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- PDB-6gc4: 50S ribosomal subunit assembly intermediate state 3 -

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Basic information

Entry
Database: PDB / ID: 6gc4
Title50S ribosomal subunit assembly intermediate state 3
Components
  • (50S ribosomal protein ...) x 24
  • 23S ribosomal RNA
  • 5S ribosomal RNA
KeywordsRIBOSOME / assembly precursor 48S 50S biogenesis in vitro reconstitution
Function / homology
Function and homology information


transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / DnaA-L2 complex / translation repressor activity / negative regulation of DNA-templated DNA replication initiation / ribosome assembly / mRNA regulatory element binding translation repressor activity ...transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / DnaA-L2 complex / translation repressor activity / negative regulation of DNA-templated DNA replication initiation / ribosome assembly / mRNA regulatory element binding translation repressor activity / response to reactive oxygen species / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / regulation of cell growth / DNA-templated transcription termination / response to radiation / mRNA 5'-UTR binding / ribosomal large subunit assembly / large ribosomal subunit rRNA binding / ribosome binding / large ribosomal subunit / cytoplasmic translation / 5S rRNA binding / cytosolic large ribosomal subunit / transferase activity / tRNA binding / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / translation / response to antibiotic / mRNA binding / negative regulation of DNA-templated transcription / DNA binding / RNA binding / zinc ion binding / cytosol / cytoplasm
Similarity search - Function
Ribosomal protein L25, short-form / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / : / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / Ribosomal protein L17 signature. / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal ...Ribosomal protein L25, short-form / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / : / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / Ribosomal protein L17 signature. / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L28/L24 superfamily / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L32p, bacterial type / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / Ribosomal protein L28 / Ribosomal protein L5, bacterial-type / Ribosomal protein L6, bacterial-type / Ribosomal protein L18, bacterial-type / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein L20 signature. / Ribosomal protein L27, conserved site / Ribosomal protein L27 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 L28 family / Ribosomal protein L28/L24 / : / Ribosomal protein L30, bacterial-type / Ribosomal protein L18 / Ribosomal L18 of archaea, bacteria, mitoch. and chloroplast / L28p-like / Ribosomal protein L20 / Ribosomal protein L20 / Ribosomal protein L20, C-terminal / Ribosomal protein L21 / Ribosomal protein L27 / Ribosomal L27 protein / 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 L30, conserved site / Ribosomal protein L5, conserved site / Ribosomal protein L29, conserved site / Ribosomal protein L30 signature. / Ribosomal protein L5, N-terminal / Ribosomal protein L5 signature. / Ribosomal protein L15, conserved site / Ribosomal protein L5, C-terminal / Ribosomal protein L5 / Ribosomal protein L5 domain superfamily / Ribosomal protein L2, conserved site / Ribosomal protein L2 signature. / Ribosomal protein L29 signature. / Ribosomal protein L5 / Ribosomal protein L6, alpha-beta domain / Ribosomal protein L6 / ribosomal L5P family C-terminus / Ribosomal protein L6, alpha-beta domain superfamily / Ribosomal protein L6 / Ribosomal protein L2, domain 3 / Ribosomal protein L14P, conserved site / Ribosomal protein L15 signature. / Ribosomal protein L22/L17, conserved site / Ribosomal protein L29/L35 / Ribosomal protein L29/L35 superfamily / Ribosomal L29 protein / Ribosomal protein L13, conserved site / Ribosomal protein L13 signature. / Ribosomal protein L24/L26, conserved site
Similarity search - Domain/homology
: / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 ...: / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL9 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL5 / Large ribosomal subunit protein bL25
Similarity search - Component
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.
History
DepositionApr 17, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 4, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 18, 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
V: 50S ribosomal protein L25
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
X: 50S ribosomal protein L28
Y: 50S ribosomal protein L29
0: 50S ribosomal protein L32
2: 50S ribosomal protein L34
O: 50S ribosomal protein L18
F: 50S ribosomal protein L5
B: 5S ribosomal RNA
Z: 50S ribosomal protein L30
H: 50S ribosomal protein L9
W: 50S ribosomal protein L27


Theoretical massNumber of molelcules
Total (without water)1,294,48126
Polymers1,294,48126
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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50S ribosomal protein ... , 24 types, 24 molecules VCDEGJKLNPQRSTUXY02OFZHW

#1: Protein 50S ribosomal protein L25 / / Large ribosomal subunit protein bL25


Mass: 10713.465 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P68919
#3: Protein 50S ribosomal protein L2 / / Large ribosomal subunit protein uL2


Mass: 29663.244 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P60422
#4: 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
#5: 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
#6: 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
#7: 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
#8: 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
#9: 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
#10: 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
#11: 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
#12: 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
#13: 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
#14: 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
#15: 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
#16: 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
#17: Protein 50S ribosomal protein L28 / / Large ribosomal subunit protein bL28


Mass: 8896.354 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P0A7M2
#18: 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
#19: 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
#20: 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
#21: Protein 50S ribosomal protein L18 / / Large ribosomal subunit protein uL18


Mass: 12663.471 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P0C018
#22: Protein 50S ribosomal protein L5 / / Large ribosomal subunit protein uL5


Mass: 20073.234 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P62399
#24: 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
#25: Protein/peptide 50S ribosomal protein L9 / / Large ribosomal subunit protein bL9


Mass: 5467.367 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P0A7R1
#26: Protein 50S ribosomal protein L27 / / Large ribosomal subunit protein bL27


Mass: 8275.498 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P0A7L8

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RNA chain , 2 types, 2 molecules AB

#2: 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: 1036415628
#23: RNA chain 5S ribosomal RNA /


Mass: 38483.926 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: GenBank: 1373146531

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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 assembly intermediate state 3 / Type: RIBOSOME / Entity ID: all / 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 / Nominal magnification: 80645 X
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: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategory
1Signatureparticle selection
2Leginonimage acquisition
4SPIDER14CTF correction
7UCSF Chimeramodel fitting
9SPIDER14initial Euler assignment
12SPIDER143D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 154462
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 26665 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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