As an example, this should generate 1,800-1,900 motivelist entries per sphere:

    sg_motl_batch_sphere('tomolist.mat', 'allmotl_1.star', 'sphere', 8, 3.5, 1, 16, []);

    Note that we are oversampling, that is, creating more objects than there are truly proteins of interest in the tomogram. This ensures that every true particle is picked at least once and we will subsequently cull unecessary objects.

Visualizing Motivelist

  1. The Place Objects tool in Chimera only opens AV3-format .em files, so we will need to convert our STOPGAP motivelist using the sg_motl_stopgap_to_av3 function. As an example:

    sg_motl_stopgap_to_av3('allmotl_1.star');

  2. In Chimera, open the Place Object function (Main Window > Tools > Utilities > Place Object). Browse for and open the .em motivelist file. Click Apply. This will display the particle positions as spheres, colored by their class number. In this case, all are the same class.

    NOTE: Since this is an oversampled dataset, there are quite a few particle positions; it may take a while to load. It will also be a bit slow when adjusting the view.

  3. To visualize particles with angular information, you can change the Object Style dropdown. Hexagons with voxel-size 0.1 work well here. Click Apply after specifying display options. Notice that the particles are evenly spaced with their planes along the surface of the spheres but their in-plane angles (phi) are randomized.

  4. You may close Chimera.