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- EMDB-5642: Cryo-EM structures of the late-stage assembly intermediates of 50... -

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Entry
Database: EMDB / ID: EMD-5642
TitleCryo-EM structures of the late-stage assembly intermediates of 50S ribosome subunit from YlqF-deficient Bacillus subtilis strain
Map data
SampleAssembly intermediate of 50S ribosome subunit from YlqF-deficient Bacillus subtilis strain:
ribosome-prokaryote
KeywordsRibosome biogenesis / ribosome assembly intermediate / RNA folding
Function / homology
Function and homology information


positive regulation of rRNA processing / nucleoid / maturation of LSU-rRNA / ribosome assembly / cytoplasmic translation / large ribosomal subunit rRNA binding / rRNA processing / large ribosomal subunit / cytosolic large ribosomal subunit / ribosomal large subunit assembly ...positive regulation of rRNA processing / nucleoid / maturation of LSU-rRNA / ribosome assembly / cytoplasmic translation / large ribosomal subunit rRNA binding / rRNA processing / large ribosomal subunit / cytosolic large ribosomal subunit / ribosomal large subunit assembly / 5S rRNA binding / regulation of translation / tRNA binding / transferase activity / negative regulation of translation / ribosome / rRNA binding / structural constituent of ribosome / translation / mRNA binding / response to antibiotic / RNA binding / DNA binding / cytosol
Ribosomal protein L13, conserved site / Ribosomal protein L5, N-terminal / Ribosomal protein L1, conserved site / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L21-like / Ribosomal protein L15 / Ribosomal protein L5, C-terminal / Ribosomal protein L20, C-terminal / Ribosomal protein L2 signature. ...Ribosomal protein L13, conserved site / Ribosomal protein L5, N-terminal / Ribosomal protein L1, conserved site / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L21-like / Ribosomal protein L15 / Ribosomal protein L5, C-terminal / Ribosomal protein L20, C-terminal / Ribosomal protein L2 signature. / Ribosomal protein L29/L35 superfamily / L21-like superfamily / Ribosomal L18e/L15P superfamily / Ribosomal protein L17 superfamily / Ribosomal protein L22/L17 superfamily / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L4 domain superfamily / Ribosomal protein L5 domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11, N-terminal / Ribosomal protein L6, bacterial-type / Ribosomal protein L3, conserved site / Ribosomal protein L3, bacterial/organelle-type / Ribosomal protein L14P, conserved site / Ribosomal protein L6, alpha-beta domain / Ribosomal protein L11, C-terminal / Ribosomal protein L11, conserved site / Ribosomal protein L2, conserved site / Ribosomal protein L5, conserved site / Ribosomal protein L5, bacterial-type / Ribosomal protein L34, conserved site / Ribosomal protein L18e/L15P / Ribosomal Proteins L2, RNA binding domain / Ribosomal protein L2, C-terminal / Ribosomal protein L6, alpha-beta domain superfamily / Ribosomal protein L14 superfamily / Ribosomal protein L21, conserved site / Ribosomal protein L19 / Ribosomal protein L1p/L10e family / Ribosomal proteins 50S-L15, 50S-L18e, 60S-L27A / Ribosomal prokaryotic L21 protein / Ribosomal L29 protein / Ribosomal L18 of archaea, bacteria, mitoch. and chloroplast / Ribosomal protein L17 / Ribosomal L32p protein family / Ribosomal protein L4/L1 family / Ribosomal protein L11, N-terminal domain / Ribosomal Proteins L2, C-terminal domain / Ribosomal proteins 50S L24/mitochondrial 39S L24 / Ribosomal protein L14 signature. / Ribosomal protein L23 signature. / Ribosomal protein L5 signature. / ribosomal L5P family C-terminus / Ribosomal protein L13 / Ribosomal protein L13 superfamily / Ribosomal protein L23 / Ribosomal protein L30, ferredoxin-like fold domain superfamily / Ribosomal protein L19 superfamily / Ribosomal Protein L26/L24, KOW domain / Ribosomal Proteins L2, RNA binding domain / Ribosomal protein L22p/L17e / Ribosomal protein L14p/L23e / Ribosomal protein L5 / Ribosomal protein L34 / Ribosomal protein L3 / Ribosomal