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- EMDB-29485: E. coli 70S ribosome with an improved MS2 tag inserted in H98 (30... -

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Basic information

Entry
Database: EMDB / ID: EMD-29485
TitleE. coli 70S ribosome with an improved MS2 tag inserted in H98 (30S focused map)
Map dataE. coli 70S ribosome with an improved MS2 tag inserted in H98 (30S focused map)
Sample
  • Complex: E. coli 70S ribosome
    • Complex: 50S SubunitProkaryotic large ribosomal subunit
    • Complex: 30S SubunitProkaryotic small ribosomal subunit
KeywordsMS2-tag / H98 / ribosome
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.98 Å
AuthorsNissley AJ / Cate JHD
Funding support United States, 1 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)CHE-200218 United States
CitationJournal: RNA / Year: 2023
Title: Interactions between terminal ribosomal RNA helices stabilize the large ribosomal subunit.
Authors: Amos J Nissley / Tammam S Kamal / Jamie H D Cate /
Abstract: The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding. In vivo assembled ribosomes have been isolated using MS2 tags ...The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding. In vivo assembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and function in vitro. RNA tags in the 50S subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth or in vitro ribosome activity. Here, we find that 50S subunits with MS2 tags inserted in H98 are destabilized compared to wild-type (WT) 50S subunits. We identify the loss of RNA-RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes.
History
DepositionJan 18, 2023-
Header (metadata) releaseAug 2, 2023-
Map releaseAug 2, 2023-
UpdateOct 4, 2023-
Current statusOct 4, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29485.png

Downloads & links

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Map

FileDownload / File: emd_29485.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationE. coli 70S ribosome with an improved MS2 tag inserted in H98 (30S focused map)
Voxel sizeX=Y=Z: 0.8248 Å
Density
Contour LevelBy AUTHOR: 0.654
Minimum - Maximum-2.5659409 - 6.446554
Average (Standard dev.)0.0043847864 (±0.15431066)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 369.5104 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: E. coli 70S ribosome with an improved MS2...

Fileemd_29485_half_map_1.map
AnnotationE. coli 70S ribosome with an improved MS2 tag inserted in H98 (30S focused half map)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: E. coli 70S ribosome with an improved MS2...

Fileemd_29485_half_map_2.map
AnnotationE. coli 70S ribosome with an improved MS2 tag inserted in H98 (30S focused half 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 : E. coli 70S ribosome

EntireName: E. coli 70S ribosome
Components
  • Complex: E. coli 70S ribosome
    • Complex: 50S SubunitProkaryotic large ribosomal subunit
    • Complex: 30S SubunitProkaryotic small ribosomal subunit

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Supramolecule #1: E. coli 70S ribosome

SupramoleculeName: E. coli 70S ribosome / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #2: 50S Subunit

SupramoleculeName: 50S Subunit / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #3: 30S Subunit

SupramoleculeName: 30S Subunit / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Escherichia coli (E. coli)

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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

Concentration0.27 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHEPES
100.0 mMPotassium ChlorideKCl
10.0 mMMagnesium AcetateMg(CH3COO)2
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 11736 / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2475999
Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 1.98 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 601617
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT

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