Journal: Nature Communications

Article Title: Structure and mechanism of the two-component α-helical pore-forming toxin YaxAB

doi: 10.1038/s41467-018-04139-2

Figure Lengend Snippet: Cryo-EM structure of the YaxAB complex reveals an α-helical transmembrane pore. a Negative-stain TEM micrographs of YaxAB complexes in solution. Left: sample in absence of detergent. Right: sample in presence of 0.1% Cymal-6. Scale bar = 50 nm. b Final sharpened cryo-EM density of YaxAB together with the fitted pore model, shown from side view. The chosen contour level (4.5σ) corresponds to the 1.21 Å 3 /Dalton convention according to Harpaz et al. . Fitted YaxA and YaxB models are colored blue and pink, respectively. The amphipol belt, illustrated in gold, is clearly visible and demarcates the putative transmembrane region. c Top view of the pore complex, overlaid with the final cryo-EM map. YaxA and YaxB form outer and inner rings, respectively (left). A zoom of the amphipol-enclosed portion of the complex (right) is contoured at 6.5σ to better distinguish individual helices. Labels of the secondary structure elements adhere to the nomenclature shown in Fig. . d Pore diameter plotted against the coordinate along the vertical axis. Calculations have been performed using the program HOLE (left). The red line indicates the narrowest point in the channel. Two major constrictions along the YaxAB model are emphasized (right)

Article Snippet: The sharpened cryo-EM map of the YaxAB complex has been deposited in the Electron Microscopy Databank under EMD-3885.

Techniques: Cryo-EM Sample Prep, Staining

Journal: Nature Communications

Article Title: Structure and mechanism of the two-component α-helical pore-forming toxin YaxAB

doi: 10.1038/s41467-018-04139-2

Figure Lengend Snippet: YaxA and YaxB interact via cis and trans interfaces within the YaxAB complex. a Cryo-EM map (left) and modeled structure (right) of the cis interface between YaxA (blue) and YaxB (pink) within a radial spoke ( cis ). Boxes labeled I and II indicate contact regions within the protomer pair that are displayed enlarged (right). Residues in interacting proximity are shown as sticks; hydrophobic side chains are colored in gold. Given the medium resolution of the cryo-EM map, the side chains were modeled as ideal rotamers in phenix.real_space_refine. b Depiction as in a , but of the trans interface between YaxA and YaxB from two adjacent radial spokes. Box III indicates the contact region within the protomer pair, colored as in a

Article Snippet: The sharpened cryo-EM map of the YaxAB complex has been deposited in the Electron Microscopy Databank under EMD-3885.

Techniques: Cryo-EM Sample Prep, Labeling

Journal: The EMBO Journal

Article Title: The complete structure of the chloroplast 70S ribosome in complex with translation factor pY

doi: 10.15252/embj.201695959

Figure Lengend Snippet: Structure of the chloroplast 70S ribosome. 50S subunit proteins are in blue, 23S rRNA in cyan, 5S rRNA in green, 4.5S rRNA in red, 30S subunit proteins in gold, 16S rRNA in pale yellow, E‐site tRNA in pink and translation factor pY in green. Plastid‐specific ribosomal proteins cS22, cS23, bTHXc, cL37 and cL38 are shown in red. Protein and rRNA elements conserved between chloroplast and bacterial 70S ribosome are in blue and grey, respectively. Chloroplast‐specific rRNA elements are shown in purple. Plastid‐specific ribosomal proteins and additional protein extensions are in red and yellow, respectively. Translation factor pY is shown in green. Structural landmarks of the 70S ribosome are indicated.

Article Snippet: The 3.4 Å cryo‐EM map of the chloroplast 70S ribosome, the 3.2 Å cryo‐EM map of the 50S subunit and the 3.6 Å cryo‐EM map of the 30S subunit have been deposited in the Electron Microscopy Databank with accession codes EMD‐3533, EMD‐3531 and EMD‐3532, respectively.

Techniques:

Journal: The EMBO Journal

Article Title: The complete structure of the chloroplast 70S ribosome in complex with translation factor pY

doi: 10.15252/embj.201695959

Figure Lengend Snippet: A–C Surface rendering of the final high‐resolution maps of the 30S subunit (A), the 70S ribosome (B) and the 50S 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 α‐helix (yellow) and (O) protein β‐sheet (green).

