1. Volume 39, Issue 4, Pages 560-569 (August 2010)
The Three-Dimensional Organization of Polyribosomes in Intact Human Cells 
Florian Brandt, Lars-Anders Carlson, F. Ulrich Hartl, Wolfgang Baumeister, Kay Grünewald 
Molecular Cell 
Volume 39, Issue 4, Pages (August 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1
Thin Protrusions of a Glioblastoma Cell Line Contain Accumulations of Ribosomes and Are Accessible to Cryo-Electron Tomography
(A) In a phase contrast microscopic image of adherent U-87 MG cells, thin peripheral protrusions can be seen with <1 μm in thickness (white arrows). Scale bar, 50 μm.
(B) Overview tomograms were recorded at peripheral protrusions of U-87 MG cells grown on cryo-EM grids. Various cytoskeletal components, vesicles, and ribosomes in polysomes can be distinguished visually (ps, polysomes; mtub, microtubule; IF, intermediate filaments; pm, plasma membrane). Scale bar, 100 nm.
(C) The panels show sedimentation profiles of lysates from U-87 MG cells on sucrose density gradients. As indicated, polysomes (ps) were observed in lysates after treatment with CHX (upper panel). Upon Prm treatment of control cells (middle panel), polysomes were largely abrogated and a shift toward monosomal 80S ribosomes was observed. Addition of EDTA after cell lysis (lower panel) further dissociated monosomes into the large and small subunits.
(D and E) Subtomograms of (D) untreated cells or (E) Prm-treated cells were reconstructed at sites where ribosomes were accumulated (left panels). The volumes were sampled and correlated with a resolution-adjusted ribosome reference in order to produce 3D CCFs (right panels). White peaks indicate the presence of densities similar to the external template. Scale bar, 50 nm.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 2
In Situ Structure Determination of Ribosomes from Cellular Tomograms Indicates the Presence of Ribosome-Associated Complexes
(A) Start reference for particle alignment, low-pass filtered beyond 10 nm.
(B and C) Gallery view of tomographic averages of (B) 1911 ribosomal particles from untreated cells as compared to (C) an average of 1568 ribosomes from Prm-treated cells.
(D) The calculated difference map between the two averages indicates less density near the peptidyl transferase center (PTC) tunnel in the Prm-treated negative control.
(E) Fourier shell correlation resolution test of 1911 untreated ribosomes after one and ten iterations of alignment as indicated. In comparison, the power spectrum (P start, gray line) of the initial reference is plotted.
(F) Average structures of untreated ribosomes (dark gray) and Prm-treated ribosomes in top view (LSU, blue mesh; SSU, yellow mesh). The L1 arm, the CP, the L7/L12 stalk, the mRNA entry (m), the SSU's head (h), and the beak (bk) structure are indicated.
(G–I) In a segmented map of Prm-treated ribosomes and the difference map with the untreated average (red), in addition to the features in (F), several surface-exposed expansion segments (ES), the elongation factor binding site (EF), the peptidyl transferase center (PTC), and the peptide tunnel exit (PT) are indicated in front, cut front, and back view. To localize the peptide tunnel, a map of a nontranslating human ribosome was docked (cut view, light blue, EMDB accession number 1093; Spahn et al., 2004b). At the PT, surface areas of proteins Rpl17 (dark red), Rpl19 (orange), Rpl23a (dark green), Rpl26 (teal), Rpl31 (orange), and Rpl35 (light green) are indicated in the back view (right panel).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

4. Figure 3
Hierarchical Clustering of Next Neighbor Distance Distributions
(A) An x, y plot (left) and an x, z plot (right) show the distribution of Δ(x,y,z)i,k center-to-center 3D distance vectors (black dots) from all 1911 untreated ribosomes to their next neighbors.
(B) A (φ,ψ) plot indicates a rather isotropical distribution of (φ,ψ,θ) angle sets (black dots) of all ribosomes.
(C) Distribution of relative angle sets (φ,ψ,θ)i,k (black dots) calculated between any particle (as in A and B) and its next neighbor.
(D) As in (A), NND vectors were plotted for 1568 ribosomes in Prm-treated control cells.
(E and F) As in (B and C) all absolute (E) and all relative (F) neighbor orientations are plotted for Prm-treated ribosomes.
(G–J) Of 1911 untreated ribosomes (as in A–C), 1675 with any neighbor closer than 80 nm were used in a hierarchical analysis. (G) Dendrogram of clusters of center distance vectors and relative orientations. (H) Cluster populations (numbers 1–168) of next (blue) and second next (red) neighbors. (I) An x, z plot shows the clustered distribution of Δ(x,y,z)i,k center-to-center 3D distance vectors from each ribosome to its next neighbor. The six largest clusters from (H, blue) are plotted and colored as indicated. (J) (φ,ψ) distribution of the six largest clusters of next neighbor orientations as in (I). The mean relative orientations (x, black) were derived from clustering in quaternion space.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

5. Figure 4
Relative Orientation Criteria for Classification of Polysomal Particles
(A) The x, z plot shows the relative distribution of three 3D distance vectors from selected particles i to their next neighbor at the i+1 position (black, center-to-center vectors; yellow, center-to-SSU vectors; blue, center-to-LSU vectors). The four configuration classes plotted here were found by clustering the NND and relative angles.
(B) Z slices (step size 2.46 nm) of averaged density maps of particles in the neighbor configurations as in (A). The neighbors' densities appear diffuse due to residual polysome flexibility. Scale bars, 25 nm.
(C) The four dominant relative configurations of i+1 and i−1 neighbors as in (A) are depicted as schematic with isosurface representations in cluster average coordinates and angles in top view (left panels) or 180° turned about the y axis (right panels).
(D) To extrapolate model polysomes, the most probable neighbor configurations were replicated n = 4 times and isosurface models of 80S ribosomes were placed as in (C). Compact helical (left) and less compact helical polysomes (middle) are predicted in “unimodal” polysomes. Spiral polysomes are predicted for 1:1 alternating models of four consecutive pairs of ribosomes (right panels).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

6. Figure 5
3D Modeling of Polysomal Ribosomes in the Cellular Environment
(A–C) The panels show examples of subtomograms in which accumulations of ribosomes (arrow) were detected by template matching, positional refinement, and classification. At regions with lower overall ribosome density, single polysomes may be discerned. (A.I-C.I) Isosurface models derived from the tomographic average were placed into experimental coordinates and orientations for visualization of the particle distribution (SSU, yellow; LSU, blue; peptide tunnel density, red). As indicated, helical (A.I), planar (B.I and B.II), or linear/spiral (C.I) organization can be deduced in these examples. Note that the large subunits are predominantly oriented toward the cytosol. Plasma membrane (pm), intermediary filaments (IF), and microtubules (mtub) are indicated when visible.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions