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- EMDB-17145: polysome/di-ribosome class III in chloramphenicol-treated Mycopla... -

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Basic information

Entry
Database: EMDB / ID: EMD-17145
Titlepolysome/di-ribosome class III in chloramphenicol-treated Mycoplasma pneumoniae cells
Map data
Sample
  • Cell: Mycoplasma pneumoniae M129 cells treated with chloramphenicol
KeywordsIn situ / cryo-electron tomography / bacterial ribosome / Chloramphenicol / antibiotic / polysome / RIBOSOME
Function / homology
Function and homology information


RNA polymerase II CTD heptapeptide repeat P3 isomerase activity / RNA polymerase II CTD heptapeptide repeat P6 isomerase activity / peptidylprolyl isomerase / protein transport / protein folding / regulation of translation / large ribosomal subunit / transferase activity / ribosomal small subunit assembly / ribosomal small subunit biogenesis ...RNA polymerase II CTD heptapeptide repeat P3 isomerase activity / RNA polymerase II CTD heptapeptide repeat P6 isomerase activity / peptidylprolyl isomerase / protein transport / protein folding / regulation of translation / large ribosomal subunit / transferase activity / ribosomal small subunit assembly / ribosomal small subunit biogenesis / small ribosomal subunit / small ribosomal subunit rRNA binding / 5S rRNA binding / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / translation / ribonucleoprotein complex / cell division / response to antibiotic / mRNA binding / RNA binding / zinc ion binding / cytosol / cytoplasm
Similarity search - Function
Trigger factor / Trigger factor, C-terminal / Trigger factor, ribosome-binding, bacterial / Trigger factor ribosome-binding domain superfamily / Bacterial trigger factor protein (TF) / Bacterial trigger factor protein (TF) C-terminus / Trigger factor, C-terminal domain superfamily / Trigger factor/SurA domain superfamily / Ribosomal protein L1, bacterial-type / Ribosomal protein L10, eubacterial, conserved site ...Trigger factor / Trigger factor, C-terminal / Trigger factor, ribosome-binding, bacterial / Trigger factor ribosome-binding domain superfamily / Bacterial trigger factor protein (TF) / Bacterial trigger factor protein (TF) C-terminus / Trigger factor, C-terminal domain superfamily / Trigger factor/SurA domain superfamily / Ribosomal protein L1, bacterial-type / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / : / : / Ribosomal protein S14, type Z / FKBP-type peptidyl-prolyl cis-trans isomerase domain profile. / FKBP-type peptidyl-prolyl cis-trans isomerase domain / FKBP-type peptidyl-prolyl cis-trans isomerase / Ribosomal protein L1, conserved site / Ribosomal protein L1 signature. / Ribosomal protein L1 / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Peptidyl-prolyl cis-trans isomerase domain superfamily / Ribosomal protein L11, bacterial-type / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L1p/L10e family / Ribosomal protein L31 type A / Ribosomal protein S21 / Ribosomal protein L31 signature. / Ribosomal protein L31 / Ribosomal protein L31 superfamily / Ribosomal protein L31 / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / Ribosomal protein L10-like domain superfamily / Ribosomal protein L10P / Ribosomal protein L10 / Ribosomal protein L16 signature 1. / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L16, conserved site / Ribosomal protein L16 signature 2. / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / : / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L11, N-terminal / Ribosomal protein L11, N-terminal domain / Ribosomal protein L17 signature. / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11/L12 / Ribosomal protein L11, RNA binding domain / Ribosomal protein L36 signature. / Ribosomal protein L28/L24 superfamily / Ribosomal protein L32p, bacterial type / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / Ribosomal protein L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / : / Ribosomal protein L28 / Ribosomal protein L35, non-mitochondrial / Ribosomal protein L5, bacterial-type / Ribosomal protein L18, bacterial-type / : / Ribosomal protein L6, bacterial-type / Ribosomal protein S3, bacterial-type / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein S19, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein S14/S29 / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S9, bacterial/plastid / Ribosomal protein L36 / Ribosomal protein L36 superfamily / Ribosomal protein L36 / Ribosomal protein S11, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein S4, bacterial-type / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / 30S ribosomal protein S17 / Ribosomal protein S5, bacterial-type / Ribosomal protein L27, conserved site
Similarity search - Domain/homology
Small ribosomal subunit protein bS16 / Large ribosomal subunit protein uL16 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Large ribosomal subunit protein bL36 / Small ribosomal subunit protein bS21 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL28 / Small ribosomal subunit protein uS15 / Large ribosomal subunit protein uL13 ...Small ribosomal subunit protein bS16 / Large ribosomal subunit protein uL16 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Large ribosomal subunit protein bL36 / Small ribosomal subunit protein bS21 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL28 / Small ribosomal subunit protein uS15 / Large ribosomal subunit protein uL13 / Small ribosomal subunit protein uS9 / Small ribosomal subunit protein bS20 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein bL35 / Trigger factor / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL9 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Small ribosomal subunit protein uS12 / Large ribosomal subunit protein uL11 / Small ribosomal subunit protein uS2 / Large ribosomal subunit protein uL22 / Small ribosomal subunit protein uS19 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL3 / Small ribosomal subunit protein uS10 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL33A / Large ribosomal subunit protein bL31 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL1 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS13 / Large ribosomal subunit protein uL15 / Small ribosomal subunit protein uS5 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL6 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS14 / Large ribosomal subunit protein uL5 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL14 / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL17
Similarity search - Component
Biological speciesMycoplasmoides pneumoniae M129 (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 8.7 Å
AuthorsXue L / Spahn C / Schacherl M / Mahamid J
Funding support United States, Germany, 2 items
OrganizationGrant numberCountry
Chan Zuckerberg InitiativeVisual Proteomics United States
German Research Foundation (DFG) Germany
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Structural insights into context-dependent inhibitory mechanisms of chloramphenicol in cells.
Authors: Liang Xue / Christian M T Spahn / Magdalena Schacherl / Julia Mahamid /
Abstract: Ribosome-targeting antibiotics represent an important class of antimicrobial drugs. Chloramphenicol (Cm) is a well-studied ribosomal peptidyl transferase center (PTC) binder and growing evidence ...Ribosome-targeting antibiotics represent an important class of antimicrobial drugs. Chloramphenicol (Cm) is a well-studied ribosomal peptidyl transferase center (PTC) binder and growing evidence suggests that its inhibitory action depends on the sequence of the nascent peptide. How such selective inhibition on the molecular scale manifests on the cellular level remains unclear. Here, we use cryo-electron tomography to analyze the impact of Cm inside the bacterium Mycoplasma pneumoniae. By resolving the Cm-bound ribosomes to 3.0 Å, we elucidate Cm's coordination with natural nascent peptides and transfer RNAs in the PTC. We find that Cm leads to the accumulation of a number of translation elongation states, indicating ongoing futile accommodation cycles, and to extensive ribosome collisions. We, thus, suggest that, beyond its direct inhibition of protein synthesis, the action of Cm may involve the activation of cellular stress responses. This work exemplifies how in-cell structural biology can expand the understanding of mechanisms of action for extensively studied antibiotics.
History
DepositionApr 17, 2023-
Header (metadata) releaseOct 30, 2024-
Map releaseOct 30, 2024-
UpdateFeb 26, 2025-
Current statusFeb 26, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17145.png

