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- EMDB-17147: free 50S with additional S4-LSU in native untreated Mycoplasma pn... -

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Basic information

Entry
Database: EMDB / ID: EMD-17147
Titlefree 50S with additional S4-LSU in native untreated Mycoplasma pneumoniae cells
Map data
Sample
  • Cell: Native untreated Mycoplasma pneumoniae M129 cells
KeywordsIn situ / Ribosome heterogeneity / S4 / cryo-ET / RIBOSOME
Biological speciesMycoplasmoides pneumoniae M129 (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 7.9 Å
AuthorsXue L / Mahamid J
Funding supportEuropean Union, Germany, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)760067European Union
German Research Foundation (DFG) Germany
CitationJournal: 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.
History
DepositionApr 17, 2023-
Header (metadata) releaseOct 30, 2024-
Map releaseOct 30, 2024-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17147.png

Downloads & links

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Map

FileDownload / File: emd_17147.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.4 Å/pix.
x 200 pix.
= 480. Å		2.4 Å/pix.
x 200 pix.
= 480. Å		2.4 Å/pix.
x 200 pix.
= 480. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.6
Minimum - Maximum-0.57092667 - 1.6986039
Average (Standard dev.)0.015714282 (±0.10613408)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 480.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17147_msk_1.map
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Half map: #1

Fileemd_17147_half_map_1.map
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Half map: #2

Fileemd_17147_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Sample components

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Entire : Native untreated Mycoplasma pneumoniae M129 cells

EntireName: Native untreated Mycoplasma pneumoniae M129 cells
Components
  • Cell: Native untreated Mycoplasma pneumoniae M129 cells

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Supramolecule #1: Native untreated Mycoplasma pneumoniae M129 cells

SupramoleculeName: Native untreated Mycoplasma pneumoniae M129 cells / type: cell / ID: 1 / Parent: 0
Details: cryo-electron tomograms of untreated Mycoplasma pneumoniae cells growing in the fast-growing phase
Source (natural)Organism: Mycoplasmoides 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
Details: The modified Hayflick medium: 14.7g/L Difco PPLO(Becton Dickinson), 20% (v/v) Gibco horse serum(New Zealand origin), 100 mM HEPES-Na; pH 7.4, 1% (w/w) glucose, 0.002% (w/w) phenol red, 1000 U/mL penicillin G.
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE
Details: Back-side blotting for 2-3 second before plunging using a manual plunger without an environmental chamber..
DetailsMycoplasma pneumoniae M129 cells grown on gold Quantifoil grids at 37 degrees Celsius before plunge freezing.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 3.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.7) / Number subtomograms used: 5791
ExtractionNumber tomograms: 356 / Number images used: 24157 / Software: (Name: PyTom (ver. 0.9.7.1), Warp (ver. 1.0.7))
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 3.0.7)
Details: 3D classification in RELION using a spherical mask focusing on the extra S4 on the large ribosomal subunit. Multiple jobs were performed.
Final angle assignmentType: OTHER / Software: (Name: RELION (ver. 3.0.7), Warp (ver. 1.0.7))
FSC plot (resolution estimation)

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