protein L11, RNA binding domain / Ribosomal protein L30p/L7e / Ribosomal protein L6 / Ribosomal protein L20 / KOW motif / Ribosomal protein L22/L17, conserved site / Ribosomal protein L19, conserved site / Ribosomal protein L22 signature. / Ribosomal protein L34 / Ribosomal protein L4/L1e / Ribosomal protein L29, conserved site / Ribosomal protein L19 / Ribosomal protein L29/L35 / Ribosomal protein L21 / Ribosomal protein L15, conserved site / Ribosomal protein L22/L17 / Ribosomal protein L23/L25, conserved site / Ribosomal protein L11/L12 / Ribosomal protein L6 / Ribosomal protein L3 / Ribosomal protein L17 / Ribosomal protein L14P / Ribosomal protein L2 / Ribosomal protein L3 signature. / Ribosomal protein L15 signature. / Ribosomal protein L6 signature 1. / Ribosomal protein L29 signature. / Ribosomal protein L30 signature. / Ribosomal protein L13 signature. / Ribosomal protein L34 signature. / Ribosomal protein L20 signature. / Ribosomal protein L19 signature. / Ribosomal protein L24 signature. / Ribosomal protein L17 signature. / Ribosomal protein L21 signature.
50S ribosomal protein L22 / 50S ribosomal protein L11 / 50S ribosomal protein L13 / 50S ribosomal protein L20 / 50S ribosomal protein L18 / 50S ribosomal protein L6 / 50S ribosomal protein L23 / 50S ribosomal protein L4 / 50S ribosomal protein L3 / 50S ribosomal protein L2 ...50S ribosomal protein L22 / 50S ribosomal protein L11 / 50S ribosomal protein L13 / 50S ribosomal protein L20 / 50S ribosomal protein L18 / 50S ribosomal protein L6 / 50S ribosomal protein L23 / 50S ribosomal protein L4 / 50S ribosomal protein L3 / 50S ribosomal protein L2 / 50S ribosomal protein L29 / 50S ribosomal protein L21 / 50S ribosomal protein L17 / 50S ribosomal protein L30 / 50S ribosomal protein L15 / 50S ribosomal protein L5 / 50S ribosomal protein L14 / 50S ribosomal protein L24 / 50S ribosomal protein L34 / 50S ribosomal protein L32 / 50S ribosomal protein L19 / 50S ribosomal protein L1
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 13.3 Å
AuthorsLi N / Chen Y / Guo Q / Zhang Y / Yuan Y / Ma C / Deng H / Lei J / Gao N
CitationJournal: Nucleic Acids Res. / Year: 2013
Title: Cryo-EM structures of the late-stage assembly intermediates of the bacterial 50S ribosomal subunit.
Authors: Ningning Li / Yuling Chen / Qiang Guo / Yixiao Zhang / Yi Yuan / Chengying Ma / Haiteng Deng / Jianlin Lei / Ning Gao /
Abstract: Ribosome assembly is a process fundamental for all cellular activities. The efficiency and accuracy of the subunit assembly are tightly regulated and closely monitored. In the present work, we ...Ribosome assembly is a process fundamental for all cellular activities. The efficiency and accuracy of the subunit assembly are tightly regulated and closely monitored. In the present work, we characterized, both compositionally and structurally, a set of in vivo 50S subunit precursors (45S), isolated from a mutant bacterial strain. Our qualitative mass spectrometry data indicate that L28, L16, L33, L36 and L35 are dramatically underrepresented in the 45S particles. This protein spectrum shows interesting similarity to many qualitatively analyzed 50S precursors from different genetic background, indicating the presence of global rate-limiting steps in the late-stage assembly of 50S subunit. Our structural data reveal two major intermediate states for the 45S particles. Consistently, both states severally lack those proteins, but they also differ in the stability of the functional centers of the 50S subunit, demonstrating that they are translationally inactive. Detailed analysis indicates that the orientation of H38 accounts for the global conformational differences in these intermediate structures, and suggests that the reorientation of H38 to its native position is rate-limiting during the late-stage assembly. Especially, H38 plays an essential role in stabilizing the central protuberance, through the interaction with the 5S rRNA, and the correctly orientated H38 is likely a prerequisite for further maturation of the 50S subunit.
Validation ReportPDB-ID: 3j3v

