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- EMDB-13283: 70S ribosome class with unexplained density near P site in Mycopl... -

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Basic information

Entry
Database: EMDB / ID: EMD-13283
Title70S ribosome class with unexplained density near P site in Mycoplasma pneumoniae cells
Map data
Sample
  • Cell: Cryo-electron tomograms of untreated Mycoplasma pneumoniae cells
Biological speciesMycoplasma pneumoniae M129 (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 16.0 Å
AuthorsXue L / Lenz S / Rappsilber J / Mahamid J
Funding support Germany, United Kingdom, 4 items
OrganizationGrant numberCountry
European Research Council (ERC)760067 Germany
German Research Foundation (DFG)426290502 Germany
Wellcome Trust103139 United Kingdom
Wellcome Trust203149 United Kingdom
Citation
Journal: Nature / Year: 2022
Title: Visualizing translation dynamics at atomic detail inside a bacterial cell.
Authors: Liang Xue / Swantje Lenz / Maria Zimmermann-Kogadeeva / Dimitry Tegunov / Patrick Cramer / Peer Bork / Juri Rappsilber / Julia Mahamid /
Abstract: Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells. Here we use advances in cryo-electron tomography and sub-tomogram analysis to ...Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells. Here we use advances in cryo-electron tomography and sub-tomogram analysis to visualize the structural dynamics of translation inside the bacterium Mycoplasma pneumoniae. To interpret the functional states in detail, we first obtain a high-resolution in-cell average map of all translating ribosomes and build an atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves 13 ribosome states that differ in their conformation and composition. These recapitulate major states that were previously resolved in vitro, and reflect intermediates during active translation. On the basis of these states, we animate translation elongation inside native cells and show how antibiotics reshape the cellular translation landscapes. During translation elongation, ribosomes often assemble in defined three-dimensional arrangements to form polysomes. By mapping the intracellular organization of translating ribosomes, we show that their association into polysomes involves a local coordination mechanism that is mediated by the ribosomal protein L9. We propose that an extended conformation of L9 within polysomes mitigates collisions to facilitate translation fidelity. Our work thus demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.
#1: Journal: Biorxiv / Year: 2021
Title: Visualizing translation dynamics at atomic detail inside a bacterial cell
Authors: Xue L / Lenz S / Zimmermann-Kogadeeva M / Tegunov D / Cramer P / Bork P / Rappsilber J / Mahamid J
History
DepositionJul 30, 2021-
Header (metadata) releaseMay 25, 2022-
Map releaseMay 25, 2022-
UpdateOct 19, 2022-
Current statusOct 19, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13283.png

Downloads & links

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Map

FileDownload / File: emd_13283.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.7005 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.09569202 - 0.8454593
Average (Standard dev.)0.023433996 (±0.09907436)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 435.328 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13283_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_13283_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_13283_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-electron tomograms of untreated Mycoplasma pneumoniae cells

EntireName: Cryo-electron tomograms of untreated Mycoplasma pneumoniae cells
Components
  • Cell: Cryo-electron tomograms of untreated Mycoplasma pneumoniae cells

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Supramolecule #1: Cryo-electron tomograms of untreated Mycoplasma pneumoniae cells

SupramoleculeName: Cryo-electron tomograms of untreated Mycoplasma pneumoniae cells
type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1-#55
Details: Ribosome sub-tomograms extracted in silico from tomograms of intact cell, refinement with a 70S mask
Source (natural)Organism: Mycoplasma pneumoniae M129 (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE-PROPANE / Instrument: HOMEMADE PLUNGER
Details: back-side blotting for 2-3 seconds before plunging using a manual plunger without an environmental chamber.
DetailsMycoplasma pneumoniae M129 cells grown on gold Quantifoil grids at 37 Celsius before plunge freezing.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.75 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 3.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 356 / Number images used: 77539 / Software: (Name: PyTom, Warp (ver. 3.0.7 beta or 3.0.9))
CTF correctionSoftware - Name: RELION (ver. 3.0.8)
Final 3D classificationSoftware - Name: RELION (ver. 3.0.8)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number subtomograms used: 1484
FSC plot (resolution estimation)

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