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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
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: To Be Published
Title: Structural evidence of context-dependent action of chloramphenicol in cells
Authors: Xue L / Spahn C / Schacherl M / Mahamid J
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_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

Size
Brightness
Contrast
Others
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

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.1 Å
Density

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
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_17145_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_17145_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 : 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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