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- EMDB-5643: Cryo-EM structures of the late-stage assembly intermediates of 50... -

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Basic information

Entry
Database: EMDB / ID: EMD-5643
TitleCryo-EM structures of the late-stage assembly intermediates of 50S ribosome subunit from YlqF-deficient Bacillus subtilis strain.
Map data3D classification of the assembly intermediates of 50S ribosome subunit from YlqF-deficient Bacillus subtilis strain
Sample
  • Sample: Assembly intermediate of 50S ribosome subunit from YlqF-deficient Bacillus subtilis strain
  • Complex: assembly intermediate of 50S ribosome subunit from YlqF-deficient Bacillus subtilis strain
KeywordsRibosome biogenesis / ribosome assembly intermediate / RNA folding
Function / homology
Function and homology information


positive regulation of rRNA processing / nucleoid / rRNA processing / large ribosomal subunit rRNA binding / large ribosomal subunit / regulation of translation / cytoplasmic translation / cytosolic large ribosomal subunit / transferase activity / negative regulation of translation ...positive regulation of rRNA processing / nucleoid / rRNA processing / large ribosomal subunit rRNA binding / large ribosomal subunit / regulation of translation / cytoplasmic translation / cytosolic large ribosomal subunit / transferase activity / negative regulation of translation / tRNA binding / rRNA binding / ribosome / structural constituent of ribosome / translation / ribonucleoprotein complex / response to antibiotic / mRNA binding / DNA binding / RNA binding / cytoplasm
Similarity search - Function
Ribosomal protein L1, bacterial-type / Ribosomal protein L1, conserved site / Ribosomal protein L1 / Ribosomal protein L1 signature. / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L1p/L10e family / Ribosomal protein L11, bacterial-type / Ribosomal protein L11, conserved site ...Ribosomal protein L1, bacterial-type / Ribosomal protein L1, conserved site / Ribosomal protein L1 / Ribosomal protein L1 signature. / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L1p/L10e family / Ribosomal protein L11, bacterial-type / Ribosomal protein L11, conserved site / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L11 signature. / : / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / Ribosomal protein L11, N-terminal / Ribosomal protein L17 signature. / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11/L12 / Ribosomal protein L11, N-terminal domain / Ribosomal protein L11, RNA binding domain / Ribosomal protein L32p, bacterial type / Ribosomal protein L6, bacterial-type / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein L20 signature. / Ribosomal protein L14P, bacterial-type / Ribosomal protein L34, conserved site / Ribosomal protein L34 signature. / Ribosomal protein L22, bacterial/chloroplast-type / Ribosomal protein L2, bacterial/organellar-type / : / Ribosomal protein L20 / Ribosomal protein L20 / Ribosomal protein L20, C-terminal / Ribosomal protein L21 / Ribosomal protein L19 / Ribosomal protein L19 superfamily / Ribosomal protein L19 / Ribosomal proteins 50S L24/mitochondrial 39S L24 / Ribosomal protein L17 / Ribosomal protein L17 superfamily / Ribosomal protein L17 / Ribosomal protein L21-like / L21-like superfamily / Ribosomal prokaryotic L21 protein / Ribosomal L32p protein family / Ribosomal protein L24 / Ribosomal protein L32p / Ribosomal protein L34 / Ribosomal protein L34 / Ribosomal protein L13, bacterial-type / Ribosomal protein L23/L25, conserved site / Ribosomal protein L3, bacterial/organelle-type / Ribosomal protein L15, bacterial-type / 50S ribosomal protein uL4 / Ribosomal protein L23 signature. / Ribosomal protein L29, conserved site / Ribosomal protein L15, conserved site / Ribosomal protein L2, conserved site / Ribosomal protein L2 signature. / Ribosomal protein L29 signature. / Ribosomal protein L2, domain 3 / Ribosomal protein L6, alpha-beta domain / Ribosomal protein L6 / Ribosomal protein L6, alpha-beta domain superfamily / Ribosomal protein L14P, conserved site / Ribosomal protein L6 / Ribosomal protein L15 signature. / Ribosomal protein L29/L35 / Ribosomal protein L29/L35 superfamily / Ribosomal protein L22/L17, conserved site / Ribosomal L29 protein / Ribosomal protein L24/L26, conserved site / Ribosomal protein L13, conserved site / Ribosomal protein L13 signature. / KOW (Kyprides, Ouzounis, Woese) motif. / Ribosomal Proteins L2, C-terminal domain / Ribosomal protein L2, C-terminal / Ribosomal Proteins L2, C-terminal domain / Ribosomal Proteins L2, RNA binding domain / Ribosomal Proteins L2, RNA binding domain / Ribosomal protein L2 / Ribosomal protein L14 signature. / Ribosomal protein L22 signature. / Ribosomal protein L15 / Ribosomal Proteins L2, RNA binding domain / Ribosomal protein L25/L23 / Ribosomal protein L14p/L23e / Ribosomal protein L14P / Ribosomal protein L14 superfamily / Ribosomal protein L23 / Ribosomal protein L26/L24, KOW domain / Ribosomal proteins 50S-L15, 50S-L18e, 60S-L27A / Ribosomal protein L3, conserved site / Ribosomal protein L24 signature.
Similarity search - Domain/homology
Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL22 ...Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein uL1
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.7 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.
History
DepositionApr 27, 2013-
Header (metadata) releaseJun 12, 2013-
Map releaseJun 12, 2013-
UpdateAug 28, 2013-
Current statusAug 28, 2013Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j3w
  • Surface level: 2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5643_1.jpg

