Journal club: Ramrath et al, 2013
C9940 3-Dimensional Transmission Electron Microscopy
S1007 Doing structural biology with the electron microscope
May 11, 2015
Elongation of the polypeptide chain by one amino acid
peptidyl transfer >> translocation >>
aa-tRNA selection >>
and accommodation
Adapted from Joachim Frank
Small subunit
Large subunit
Ehrenberg, Nature, Vol. 466
Figure 1
Yamamoto et al., (2014) Nature Microbiology, Vol12
Figure 2
Justus Loerke et al. (2010) Methods in Enzymology, Volume 483
Figure 2
“Normal” reconstruction
and refinement of orientations
Justus Loerke et al. (2010) Methods in Enzymology, Volume 483
Figure 2
Question:
What's special about this complex?
Ramrath et al, 2013
Abbreviations used

ASL: anticodon stem loop

ASF: A-site finger (Helix H38)
Overview of the canonical TIPOSTcomplex.
David J. F. Ramrath et al. PNAS 2013;110:20964-20969
Fig. 1. Overview of the canonical TI POST complex. (A and B) Cryo-EM density of the ligands ap/P- and
pe/E-tRNAs (green and orange), mRNA (magenta), and EF-G (red) in surface representation within the
70S complex [50S (blue) and 30S (yellow)] (A) or with a mesh representation of both subunits together
with docked models of the 30S [16S (yellow) and S-proteins (gray)] and 50S [23S and 5S (blue) and Lproteins
(orange)] (B). (C) View from the intersubunit space onto the 30S subunit in the presented
authentic TI POST complex. Elements are colored according to their structural displacement compared
with the classic conformation (30) upon a common 50S alignment.
David J. F. Ramrath et al. PNAS 2013;110:20964-20969
Fig. S1.
Resolution curves of the authentic TI POST . According to the 0.5 FSC cut-off, the resolution of
the authentic TI POST complex is 6.8 Å.
Overview of the canonical TIPOSTcomplex.
David J. F. Ramrath et al. PNAS 2013;110:20964-20969
Fig. 1. Overview of the canonical TI POST complex. (A and B) Cryo-EM density of the ligands ap/P- and
pe/E-tRNAs (green and orange), mRNA (magenta), and EF-G (red) in surface representation within the
70S complex [50S (blue) and 30S (yellow)] (A) or with a mesh representation of both subunits together
with docked models of the 30S [16S (yellow) and S-proteins (gray)] and 50S [23S and 5S (blue) and Lproteins
(orange)] (B). (C) View from the intersubunit space onto the 30S subunit in the presented
authentic TI POST complex. Elements are colored according to their structural displacement compared
with the classic conformation (30) upon a common 50S alignment.
David J. F. Ramrath et al. PNAS 2013;110:20964-20969
Fig. S2. Formation of a unique
intersubunit bridge. (A) Cryo-EM
density of the authentic TI POST
shown as gray transparent surface with
docked models of the ribosome [30S
(yellow), 50S (blue), S13 (cyan), S19
(magenta)], EF-G (red), and tRNAs
[ap/P-tRNA (green), pe/E-tRNA
(orange)]. The map displays a
continuous density between L5 (red),
the ASF (dark blue), and S19
(magenta). (B) Cartoon representation
of the ribosomal conformation in the
classic unrotated classic
pretranslocational state (15) with an
opened L1-stalk indicating the contact
between the ASF and S13. (C) Cartoon
representation of the authentic TI
POST indicating the structural changes
of the 30S-head, ASF, and L1-stalk.
The positions of L1-stalk and ASF as
shown in B are displayed in
transparent gray.
David J. F. Ramrath et al. PNAS 2013;110:20964-20969
Fig. S3. Interactions of the 30S subunit and different bound tRNAs. Ribbon representation
of the tRNA binding region on the 30S subunit. Residues that contact tRNAs in the A
(magenta), P (blue), and E site (red) (25) are shown as spheres together with two tRNAs in
chimeric intrasubunit (ap/P and pe/E), classic (A/A andP/P), intersubunit hybrid (A/P and
P/E), or classic posttranslcoated (P/P and E/E) tRNA binding sites (14, 15).
Ehrenberg, Nature, Vol. 466
Figure 1
David J. F. Ramrath et al. PNAS 2013;110:20964-20969
Fig. S3. Interactions of the 30S subunit and different bound tRNAs. Ribbon representation
of the tRNA binding region on the 30S subunit. Residues that contact tRNAs in the A
(magenta), P (blue), and E site (red) (25) are shown as spheres together with two tRNAs in
chimeric intrasubunit (ap/P and pe/E), classic (A/A andP/P), intersubunit hybrid (A/P and
P/E), or classic posttranslcoated (P/P and E/E) tRNA binding sites (14, 15).
Contacts of EF-G domain IV. (A) Cryo-EM density of the presented TIPOST complex shown as
transparent gray surface together with docked models of the 30S, mRNA, ap/P-tRNA, and EF-G
(colored as in Fig. 1) in the indicated view (Left).
David J. F. Ramrath et al. PNAS 2013;110:20964-20969
David J. F. Ramrath et al. PNAS 2013;110:20964-20969
Fig. 3. Ligand positions within the authentic TI POST complex. (A) Cryo-EM density for the ligands
within the presented authentic TI POST complex in a transparent surface representation together with
docked models of EF-G (red), ap/P-tRNA (green), pe/E-tRNA (orange), and mRNA (magenta). (B)
Superimposition of the ligands (as shown in A) together with tRNAs bound to classic A, P, and E sites
(transparent gray) (23) upon a 50S alignment. (C and D) Ribbon representation of the ribosomal ligands
within the present authentic TI POST complex (C), and an unrotated ribosomal complex with classic A,
P, and E-tRNAs (23) (D). Highlighted are the distances between the last base pair within ASL of bound
tRNAs.
Ligand positions within the
authentic TIPOSTcomplex.
Ribosome–tRNA
interactions.
David J. F. Ramrath et al. PNAS 2013;110:20964-20969
Fig. 4. Ribosome–tRNA interactions. (A) Cryo-EM density for the 30S and ligands within the
presented authentic TI POST complex in a transparent gray surface representation together with
docked models of the 30S (yellow), ap/P-tRNA (green), pe/E-tRNA (orange), and EF-G (red). (B)
Schematic of the 30Ssubunit and bound ligands [EF-G (red), ap/P-tRNA (green) and pe/E-tRNA
(orange)] from the presented authentic TI POST . (C) Cryo-EM density of the ligands within the
presented authentic TI POST (transparent gray surface) together with docked models of EF-G, pe/EtRNA,
ap/P-tRNA, and mRNA (colored as in A). (D) Superimposition of the ligands from the present
authentic TI POST complex with either tRNAs in classic A, P, and E-site (23) (transparent gray) upon
a 30S head alignment.
David J. F. Ramrath et al. PNAS 2013;110:20964-20969
Supplemental Figure 4