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- EMDB-17432: Escherichia coli paused disome complex (rotated-PRE-2 | POST) -

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Basic information

Entry
Database: EMDB / ID: EMD-17432
TitleEscherichia coli paused disome complex (rotated-PRE-2 | POST)
Map dataCryo-EM reconstruction of the E. coli disome complex (rotated-PRE-2 | POST)
Sample
  • Complex: Escherichia coli paused disome complex
Keywordsribosome / polysome / tranlsation / elongation / pausing / disome / collision / PURE system
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.6 Å
AuthorsFluegel T / Schacherl M
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG) Germany
CitationJournal: Nat Commun / Year: 2024
Title: Transient disome complex formation in native polysomes during ongoing protein synthesis captured by cryo-EM.
Authors: Timo Flügel / Magdalena Schacherl / Anett Unbehaun / Birgit Schroeer / Marylena Dabrowski / Jörg Bürger / Thorsten Mielke / Thiemo Sprink / Christoph A Diebolder / Yollete V Guillén ...Authors: Timo Flügel / Magdalena Schacherl / Anett Unbehaun / Birgit Schroeer / Marylena Dabrowski / Jörg Bürger / Thorsten Mielke / Thiemo Sprink / Christoph A Diebolder / Yollete V Guillén Schlippe / Christian M T Spahn /
Abstract: Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex ...Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex vivo-derived E. coli polysomes in the PURE in vitro translation system and analyzed the actively elongating polysomes by cryo-EM. We find that 31% of 70S ribosomes assemble into disome complexes that represent eight distinct functional states including decoding and termination intermediates, and a pre-nucleophilic attack state. The functional diversity of disome complexes together with RNase digest experiments suggests that paused disome complexes transiently form during ongoing elongation. Structural analysis revealed five disome interfaces between leading and queueing ribosomes that undergo rearrangements as the leading ribosome traverses through the elongation cycle. Our findings reveal at the molecular level how bL9's CTD obstructs the factor binding site of queueing ribosomes to thwart harmful collisions and illustrate how translation dynamics reshape inter-ribosomal contacts.
History
DepositionMay 23, 2023-
Header (metadata) releaseMar 6, 2024-
Map releaseMar 6, 2024-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17432.png

Downloads & links

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Map

FileDownload / File: emd_17432.map.gz / Format: CCP4 / Size: 48.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of the E. coli disome complex (rotated-PRE-2 | POST)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.18 Å/pix.
x 234 pix.
= 744.12 Å		3.18 Å/pix.
x 234 pix.
= 744.12 Å		3.18 Å/pix.
x 234 pix.
= 744.12 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.18 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.0
Minimum - Maximum-4.9226866 - 14.460637999999999
Average (Standard dev.)-0.000000000614605 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions234234234
Spacing234234234
CellA=B=C: 744.12 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17432_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM reconstruction of the E. coli disome complex...

Fileemd_17432_half_map_1.map
AnnotationCryo-EM reconstruction of the E. coli disome complex (rotated-PRE-2 | POST). Half map A.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM reconstruction of the E. coli disome complex...

Fileemd_17432_half_map_2.map
AnnotationCryo-EM reconstruction of the E. coli disome complex (rotated-PRE-2 | POST). Half map B.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Escherichia coli paused disome complex

EntireName: Escherichia coli paused disome complex
Components
  • Complex: Escherichia coli paused disome complex

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Supramolecule #1: Escherichia coli paused disome complex

SupramoleculeName: Escherichia coli paused disome complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#60
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE 600

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 75 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 1.13 sec. / Average electron dose: 45.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1883839
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3) / Number images used: 5677
FSC plot (resolution estimation)

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