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Expanded View Figures
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Figure EV1. Resolution and quality of the cryo-EM reconstructions.
A–C Surface rendering of the final high-resolution maps of the 30S subunit (A), the 70S ribosome (B) and the 50 S subunit (C).
D–I Local resolution plots showing the surface (D–F) and a cross section (G–I) of the cryo-EM maps. Local resolution maps of the 30S (D, G), 70S (E, H) and 50S (F, I) are
shown from the same view as in panels (A, B and C), respectively.
J–L Fourier shell correlation (FSC) curves of the 30S (J), the 70S (K) and 50S (L) cryo-EM reconstructions. The indicated resolutions are according to the FSC = 0.143
criterion (“gold-standard”).
M–O Examples for the quality of the density: (M) ribosomal protein (salmon) interacting with rRNA (grey), (N) protein a-helix (yellow) and (O) protein b-sheet (green).
ª2016 The Authors The EMBO Journal
Philipp Bieri et al Structure of the chloroplast 70S ribosome The EMBO Journal
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Figure EV1.
The EMBO Journal ª2016 The Authors
The EMBO Journal Structure of the chloroplast 70S ribosome Philipp Bieri et al
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Figure EV2.De novo built plastid-specific ribosomal proteins.
A–C The density indicates clear side chain features and allows unambiguous tracing of cL37 (A), cL38 (B) and bTHXc (C).
D–F Binding sites of cL37 (D), cL38 (E) and bTHXc (F) in the chloroplast 70S ribosome.
G–I Corresponding sites to panels (D, E and F), respectively, in the bacterial 70S ribosome (PDB 4YBB; Noeske et al,2015).
ª2016 The Authors The EMBO Journal
Philipp Bieri et al Structure of the chloroplast 70S ribosome The EMBO Journal
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Figure EV3. The chloroplast 4.5S ribosomal RNA.
A Primary transcript of the chloroplast rRNA operon. The canonical sequences of ribosomal RNAs are indicated by coloured boxes. The 4.5S rRNA is separated from
the 23S rRNA by a 115-nucleotide RNA spacer.
B, C Comparison of the chloroplast 50S subunit (B) with the bacterial 50S subunit (PDB 4YBB; Noeske et al,2015 ) (C) indicating the structural rearrangement of the
ribosomal proteins that interact with the 23S rRNA (blue) and the 4.5S rRNA (red).
D, E Secondary structure diagram of the chloroplast 4.5S rRNA (D) and the 30end of the bacterial 23S rRNA (E). The interactions of the 50and 30ends of the 4.5S rRNA
with the 23S rRNA are shown. Watson–Crick base pairs are indicated by lines (-), GU base pairs by dots () and non-standard base pairs by rings (○).
F, G Model of the chloroplast 4.5S rRNA (F) and the 30end of the bacterial 23S rRNA (G). The same nucleotides are shown in the models as represented in the secondary
structure diagrams (D, E).
The EMBO Journal ª2016 The Authors
The EMBO Journal Structure of the chloroplast 70S ribosome Philipp Bieri et al
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Figure EV4. The hidden breaks of the 23S rRNA.
A Analysis of ribosomal RNA by agarose gel electrophoresis. RNA was extracted from chloroplast 70S ribosome sample and separated on a 2% (w/v) agarose gel (L:
high range RNA ladder; 70S: RNA of chloroplast 70S sample).
B Schematic of rRNA processing and assembly in the chloroplast. Because of two specific cleavage sites on the 23S rRNA, called “hidden breaks”, the 23S rRNA gets
separated into three fragments: A (0.5kb), B (1.2kb) and C (1.1kb).
C–E Views of the 30S (C) and the 50S subunits (D) from the subunit interface and of the 50S subunit (E) from the solvent accessible side. The rRNA is shown as spheres
and coloured according to the elements indicated in panel (B). The positions of the hidden breaks on the 23S rRNA are marked with triangles.
F–H The hidden break indicated with a triangle between fragments A and B is introduced in the connection between helices H2and H24.
I–K The hidden break indicated with a triangle between fragments B and C is positioned at the stem loop of helix H63. The binding site of the helicase RH39 on helix
H62 is coloured black. The electron density map shown in panels (H and K) is low-pass filtered to 4Å, and the nucleotides at the hidden break sites are labelled.
The exact positions of the hidden breaks on the 23S rRNA sequence shown in panels (F and I) were stated in a previous publication (Liu et al,2015).
ª2016 The Authors The EMBO Journal
Philipp Bieri et al Structure of the chloroplast 70S ribosome The EMBO Journal
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Figure EV4.
The EMBO Journal ª2016 The Authors
The EMBO Journal Structure of the chloroplast 70S ribosome Philipp Bieri et al
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Figure EV5. Plastid translation factor pY.
A–D The 70S map with density for pY in the mRNA channel is shown in grey (A) and the empty 70S map is shown in yellow (B). The 70 maps were overlaid using only
the density of the 50S. Comparison of the overlaid maps, shown in 30S subunit view (C) and top view (D). The rotation of the 30S subunit is indicated with arrows.
E, F The body and the head domains of the 30S model were independently fitted into the cryo-EM map of the rotated state. The rotation angle of the 30S body rotation
(ratcheting) (E) and the 30S head rotation (swivelling) (F) were measured using PyMOL, and the rotation axes are shown in red and blue, respectively.
G Intersubunit bridges are affected by the 30S rotation. A selected residue of each intersubunit bridge is represented as white sphere in the non-rotated state. The
same residues are coloured in the rotated state either in blue, if the contact with the large subunit is maintained, or in purple, if the contact is lost. The
intersubunit bridges and the differences between the rotated and the non-rotated state are described in Appendix Table S4.
H Distances (d1,d2and d3) between backbone phosphates of selected rRNA residues (spheres) in the non-rotated (white) and the rotated state (purple) indicating a
slight opening of the mRNA channel.
I, J tRNAs in the P/P- and E/E-state from a crystal structure of the bacterial 70S ribosome in complex with mRNA and tRNAs (PDB 4V51; Selmer et al,2006) are
overlaid and shown in blue and purple, respectively. A tRNA (black) was fitted as rigid body into the 70S maps of the non-rotated (I) and of the rotated state (J).
ª2016 The Authors The EMBO Journal
Philipp Bieri et al Structure of the chloroplast 70S ribosome The EMBO Journal
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