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- EMDB-15796: Translating 70S ribosome in the unrotated state (A and P tRNAs) -

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Basic information

Entry
Database: EMDB / ID: EMD-15796
TitleTranslating 70S ribosome in the unrotated state (A and P tRNAs)
Map dataPost-processed (localfilter from cryoSPARC) map from Homogeneous Refinement
Sample
  • Complex: 70S ribosomeRibosome
Keywords70S / bacterial / translation / high-resolution / RIBOSOME
Biological speciesEscherichia coli B (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.2 Å
AuthorsFromm SA / O'Connor KM / Purdy M / Bhatt PR / Loughran G / Atkins JF / Jomaa A / Mattei S
Funding support Ireland, European Union, 4 items
OrganizationGrant numberCountry
Other government
Other government
Irish Research CouncilIRCLA/2019/74 Ireland
iNEXT-Discovery871037European Union
Citation
Journal: Nat Commun / Year: 2023
Title: The translating bacterial ribosome at 1.55 Å resolution generated by cryo-EM imaging services.
Authors: Simon A Fromm / Kate M O'Connor / Michael Purdy / Pramod R Bhatt / Gary Loughran / John F Atkins / Ahmad Jomaa / Simone Mattei /
Abstract: Our understanding of protein synthesis has been conceptualised around the structure and function of the bacterial ribosome. This complex macromolecular machine is the target of important ...Our understanding of protein synthesis has been conceptualised around the structure and function of the bacterial ribosome. This complex macromolecular machine is the target of important antimicrobial drugs, an integral line of defence against infectious diseases. Here, we describe how open access to cryo-electron microscopy facilities combined with bespoke user support enabled structural determination of the translating ribosome from Escherichia coli at 1.55 Å resolution. The obtained structures allow for direct determination of the rRNA sequence to identify ribosome polymorphism sites in the E. coli strain used in this study and enable interpretation of the ribosomal active and peripheral sites at unprecedented resolution. This includes scarcely populated chimeric hybrid states of the ribosome engaged in several tRNA translocation steps resolved at ~2 Å resolution. The current map not only improves our understanding of protein synthesis but also allows for more precise structure-based drug design of antibiotics to tackle rising bacterial resistance.
#1: Journal: Biorxiv / Year: 2022
Title: The translating bacterial ribosome at 1.55 angstrom resolution by open access cryo-EM
Authors: Fromm SA / O'Connor KM / Purdy M / Bhatt PR / Loughran G / Atkins JF / Jomaa A / Mattei S
History
DepositionSep 8, 2022-
Header (metadata) releaseSep 28, 2022-
Map releaseSep 28, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15796.png

Downloads & links

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Map

FileDownload / File: emd_15796.map.gz / Format: CCP4 / Size: 669.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed (localfilter from cryoSPARC) map from Homogeneous Refinement
Voxel sizeX=Y=Z: 0.731 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-1.6716723 - 3.5394778
Average (Standard dev.)0.008030669 (±0.06823565)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions560560560
Spacing560560560
CellA=B=C: 409.36002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15796_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_15796_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_15796_half_map_2.map
Annotationhalf 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 : 70S ribosome

EntireName: 70S ribosomeRibosome
Components
  • Complex: 70S ribosomeRibosome

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Supramolecule #1: 70S ribosome

SupramoleculeName: 70S ribosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#53
Source (natural)Organism: Escherichia coli B (bacteria)
Molecular weightTheoretical: 2.5 MDa

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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

Concentration0.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationName
50.0 mMHEPES
100.0 mMKOAc
25.0 mMMg(OAc)2
1.0 mMDTT
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.2 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER / Details: Fischione 1070
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 279 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: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 165000
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 19449 / Average exposure time: 5.2 sec. / Average electron dose: 40.0 e/Å2
Details: saved in EER format; 16 exposures per hole/stage movement. 11 in outer ring around the hole edge, 5 in inner ring around the hole center.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1579392
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Details: Ab-initio reconstruction in cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
Details: 3D Variability analysis in cryoSPARC; analyzed in cluster mode
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Details: as implemented in cryoSPARC Homogeneous Refinement
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 16903
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL

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