Downloads & links

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Map

FileDownload / File: emd_17145.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
3.1 Å/pix.
x 256 pix.
= 793.6 Å		3.1 Å/pix.
x 256 pix.
= 793.6 Å		3.1 Å/pix.
x 256 pix.
= 793.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 3.1 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.2
Minimum - Maximum-2.425156 - 5.4774103
Average (Standard dev.)0.021869715 (±0.23697715)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 793.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17145_msk_1.map
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Half map: #1

Fileemd_17145_half_map_1.map
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Half map: #2

Fileemd_17145_half_map_2.map
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Sample components

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Entire : Mycoplasma pneumoniae M129 cells treated with chloramphenicol

EntireName: Mycoplasma pneumoniae M129 cells treated with chloramphenicol
Components
  • Cell: Mycoplasma pneumoniae M129 cells treated with chloramphenicol

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Supramolecule #1: Mycoplasma pneumoniae M129 cells treated with chloramphenicol

SupramoleculeName: Mycoplasma pneumoniae M129 cells treated with chloramphenicol
type: cell / ID: 1 / Parent: 0
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: 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 / Instrument: HOMEMADE PLUNGER
Details: Back-side blotting for 2-3 seconds before plunging using a manual plunger without an environmental control chamber..
DetailsMycoplasma pneumoniae M129 cells were grown on gold Quantifoil grids at 37 Celsius in modified Hayflick medium. Treatment with chloramphenicol at a final concentration of 0.2 mg/ml was performed for approximately 15 minutes 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 K3 BIOQUANTUM (6k x 4k) / Number real images: 1 / Average electron dose: 3.34 e/Å2
Details: Gatan K3 camera in non-CDS counting mode, targeted dose rate on camera ~20 e/pixel/second, 10 frames per tilt image, constant exposure time for each tilt, pixel size 1.329A
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: 3.25 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 64000
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

Details30774 sub-tomograms with large box size (3.1A/voxel pixel and box 256) were extracted, which can accommodate the neighbouring ribosomes(disomes).
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.7 Å / Resolution method: FSC 0.143 CUT-OFF
Software:
Namedetails
Warp (ver. 1.0.9)Warp/M
RELION (ver. 3.0.8)

Number subtomograms used: 963
ExtractionNumber tomograms: 139 / Number images used: 30774 / Software: (Name: PyTom (ver. 0.9.7.1), Warp (ver. 1.0.9))
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.0.8) / Software - details: Class3D to remove bad particles
Final angle assignmentType: MAXIMUM LIKELIHOOD
Software:
Namedetails
RELION (ver. 3.0.8)Refine3D
Warp (ver. 1.0.9)Warp/M uses RELION alignments as inputs and do multi-particle refinement
FSC plot (resolution estimation)

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