Proteasome EM Tomography ChimeraX Tutorial

Tom Goddard
S2C2 High Resolution CryoET Image Processing Workshop
March 1-3, 2022

We will look at electron tomography 3d maps of proteasomes in fibril aggregates found in cultured rat neurons and associated with amyotrophic lateral sclerosis and frontotemporal dementia using ChimeraX (version 1.3) visualization software. We also look at how to fit atomic models of proteasomes into subtomogram average proteasome maps.

Poly-Gly-Ala aggregates (red) surrounded by proteasomes (green) within a neuron described in the followng article:

In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment.
Guo Q, Lehmer C, Martinez-Sánchez A, Rudack T, Beck F, Hartmann H, Perez-Berlanga M,
Frottin F, Hipp MS, Hartl FU, Edbauer D, Baumeister W, Fernandez-Busnadiego R.
Cell. 2018 Feb 8;172(4):696-705.e12. doi: 10.1016/j.cell.2017.12.030. Epub 2018 Feb 1.


Data sets we will look at: tomogram EMDB 4191, subtomogram averaged proteasome EMDB 3913 and 3916, atomic model PDB 5MPC.

Tomogram visualization

  1. Open tomogram from EM Databank (large 240 Mbytes)
    open 4191 from emdb
    or smaller middle 1/3 of tomogram for faster download (25 Mbytes)
    or if previous site is down

    a) Tomogram EMDB 4191 is 240 Mbytes and can be slow to download.
    b) Size 926 x 926 x 71 grid points, 32-bit float values reported in Log.
    c) ChimeraX caches fetched data under folder ~/Download/ChimeraX

  2. Switch to plane style in Volume Viewer panel.
  3. Adjust yellow curve nodes to control brightness.
  4. Adjust Plane slider to look at different slices.

  5. Invert map values, easier to see proteasomes and filaments if they have high intensity instead of low intensity.
    volume scale #1 factor -1
    Close original map
    close #1

    a) Tomogram lower map values correspond to higher density.
    b) Map values represent how much of the electron beam passed through the sample and hit the detector.
    c) It is useful for visualization to make high values correspond to high density by inverting values.
    d) The "volume scale" command creates a copy of the map in memory. It does not modify the original map.

    Marking proteasomes

  6. Zoom in and look for proteasomes, white rings with holes.

  7. Use Markers toolbar, click Plane button.
    Right click (Cmd+click on Mac) to place markers on proteasomes.

  8. Extract box around marker.
    volume box #2 center #1 isize 32

  9. To save extracted proteasome map
    save ~/Desktop/proteasome1.mrc model #3
    To save markers to an XML text file
    save ~/Desktop/proteasomes.cmm model #1
    In article Guo Q, Cell Feb 2018 10000 proteasomes in 9 tomograms found with custom MATLAB and TOM toolbox template matching algorithms.

    Surface depictions

  10. Switch to surface style in volume viewer, or use command
    volume #2 style surface level 780

  11. Hide small connected surface blobs using Map toolbar Hide Dust button, or command
    surface dust #2 size 200

  12. Use Blob mouse mode from Right Mouse toolbar.
    Click proteasome surfaces to measure size.

    a) Picked blob Log output.
    volume = 7.4896e+05
    area = 87962
    size = 325.01 111.15 115.29
    b) Blob mouse mode draws a box aligned with the principle axes of inertia and reports lengths along those axes.
    c) Blob coloring is lost when surface level is changed.

    Comparing subtomogram maps by morphing

  13. Guo Q, Cell Feb 2018 averaged proteasomes from tomogram with 4 different shapes. Two conformations were degrading ubiquitinated protein. We will look at two "ground state" conformations with no substrate.

  14. Close previous data
    Open two subtomogram average maps
    open 3913 from emdb
    open 3916 from emdb

  15. Morph from one map to the other to see differences
    volume morph #1,2

  16. The record button on the morph slider saves a movie to the Desktop.

    Fitting atomic models

  17. Guo Q, Cell Feb 2018 fit atomic structures of proteasomes degrading substrate or not bound to substrate to infer the state of the proteasomes and concluded many proteasomes are stalled, unable to digest the poly Gly-Ala fibrils.

  18. We will fit an atomic model of a proteasome into one of their subtomogram average proteasome maps at 12 Angstroms resolution.

  19. Close previous data
    Open subtomogram average map
    open 3913 from emdb

  20. Open a proteasome atomic model for fitting into subtomogram map
    open 5mpc
    Center view

  21. Subtomogram map includes only half of proteasome core.
    Delete extra proteins from atomic model.
    del /a-n,X

  22. Move selected atomic model into map with mouse
    select #2
    Use Move Model mouse mode from Right Mouse toolbar.

    a) Hold the shift key to rotate, no-shift to translate.

  23. Optimize fit use Map toolbar Fit button, or command
    fit #2 in #1
    Make surface transparent with Map toolbar Transparent button, or command
    transparency #1 30

    a) Guo's proteasome maps are half-proteasomes. The proteasome is a cylinder made of two rings and a lid complex on either end. A half proteasome has one ring and a lid.
    b) The fitmap command does a rigid rotation and translation to optimize the position of an atomic model in a map.
    c) To rotate with mouse or trackpad about the axis perpendicular to the screen click near the edge of the graphics window and drag.