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- EMDB-17133: 3.2Angstrom 30S ribosome focused-refined map in chloramphenicol-t... -

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Basic information

Entry
Database: EMDB / ID: EMD-17133
Title3.2Angstrom 30S ribosome focused-refined map in chloramphenicol-treated Mycoplasma pneumoniae cells
Map dataRELION postprocess, masked
Sample
  • Cell: Mycoplasma pneumoniae M129 cells treated with chloramphenicol
KeywordsIn situ / Ribosome / Chloramphenicol / cryo-ET
Function / homology
Function and homology information


ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / cytosolic small ribosomal subunit / small ribosomal subunit rRNA binding / tRNA binding / rRNA binding / ribosome / structural constituent of ribosome / translation ...ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / cytosolic small ribosomal subunit / small ribosomal subunit rRNA binding / tRNA binding / rRNA binding / ribosome / structural constituent of ribosome / translation / ribonucleoprotein complex / mRNA binding / RNA binding / zinc ion binding / cytosol / cytoplasm
Similarity search - Function
Ribosomal protein S14, type Z / Ribosomal protein L31 type A / Ribosomal protein L31 signature. / Ribosomal protein L31 / Ribosomal protein L31 superfamily / Ribosomal protein L31 / Ribosomal protein S21 / Ribosomal protein S19, bacterial-type / Ribosomal protein S3, bacterial-type / Ribosomal protein S13, bacterial-type ...Ribosomal protein S14, type Z / Ribosomal protein L31 type A / Ribosomal protein L31 signature. / Ribosomal protein L31 / Ribosomal protein L31 superfamily / Ribosomal protein L31 / Ribosomal protein S21 / Ribosomal protein S19, bacterial-type / Ribosomal protein S3, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S11, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein S4, bacterial-type / 30S ribosomal protein S17 / Ribosomal protein S5, bacterial-type / Ribosomal protein S14/S29 / Ribosomal protein S2, bacteria/mitochondria/plastid / Ribosomal protein S18, conserved site / Ribosomal protein S18 signature. / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein S16 / Ribosomal protein S16 domain superfamily / Ribosomal protein S16 / L28p-like / Ribosomal protein S15, bacterial-type / Ribosomal protein S12, bacterial-type / Ribosomal protein S18 / Ribosomal protein S18 / Ribosomal protein S18 superfamily / K Homology domain / K homology RNA-binding domain / Ribosomal protein S6 / Ribosomal protein S6 / Ribosomal protein S6 superfamily / Ribosomal protein S2 signature 2. / Translation elongation factor EF1B/ribosomal protein S6 / Ribosomal protein S3, conserved site / Ribosomal protein S3 signature. / Ribosomal protein S10, conserved site / Ribosomal protein S10 signature. / Ribosomal protein S14, conserved site / Ribosomal protein S14 signature. / KH domain / Type-2 KH domain profile. / K Homology domain, type 2 / Ribosomal protein S3, C-terminal / Ribosomal protein S3, C-terminal domain / Ribosomal protein S3, C-terminal domain superfamily / Ribosomal protein S15/S19, conserved site / Ribosomal protein S19 signature. / Ribosomal protein S10 / Ribosomal protein S19/S15 / Ribosomal protein S19/S15, superfamily / Ribosomal protein S19 / : / Ribosomal protein S5, N-terminal, conserved site / Ribosomal protein S5 signature. / Ribosomal protein S7, conserved site / Ribosomal protein S2, conserved site / Ribosomal protein S7 signature. / Ribosomal protein S2 / Ribosomal protein S2, flavodoxin-like domain superfamily / Ribosomal protein S2 / Ribosomal protein S17, conserved site / Ribosomal protein S17 signature. / K homology domain superfamily, prokaryotic type / Ribosomal protein S5 / Ribosomal protein S13, conserved site / Ribosomal protein S13 signature. / S5 double stranded RNA-binding domain profile. / Ribosomal protein S5, N-terminal / Ribosomal protein S13 / 30s ribosomal protein S13, C-terminal / Ribosomal protein S13/S18 / Ribosomal protein S5, C-terminal / Ribosomal protein S13 family profile. / Ribosomal protein S5, N-terminal domain / Ribosomal protein S5, C-terminal domain / Ribosomal protein S8 signature. / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S4, conserved site / Ribosomal protein S15 signature. / Ribosomal protein S4 signature. / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S4/S9, N-terminal / Ribosomal protein S14 / Ribosomal protein S14p/S29e / Ribosomal protein S4/S9 / K homology domain-like, alpha/beta / Ribosomal protein S8 / Ribosomal protein S8 superfamily / Ribosomal protein S8 / S4 RNA-binding domain profile. / Ribosomal protein S13-like, H2TH
Similarity search - Domain/homology
Small ribosomal subunit protein bS16 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein bS21 / Small ribosomal subunit protein uS15 / Small ribosomal subunit protein uS9 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 ...Small ribosomal subunit protein bS16 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein bS21 / Small ribosomal subunit protein uS15 / Small ribosomal subunit protein uS9 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein uS10 / Large ribosomal subunit protein bL31 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17
Similarity search - Component
Biological speciesMycoplasmoides pneumoniae M129 (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 3.2 Å
AuthorsXue L / Spahn C / Schacherl M / Mahamid J
Funding support United States, Germany, 2 items
OrganizationGrant numberCountry
Chan Zuckerberg InitiativeVisual Proteomics Imaging 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_17133.png

Downloads & links

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Map

FileDownload / File: emd_17133.map.gz / Format: CCP4 / Size: 255.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRELION postprocess, masked
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.33 Å/pix.
x 406 pix.
= 539.574 Å		1.33 Å/pix.
x 406 pix.
= 539.574 Å		1.33 Å/pix.
x 406 pix.
= 539.574 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.329 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0022
Minimum - Maximum-0.009426564 - 0.016385427
Average (Standard dev.)0.0000069670245 (±0.00022265867)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions406406406
Spacing406406406
CellA=B=C: 539.574 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17133_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: RELION postprocess, unmasked map

Fileemd_17133_additional_1.map
AnnotationRELION postprocess, unmasked map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: postprocess in M

Fileemd_17133_additional_2.map
Annotationpostprocess in M
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map from M

Fileemd_17133_half_map_1.map
Annotationhalf map from M
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map from M

Fileemd_17133_half_map_2.map
Annotationhalf map from M
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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: 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 / 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 the modified Hayflick medium. Treatment with chloramphenicol at the final concentration of 0.2 mg/ml was performed for about 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

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Warp (ver. 1.0.9) / Software - details: Warp/M / Details: half maps from Warp/M / Number subtomograms used: 30774
ExtractionNumber tomograms: 139 / Number images used: 30774 / Software: (Name: PyTom (ver. 0.9.7.1), Warp (ver. 1.0.9))
Final 3D classificationSoftware - 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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