emd_5643_2.tif

Downloads & links

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Map

FileDownload / File: emd_5643.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D classification of the assembly intermediates of 50S ribosome subunit from YlqF-deficient Bacillus subtilis strain
Voxel sizeX=Y=Z: 1.5 Å
Density
Contour LevelBy AUTHOR: 2.0 / Movie #1: 2
Minimum - Maximum-3.26229954 - 10.90894222
Average (Standard dev.)0.0 (±1.0)
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-3.26210.909-0.000

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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
Components
  • Sample: Assembly intermediate of 50S ribosome subunit from YlqF-deficient Bacillus subtilis strain
  • Complex: assembly intermediate of 50S ribosome subunit from YlqF-deficient Bacillus subtilis strain

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Supramolecule #1000: Assembly intermediate of 50S ribosome subunit from YlqF-deficient...

SupramoleculeName: Assembly intermediate of 50S ribosome subunit from YlqF-deficient Bacillus subtilis strain
type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: assembly intermediate of 50S ribosome subunit from YlqF-deficient...

SupramoleculeName: assembly intermediate of 50S ribosome subunit from YlqF-deficient Bacillus subtilis strain
type: complex / ID: 1 / Name.synonym: immature 50S ribosome / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-prokaryote: LSU 50S
Source (natural)Organism: Bacillus subtilis (bacteria) / Strain: Bacillus subtilis subsp. subtilis str. 168

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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: 7.5 / Details: 100mM NH4Cl, 20mM Tris-HCl, 10mM MgOAc2, 1mM TCEP
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: Blot for 20 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateDec 6, 2011
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Average electron dose: 20 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.7 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 27652
DetailsThis is one of the classified groups with the software RELION

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Atomic model buiding 1

Initial modelPDB ID:

2aw4
PDB Unreleased entry

SoftwareName: S2S, modeRNA, MODELLER, MDFF
DetailsAtom 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
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-3j3w:
Atomic model of the immature 50S subunit from Bacillus subtilis (state II-a)

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Atomic model buiding 2

Initial modelPDB ID:

2j01
PDB Unreleased entry

SoftwareName: S2S, modeRNA, MODELLER, MDFF
DetailsAtom 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
RefinementSpace: REAL / Target criteria: Cross-correlation coefficient
Output model

PDB-3j3w:
Atomic model of the immature 50S subunit from Bacillus subtilis (state II-a)

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