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- PDB-6gc6: 50S ribosomal subunit assembly intermediate state 2 -

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Basic information

Entry
Database: PDB / ID: 6gc6
Title50S ribosomal subunit assembly intermediate state 2
Components
  • (50S ribosomal protein ...) x 18
  • 23S ribosomal RNA
KeywordsRIBOSOME / assembly precursor 48S 50S biogenesis in vitro reconstitution
Function / homology
Function and homology information


endoribonuclease inhibitor activity / negative regulation of endoribonuclease activity / translation repressor activity / polysomal ribosome / ribosome assembly / DNA-templated transcription, termination / translation repressor activity, mRNA regulatory element binding / DNA-binding transcription repressor activity / response to reactive oxygen species / cytoplasmic translation ...endoribonuclease inhibitor activity / negative regulation of endoribonuclease activity / translation repressor activity / polysomal ribosome / ribosome assembly / DNA-templated transcription, termination / translation repressor activity, mRNA regulatory element binding / DNA-binding transcription repressor activity / response to reactive oxygen species / cytoplasmic translation / large ribosomal subunit rRNA binding / large ribosomal subunit / ribosomal large subunit assembly / cytosolic large ribosomal subunit / protein-DNA complex / response to radiation / transferase activity / negative regulation of translation / ribosome / rRNA binding / structural constituent of ribosome / translation / mRNA binding / response to antibiotic / negative regulation of transcription, DNA-templated / zinc ion binding / cytosol
Ribosomal protein L30p/L7e / Ribosomal protein L3, bacterial/organelle-type / Ribosomal protein L15 / Ribosomal protein L21-like / Ribosomal protein L4 domain superfamily / Ribosomal protein L13, conserved site / Ribosomal protein L2, conserved site / Ribosomal protein L2, C-terminal / Ribosomal Proteins L2, RNA binding domain / Ribosomal protein L18e/L15P ...Ribosomal protein L30p/L7e / Ribosomal protein L3, bacterial/organelle-type / Ribosomal protein L15 / Ribosomal protein L21-like / Ribosomal protein L4 domain superfamily / Ribosomal protein L13, conserved site / Ribosomal protein L2, conserved site / Ribosomal protein L2, C-terminal / Ribosomal Proteins L2, RNA binding domain / Ribosomal protein L18e/L15P / Ribosomal protein L34, conserved site / Ribosomal protein L6, alpha-beta domain / Ribosomal protein L14P, conserved site / Ribosomal protein L3, conserved site / Ribosomal protein L29/L35 superfamily / Ribosomal protein L6, bacterial-type / Ribosomal protein L22/L17, conserved site / Ribosomal protein L21, conserved site / Ribosomal protein L19, conserved site / Ribosomal protein L30, conserved site / Ribosomal protein L30, ferredoxin-like fold domain / Ribosomal protein L2, domain 3 / Ribosomal protein L2, domain 2 / Ribosomal protein L25/L23 / 50S ribosomal protein uL4 / Ribosomal protein L23/L15e core domain superfamily / Ribosomal protein L20, C-terminal / L21-like superfamily / Nucleic acid-binding, OB-fold / Ribosomal protein L4/L1 family / Ribosomal L29 protein / Ribosomal proteins 50S L24/mitochondrial 39S L24 / KOW motif / Ribosomal protein L22p/L17e / Ribosomal prokaryotic L21 protein / Ribosomal protein L20 / Ribosomal protein L19 / Ribosomal protein L17 / Ribosomal proteins 50S-L15, 50S-L18e, 60S-L27A / Ribosomal protein L13 / Ribosomal protein L6 / Ribosomal protein L3 / Ribosomal L18e/L15P superfamily / Ribosomal Proteins L2, RNA binding domain / Ribosomal protein L34 / Ribosomal L32p protein family / Ribosomal Protein L26/L24, KOW domain / Ribosomal protein L19 superfamily / Ribosomal protein L30, ferredoxin-like fold domain superfamily / Ribosomal protein L13 superfamily / Ribosomal protein L14 superfamily / Ribosomal protein L6, alpha-beta domain superfamily / Ribosomal protein L22/L17 superfamily / Ribosomal protein L17 superfamily / Nucleotide-binding alpha-beta plait domain superfamily / Ribosomal protein L29, conserved site / Zinc-binding ribosomal protein / Ribosomal protein L6, conserved site / Translation protein, beta-barrel domain superfamily / Ribosomal protein L14P / Ribosomal protein L34 / Ribosomal protein L17 / Ribosomal protein L3 / Ribosomal protein L6 / Ribosomal protein L23/L25, conserved site / Ribosomal protein L22/L17 / Ribosomal protein L15, conserved site / Ribosomal protein L21 / Ribosomal protein L19 / Ribosomal protein L4/L1e / Ribosomal protein L2 / Ribosomal protein L29/L35 / Ribosomal protein L32p / Ribosomal protein L13, bacterial-type / Translation protein SH3-like domain superfamily / Ribosomal protein L30, bacterial-type / Ribosomal protein L24 / Ribosomal protein L24/L26, conserved site / KOW / Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein L13 / Ribosomal protein L14P, bacterial-type / Ribosomal protein L20 / Ribosomal protein L15, bacterial-type / Ribosomal protein L22, bacterial/chloroplast-type
50S ribosomal protein L15 / 50S ribosomal protein L19 / 50S ribosomal protein L20 / 50S ribosomal protein L34 / 50S ribosomal protein L13 / 50S ribosomal protein L32 / gb:1063812051: / 50S ribosomal protein L22 / 50S ribosomal protein L4 / 50S ribosomal protein L24 ...50S ribosomal protein L15 / 50S ribosomal protein L19 / 50S ribosomal protein L20 / 50S ribosomal protein L34 / 50S ribosomal protein L13 / 50S ribosomal protein L32 / gb:1063812051: / 50S ribosomal protein L22 / 50S ribosomal protein L4 / 50S ribosomal protein L24 / 50S ribosomal protein L3 / 50S ribosomal protein L2 / 50S ribosomal protein L6 / 50S ribosomal protein L30 / 50S ribosomal protein L21 / 50S ribosomal protein L17 / 50S ribosomal protein L23 / 50S ribosomal protein L14 / 50S ribosomal protein L29
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsNikolay, R. / Hilal, T. / Qin, B. / Loerke, J. / Buerger, J. / Mielke, T. / Spahn, C.M.T.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research FoundationFOR1805 Germany
German Research FoundationSFB740 Germany
CitationJournal: Mol. Cell / Year: 2018
Title: Structural Visualization of the Formation and Activation of the 50S Ribosomal Subunit during In Vitro Reconstitution.
Authors: Rainer Nikolay / Tarek Hilal / Bo Qin / Thorsten Mielke / Jörg Bürger / Justus Loerke / Kathrin Textoris-Taube / Knud H Nierhaus / Christian M T Spahn /
Abstract: The assembly of ribosomal subunits is an essential prerequisite for protein biosynthesis in all domains of life. Although biochemical and biophysical approaches have advanced our understanding of ...The assembly of ribosomal subunits is an essential prerequisite for protein biosynthesis in all domains of life. Although biochemical and biophysical approaches have advanced our understanding of ribosome assembly, our mechanistic comprehension of this process is still limited. Here, we perform an in vitro reconstitution of the Escherichia coli 50S ribosomal subunit. Late reconstitution products were subjected to high-resolution cryo-electron microscopy and multiparticle refinement analysis to reconstruct five distinct precursors of the 50S subunit with 4.3-3.8 Å resolution. These assembly intermediates define a progressive maturation pathway culminating in a late assembly particle, whose structure is more than 96% identical to a mature 50S subunit. Our structures monitor the formation and stabilization of structural elements in a nascent particle in unprecedented detail and identify the maturation of the rRNA-based peptidyl transferase center as the final critical step along the 50S assembly pathway.
Validation Report
SummaryFull reportAbout validation report
History
DepositionApr 17, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 4, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Assembly

