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A molecular census of 26S proteasomes in intact neurons

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Science  23 Jan 2015:
Vol. 347, Issue 6220, pp. 439-442
DOI: 10.1126/science.1261197

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  1. Fig. 1 Raw image and quality assessment.

    (A) Slice from a representative cryotomogram of a cultured hippocampal neuronal cell. One single-capped (middle) and two double-capped 26S proteasomes (left and right) are indicated by a red frame. The insets display magnified 1.7-nm Z slices through each proteasome volume. Scale bars: overview, 100 nm; insets, 25 nm. (B) Histogram depicting the CCCs of all single-capped (blue bars) and double-capped (red bars) 26S proteasomes. The histograms were fitted with a Gaussian distribution (blue: single-capped 26S proteasomes, mean CCC ~0.28; red: double-capped 26S proteasomes, mean CCC ~0.28). The dashed gray line indicates the average threshold (~0.12), below which template matching results have been discarded. (C) Histogram showing the FCR of each single-capped 26S proteasome [blue in (B)] and the atomic model of the S. cerevisiae single-capped 26S proteasome (PDB, 4cr2). The mean cross resolution is at 47 Å.

  2. Fig. 2 Subtomogram averaging and classification workflow.

    (A) Global average from all particles displayed as center slice (left) and isosurface view (middle), as well as isosurface representation of the variance map (right; variance in violet). (B) First classification round separating single-capped from double-capped 26S proteasomes. The difference map between the two classes (right; difference shown in red) shows the additional RP. The in silico cutting was performed along the blue dashed line between the two 20S β rings. (C) Resulting average displayed as slice (left) and isosurface (middle), as well as corresponding variance map (right). (D) Second classification of the cut particles into two classes. Class 2 (left, light blue isosurface) and Class 1 (middle, light green isosurface) mainly differ at Rpn1, Rpn6, and the substrate entry location (right, red isosurface). Scale bars for the slices, 25 nm.

  3. Fig. 3 Rigid body fitting of atomic subunit models into EM densities.

    (A) Fitted atomic models of the S. cerevisiae 26S proteasome subunits in the GS and SPS EM densities (Fig. 2D). The subunits Rpn9/5/6/7/3/12 are colored in different shades of green, Rpn8/Rpn11 in light/dark magenta, Rpn10 and Rpn13 in purple, Rpn1 in brown, Rpn2 in yellow, the AAA-ATPase hexamer in blue, and the CP in red (13). Red arrows indicate selected Rpns and the unassigned density in the SPS. (B) Single-capped and double-capped 26S proteasomes displayed as green (GS) and blue (SPS) isosurfaces overlaid on a slice of a representative tomogram. Scale bar, 500 nm. (C) Chart showing the overall distribution of single-capped and double-capped 26S proteasomes (left) and of the different states within single-capped (middle) and double-capped (right) 26S proteasomes.

  4. Fig. 4 Subclassification within GS and SPS.

    (A) Classification of GS class into four subclasses. (Left) Variance map (top), the GS average (middle) and corresponding down-sampled atomic model (bottom). (Center) Subclasses GS1 to GS4 displayed as isosurfaces; their relative abundances are indicated. (Right) Differences between GS1 to GS4 and the down-sampled atomic model (red). (B) Classification of SPS class into three subclasses, SPS1 to SPS3, displayed as in (A).