Article Snippet: The 3.4 Å cryo‐EM map of the chloroplast 70S ribosome, the 3.2 Å cryo‐EM map of the 50S subunit and the 3.6 Å cryo‐EM map of the 30S subunit have been deposited in the Electron Microscopy Databank with accession codes EMD‐3533, EMD‐3531 and EMD‐3532, respectively.

Techniques: Cryo-EM Sample Prep

Journal: The EMBO Journal

Article Title: The complete structure of the chloroplast 70S ribosome in complex with translation factor pY

doi: 10.15252/embj.201695959

Figure Lengend Snippet: 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 , ).

Article Snippet: The 3.4 Å cryo‐EM map of the chloroplast 70S ribosome, the 3.2 Å cryo‐EM map of the 50S subunit and the 3.6 Å cryo‐EM map of the 30S subunit have been deposited in the Electron Microscopy Databank with accession codes EMD‐3533, EMD‐3531 and EMD‐3532, respectively.

Techniques: Binding Assay

Journal: The EMBO Journal

Article Title: The complete structure of the chloroplast 70S ribosome in complex with translation factor pY

doi: 10.15252/embj.201695959

Figure Lengend Snippet: 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 (0.5 kb), B (1.2 kb) and C (1.1 kb). 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 H2 and 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 4 Å, 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 , ).

Article Snippet: The 3.4 Å cryo‐EM map of the chloroplast 70S ribosome, the 3.2 Å cryo‐EM map of the 50S subunit and the 3.6 Å cryo‐EM map of the 30S subunit have been deposited in the Electron Microscopy Databank with accession codes EMD‐3533, EMD‐3531 and EMD‐3532, respectively.

Techniques: Agarose Gel Electrophoresis, Solvent, Binding Assay, Sequencing

Journal: The EMBO Journal

Article Title: The complete structure of the chloroplast 70S ribosome in complex with translation factor pY

doi: 10.15252/embj.201695959

Figure Lengend Snippet: Binding of plastid translation factor pY, shown in lime green, to the mRNA channel of the small subunit. The small subunit is shown from the intersubunit side. The 16S rRNA is coloured in pale yellow, and ribosomal proteins are in gold. EM density for plastid pY. Secondary structure elements and N‐ and C‐termini are indicated. Molecular interaction of plastid pY with 16S rRNA. The conserved nucleotides involved in A‐site decoding are coloured in red and labelled (bacterial numbering in brackets). The bacterial nucleotides A1492 and A1493 of the empty 30S subunit (PDB 1J5E; Wimberly et al , ) and of the 70S ribosome in complex with mRNA and tRNAs (PDB 4V51; Selmer et al , ) are overlaid and coloured in purple and blue, respectively. Pro127 and Arg119 of plastid pY are indicated. Superposition of the chloroplast 70S:pY complex with bacterial A‐, P‐ and E‐site tRNAs and mRNA from the crystal structure of the Thermus thermophilus 70S ribosome (PDB 4V51; Selmer et al , ).

Article Snippet: The 3.4 Å cryo‐EM map of the chloroplast 70S ribosome, the 3.2 Å cryo‐EM map of the 50S subunit and the 3.6 Å cryo‐EM map of the 30S subunit have been deposited in the Electron Microscopy Databank with accession codes EMD‐3533, EMD‐3531 and EMD‐3532, respectively.

Techniques: Binding Assay

Journal: The EMBO Journal

Article Title: The complete structure of the chloroplast 70S ribosome in complex with translation factor pY

doi: 10.15252/embj.201695959

Figure Lengend Snippet: 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, d2 and 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 , ) 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).

Article Snippet: The 3.4 Å cryo‐EM map of the chloroplast 70S ribosome, the 3.2 Å cryo‐EM map of the 50S subunit and the 3.6 Å cryo‐EM map of the 30S subunit have been deposited in the Electron Microscopy Databank with accession codes EMD‐3533, EMD‐3531 and EMD‐3532, respectively.

Techniques: Comparison, Cryo-EM Sample Prep, Residue