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- EMDB-19098: Escherichia coli paused disome complex (local refinement of leadi... -

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Basic information

Entry
Database: EMDB / ID: EMD-19098
TitleEscherichia coli paused disome complex (local refinement of leading 70S open non-rotated PRE)
Map dataCryo-EM reconstruction of the E. coli disome complex (local refinement of leading 70S open non-rotated PRE). Full map.
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: 3.79 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
DepositionDec 11, 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_19098.png

Downloads & links

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Map

FileDownload / File: emd_19098.map.gz / Format: CCP4 / Size: 391 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of the E. coli disome complex (local refinement of leading 70S open non-rotated PRE). Full map.
Voxel sizeX=Y=Z: 1.59 Å
Density
Contour LevelBy AUTHOR: 4.5
Minimum - Maximum-6.1386466 - 20.024794
Average (Standard dev.)-0.000000002895628 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions468468468
Spacing468468468
CellA=B=C: 744.12 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19098_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_19098_half_map_1.map
AnnotationCryo-EM reconstruction of the E. coli disome complex (local refinement of leading 70S open non-rotated PRE). 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_19098_half_map_2.map
AnnotationCryo-EM reconstruction of the E. coli disome complex (local refinement of leading 70S open non-rotated PRE). 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-#33, #35-#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 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 75 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Withdrawn samples were spotted directly onto freshly glow-discharged holey carbon grids, blotted for 1-2 s, and flash frozen in liquid ethane using a Vitrobot Mark IV plunger (ThermoFisher ...Details: Withdrawn samples were spotted directly onto freshly glow-discharged holey carbon grids, blotted for 1-2 s, and flash frozen in liquid ethane using a Vitrobot Mark IV plunger (ThermoFisher Scientific) after a wait time of 40 s at 4 degrees Celcius..
DetailsIn vitro translation reactions were performed in the PURE translation system using the PURExpress delta ribosome kit (NEB, #E3313S). Translation reactions were supplemented with 0.8 U/uL RNAsin Plus RNase Inhibitor (Promega, N261B). SolA, factor mix, and RNAsin Plus were combined on ice, followed by a preincubation at 37 degrees Celcius for 2 min, and added directly to polysomes (700 nM final concentration) that had been preincubated at 37 degrees Celsius for 2 min. After 1 min reaction time, 4 uL of the reaction mixture were withdrawn for plunge freezing.

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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
TemperatureMin: 80.0 K / Max: 82.0 K
SoftwareName: EPU (ver. 2.8.1)
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 8982 / 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 / Software - Name: cryoSPARC (ver. 3.3)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.79 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3) / Number images used: 6670
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7n1p


Chain - Source name: PDB / Chain - Initial model type: experimental model
SoftwareName: Coot (ver. 0.9.6)
DetailsThe atomic model of the E. coli 70S ribosome (PDB ID: 7N1P) was initially docked into the postprocessed density maps with UCSF Chimera and manually adjusted in Coot. All models were refined over multiple rounds using the module phenix.real_space_refine in PHENIX and interactive model building and refinement in Coot, using libG restraints for the RNAs.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient

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