Deposited unit
A: 23S ribosomal RNA
C: 50S ribosomal protein L2
D: 50S ribosomal protein L3
E: 50S ribosomal protein L4
G: 50S ribosomal protein L6
J: 50S ribosomal protein L13
K: 50S ribosomal protein L14
L: 50S ribosomal protein L15
N: 50S ribosomal protein L17
P: 50S ribosomal protein L19
Q: 50S ribosomal protein L20
R: 50S ribosomal protein L21
S: 50S ribosomal protein L22
T: 50S ribosomal protein L23
U: 50S ribosomal protein L24
Y: 50S ribosomal protein L29
0: 50S ribosomal protein L32
2: 50S ribosomal protein L34
Z: 50S ribosomal protein L30


Theoretical massNumber of molelcules
Total (without water)1,184,45819
Polymers1,184,45819
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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RNA chain , 1 types, 1 molecules A

#1: RNA chain 23S ribosomal RNA /


Mass: 941612.375 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: GenBank: 1063812051

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50S ribosomal protein ... , 18 types, 18 molecules CDEGJKLNPQRSTUY02Z

#2: Protein 50S ribosomal protein L2 / / Large ribosomal subunit protein uL2


Mass: 24213.004 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P60422
#3: Protein 50S ribosomal protein L3 / / Large ribosomal subunit protein uL3


Mass: 22277.535 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P60438
#4: Protein 50S ribosomal protein L4 / / Large ribosomal subunit protein uL4


Mass: 22121.566 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P60723
#5: Protein 50S ribosomal protein L6 / / Large ribosomal subunit protein uL6


Mass: 18801.598 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P0AG55
#6: Protein 50S ribosomal protein L13 / / Large ribosomal subunit protein uL13


Mass: 16050.606 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P0AA10
#7: Protein 50S ribosomal protein L14 / / Large ribosomal subunit protein uL14


Mass: 13451.910 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P0ADY3
#8: Protein 50S ribosomal protein L15 / / Large ribosomal subunit protein uL15


Mass: 14877.273 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P02413
#9: Protein 50S ribosomal protein L17 / / Large ribosomal subunit protein bL17


Mass: 13721.938 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P0AG44
#10: Protein 50S ribosomal protein L19 / / Large ribosomal subunit protein bL19


Mass: 13028.082 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P0A7K6
#11: Protein 50S ribosomal protein L20 / / Large ribosomal subunit protein bL20


Mass: 13396.828 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P0A7L3
#12: Protein 50S ribosomal protein L21 / / Large ribosomal subunit protein bL21


Mass: 11586.374 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P0AG48
#13: Protein 50S ribosomal protein L22 / / Large ribosomal subunit protein uL22


Mass: 12253.359 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P61175
#14: Protein 50S ribosomal protein L23 / / Large ribosomal subunit protein uL23


Mass: 10546.472 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P0ADZ0
#15: Protein 50S ribosomal protein L24 / / Large ribosomal subunit protein uL24


Mass: 11078.874 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P60624
#16: Protein 50S ribosomal protein L29 / / Large ribosomal subunit protein uL29


Mass: 7286.464 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P0A7M6
#17: Protein 50S ribosomal protein L32 / / Large ribosomal subunit protein bL32


Mass: 6332.249 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P0A7N4
#18: Protein/peptide 50S ribosomal protein L34 / / Large ribosomal subunit protein bL34


Mass: 5397.463 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P0A7P5
#19: Protein 50S ribosomal protein L30 / / Large ribosomal subunit protein uL30


Mass: 6423.625 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P0AG51

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: 50S ribosomal subunit in vitro assembly intermediate state 2
Type: RIBOSOME
Entity ID: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
Source: NATURAL
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 25 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.11_2561: / Classification: refinement
EM software
IDNameVersionCategory
2Leginonimage acquisition
7UCSF Chimeramodel fitting
9SPIDER14initial Euler assignment
10SPIDER14final Euler assignment
11SPIDER14classification
12SPIDER143D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 235068
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 16754 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00267012
ELECTRON MICROSCOPYf_angle_d0.629100893
ELECTRON MICROSCOPYf_dihedral_angle_d14.87135406
ELECTRON MICROSCOPYf_chiral_restr0.03113005
ELECTRON MICROSCOPYf_plane_restr0.0054966

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