SummaryFull reportAbout validation report
DateDeposition: Apr 25, 2013 / Header (metadata) release: Jun 12, 2013 / Map release: Jun 12, 2013 / Update: Aug 28, 2013

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.7
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 2.7
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-3j3v
  • Surface level: 2.7
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_5642.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.5 Å/pix.
x 256 pix.
= 384. Å
1.5 Å/pix.
x 256 pix.
= 384. Å
1.5 Å/pix.
x 256 pix.
= 384. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.5 Å
Density
Contour LevelBy AUTHOR: 2.7 / Movie #1: 2.7
Minimum - Maximum-4.30117369 - 10.841475490000001
Average (Standard dev.)0.0 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-127-127-127
Dimensions256256256
Spacing256256256
CellA=B=C: 384.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.51.51.5
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z384.000384.000384.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-127-127-127
NC/NR/NS256256256
D min/max/mean-4.30110.841-0.000

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Supplemental data

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Sample components

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Entire Assembly intermediate of 50S ribosome subunit from YlqF-deficient...

EntireName: Assembly intermediate of 50S ribosome subunit from YlqF-deficient Bacillus subtilis strain
Number of components: 1

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Component #1: ribosome-prokaryote, assembly intermediate of 50S ribosome subuni...

Ribosome-prokaryoteName: assembly intermediate of 50S ribosome subunit from YlqF-deficient Bacillus subtilis strain
a.k.a: immature 50S ribosome / Prokaryote: LSU 50S / Recombinant expression: No
SourceSpecies: Bacillus subtilis (bacteria) / Strain: Bacillus subtilis subsp. subtilis str. 168

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionBuffer solution: 100mM NH4Cl, 20mM Tris-HCl, 10mM MgAc2, 1mM TCEP
pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Method: Blot for 20 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Dec 6, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 59000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 4000 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI EAGLE (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 21020
Details: This is one of the classified groups with the software RELION
3D reconstructionAlgorithm: Reference projections / Software: RELION / CTF correction: Each particle / Resolution: 13.3 Å
Resolution method: SSNR-MAP(an optimized approach of Spectral Single-to Noise Ratio) at 1.0 cutoff

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Atomic model buiding

Modeling #1Software: S2S, modeRNA, MODELLER, MDFF / Refinement protocol: flexible / Refinement space: REAL
Details: Atom models of the 23S and 5S rRNAs were built using the software S2S and modeRNA, with the crystal structures of the 50S subunits from E. coli (PDB ID: 2AW4) and Thermus thermophilus (PDB ID: 2J01) as template. Models of ribosomal proteins, L1, L3, L4, L6, L10, L13, L14, L15, L17, L19, L20, L21, L22, L23, L24, L27, L29, L30, L31, L32, L33, L34, L35 and L36 were downloaded from the SWISS-MODEL Repository. The others, including L2, L5, L11, L16, L18 and L28 were modeled using MODELLER with crystal structures of E. coli and T. thermophilus 50S subunits as templates.The combined atomic model of the B. subtilis 50S subunit was docked into a high resolution mature 50S density map and optimized using MDFF. This optimized model was docked into the EM density using Chimera and flexible fitted into the density using MDFF
Input PDB model: 2AW4
Modeling #2Software: S2S, modeRNA, MODELLER, MDFF / Refinement protocol: flexible / Refinement space: REAL
Details: Atom models of the 23S and 5S rRNAs were built using the software S2S and modeRNA, with the crystal structures of the 50S subunits from E. coli (PDB ID: 2AW4) and Thermus thermophilus (PDB ID: 2J01) as template. Models of ribosomal proteins, L1, L3, L4, L6, L10, L13, L14, L15, L17, L19, L20, L21, L22, L23, L24, L27, L29, L30, L31, L32, L33, L34, L35 and L36 were downloaded from the SWISS-MODEL Repository. The others, including L2, L5, L11, L16, L18 and L28 were modeled using MODELLER with crystal structures of E. coli and T. thermophilus 50S subunits as templates.The combined atomic model of the B. subtilis 50S subunit was docked into a high resolution mature 50S density map and optimized using MDFF. This optimized model was docked into the EM density using Chimera and flexible fitted into the density using MDFF
Input PDB model: 2J01
Output model

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