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- EMDB-30214: State 1 of pre50SH ribosome from RrmJ knock out E.coli stain -

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Basic information

Entry
Database: EMDB / ID: EMD-30214
TitleState 1 of pre50SH ribosome from RrmJ knock out E.coli stain
Map dataState 1
Sample
  • Complex: State 1 of pre50SH ribosome
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.64 Å
AuthorsWang W / Li WQ / Ge XL / Yan KG / Mandava CS / Sanyal S / Gao N
Funding support China, 5 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31630087 China
Ministry of Science and Technology (MoST, China)2019YFA0508900 China
National Natural Science Foundation of China (NSFC)31725007 China
Other governmentShenzhen Science and Technology Innovation Committee (JCYJ20180302174213122) China
Other governmentChina Postdoctoral Science Foundation (1131000065) China
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Loss of a single methylation in 23S rRNA delays 50S assembly at multiple late stages and impairs translation initiation and elongation.
Authors: Wei Wang / Wanqiu Li / Xueliang Ge / Kaige Yan / Chandra Sekhar Mandava / Suparna Sanyal / Ning Gao /
Abstract: Ribosome biogenesis is a complex process, and dozens of factors are required to facilitate and regulate the subunit assembly in bacteria. The 2'-O-methylation of U2552 in 23S rRNA by ...Ribosome biogenesis is a complex process, and dozens of factors are required to facilitate and regulate the subunit assembly in bacteria. The 2'-O-methylation of U2552 in 23S rRNA by methyltransferase RrmJ is a crucial step in late-stage assembly of the 50S subunit. Its absence results in severe growth defect and marked accumulation of pre50S assembly intermediates. In the present work, we employed cryoelectron microscopy to characterize a set of late-stage pre50S particles isolated from an Δ strain. These assembly intermediates (solved at 3.2 to 3.8 Å resolution) define a collection of late-stage particles on a progressive assembly pathway. Apart from the absence of L16, L35, and L36, major structural differences between these intermediates and the mature 50S subunit are clustered near the peptidyl transferase center, such as H38, H68-71, and H89-93. In addition, the ribosomal A-loop of the mature 50S subunit from Δ strain displays large local flexibility on nucleotides next to unmethylated U2552. Fast kinetics-based biochemical assays demonstrate that the Δ 50S subunit is only 50% active and two times slower than the WT 50S subunit in rapid subunit association. While the Δ 70S ribosomes show no defect in peptide bond formation, peptide release, and ribosome recycling, they translocate with 20% slower rate than the WT ribosomes in each round of elongation. These defects amplify during synthesis of the full-length proteins and cause overall defect in protein synthesis. In conclusion, our data reveal the molecular roles of U2552 methylation in both ribosome biogenesis and protein translation.
History
DepositionApr 9, 2020-
Header (metadata) releaseJul 1, 2020-
Map releaseJul 1, 2020-
UpdateJul 22, 2020-
Current statusJul 22, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.046
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.046
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30214.png

Downloads & links

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Map

FileDownload / File: emd_30214.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationState 1
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 320 pix.
= 345.6 Å		1.08 Å/pix.
x 320 pix.
= 345.6 Å		1.08 Å/pix.
x 320 pix.
= 345.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.046 / Movie #1: 0.046
Minimum - Maximum-0.107864805 - 0.2032672
Average (Standard dev.)0.0004454462 (±0.011624338)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z345.600345.600345.600
α/β/γ90.00090.00090.000
start NX/NY/NZ434333
NX/NY/NZ116118137
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1080.2030.000

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Supplemental data

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Sample components

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Entire : State 1 of pre50SH ribosome

EntireName: State 1 of pre50SH ribosome
Components
  • Complex: State 1 of pre50SH ribosome

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Supramolecule #1: State 1 of pre50SH ribosome

SupramoleculeName: State 1 of pre50SH ribosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#32
Details: State 1 of pre50SH ribosome from RrmJ knock out E.coli strains
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 1.5 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

Concentration0.15 mg/mL
BufferpH: 7.5
Details: Solutions were made fresh form concentrated to avoid microbial contamination.
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 7000.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa
Details: Grids were coated with a homemade continuous thin carbon film.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 2 seconds before plunging.
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DetailsPreliminary grid screening was performed manually.
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Frames/image: 3-28 / Number grids imaged: 1 / Number real images: 1262 / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 75000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 75000
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

DetailsThe images were normalized and manually selected.
Particle selectionNumber selected: 235600
Details: Images were binned and lowpass filtered for autopicking.
CTF correctionSoftware - Name: CTFFIND (ver. 3.0)
Details: CTF amplitude correction was performed following 3D reconstruction.
Startup modelType of model: OTHER
Details: The model was a low resolution 50S ribosome generated from our own data.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Resolution.type: BY AUTHOR / Resolution: 3.64 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 13661
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 1.4) / Details: Relion software was used in the reconstruction.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 1.4) / Details: Relion software was used in the reconstruction.
Final 3D classificationNumber classes: 6 / Avg.num./class: 5000 / Software - Name: RELION (ver. 1.4)

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Atomic model buiding 1

Initial model(PDB ID:

4ybb

,

5h5u

)
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient

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