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- PDB-8c95: Cryo-EM captures early ribosome assembly in action -

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Basic information

Entry
Database: PDB / ID: 8c95
TitleCryo-EM captures early ribosome assembly in action
Components
  • (50S ribosomal protein ...) x 14
  • 23S rRNA
  • 5S rRNA
KeywordsRIBOSOME / ribosome assembly / ribosome biogenesis / total reconstitution / RNA / ribosomal protein.
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 / translation repressor activity / ribosome assembly / mRNA regulatory element binding translation repressor activity / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly ...transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / translation repressor activity / ribosome assembly / mRNA regulatory element binding translation repressor activity / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / regulation of cell growth / DNA-templated transcription termination / response to radiation / ribosomal large subunit assembly / mRNA 5'-UTR binding / large ribosomal subunit / ribosome binding / 5S rRNA binding / large ribosomal subunit rRNA binding / cytosolic large ribosomal subunit / tRNA binding / cytoplasmic translation / rRNA binding / negative regulation of translation / ribosome / structural constituent of ribosome / translation / response to antibiotic / negative regulation of DNA-templated transcription / mRNA binding / DNA binding / RNA binding / zinc ion binding / cytoplasm / cytosol
Similarity search - Function
Ribosomal protein L25, short-form / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L5, bacterial-type / Ribosomal protein L18, bacterial-type / Ribosomal protein L20 signature. ...Ribosomal protein L25, short-form / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L5, bacterial-type / Ribosomal protein L18, bacterial-type / Ribosomal protein L20 signature. / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein L34, conserved site / Ribosomal protein L34 signature. / Ribosomal protein L22, bacterial/chloroplast-type / Ribosomal protein L30, bacterial-type / : / Ribosomal protein L18 / Ribosomal L18 of archaea, bacteria, mitoch. and chloroplast / Ribosomal protein L20 / Ribosomal protein L20 / Ribosomal protein L20, C-terminal / Ribosomal protein L21 / Ribosomal protein L27 / Ribosomal L27 protein / Ribosomal proteins 50S L24/mitochondrial 39S L24 / Ribosomal protein L21-like / L21-like superfamily / Ribosomal prokaryotic L21 protein / Ribosomal protein L24 / Ribosomal protein L34 / Ribosomal protein L34 / Ribosomal protein L13, bacterial-type / Ribosomal protein L15, bacterial-type / 50S ribosomal protein uL4 / Ribosomal protein L30, conserved site / Ribosomal protein L30 signature. / Ribosomal protein L5, conserved site / Ribosomal protein L5 signature. / Ribosomal protein L29, conserved site / Ribosomal protein L29 signature. / Ribosomal protein L15, conserved site / Ribosomal protein L15 signature. / Ribosomal protein L5, N-terminal / Ribosomal protein L5 / Ribosomal protein L5, C-terminal / ribosomal L5P family C-terminus / Ribosomal protein L5 / Ribosomal protein L5 domain superfamily / Ribosomal protein L13, conserved site / Ribosomal protein L13 signature. / Ribosomal protein L22/L17, conserved site / Ribosomal protein L22 signature. / Ribosomal L29 protein / Ribosomal protein L29/L35 / Ribosomal protein L29/L35 superfamily / Ribosomal protein L24 signature. / Ribosomal protein L24/L26, conserved site / KOW (Kyprides, Ouzounis, Woese) motif. / Ribosomal protein L13 / Ribosomal protein L13 / Ribosomal protein L13 superfamily / Ribosomal protein L30, ferredoxin-like fold domain / Ribosomal protein L30p/L7e / Ribosomal protein L15 / Ribosomal protein L30, ferredoxin-like fold domain superfamily / Ribosomal proteins 50S-L15, 50S-L18e, 60S-L27A / Ribosomal protein L26/L24, KOW domain / Ribosomal protein L22/L17 / Ribosomal protein L22/L17 superfamily / Ribosomal protein L22p/L17e / Ribosomal protein L4/L1e / Ribosomal protein L4 domain superfamily / Ribosomal protein L4/L1 family / Ribosomal protein L18e/L15P / Ribosomal L18e/L15P superfamily / Translation protein SH3-like domain superfamily / KOW motif / KOW / Ribosomal protein L2, domain 2
Similarity search - Domain/homology
: / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein uL29 ...: / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL18 / 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 (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.92 Å
AuthorsLauer, S. / Nikolay, R. / Qin, B.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Federal Ministry for Education and Research16GW0300 Germany
CitationJournal: Nat Commun / Year: 2023
Title: Cryo-EM captures early ribosome assembly in action.
Authors: Bo Qin / Simon M Lauer / Annika Balke / Carlos H Vieira-Vieira / Jörg Bürger / Thorsten Mielke / Matthias Selbach / Patrick Scheerer / Christian M T Spahn / Rainer Nikolay /
Abstract: Ribosome biogenesis is a fundamental multi-step cellular process in all domains of life that involves the production, processing, folding, and modification of ribosomal RNAs (rRNAs) and ribosomal ...Ribosome biogenesis is a fundamental multi-step cellular process in all domains of life that involves the production, processing, folding, and modification of ribosomal RNAs (rRNAs) and ribosomal proteins. To obtain insights into the still unexplored early assembly phase of the bacterial 50S subunit, we exploited a minimal in vitro reconstitution system using purified ribosomal components and scalable reaction conditions. Time-limited assembly assays combined with cryo-EM analysis visualizes the structurally complex assembly pathway starting with a particle consisting of ordered density for only ~500 nucleotides of 23S rRNA domain I and three ribosomal proteins. In addition, our structural analysis reveals that early 50S assembly occurs in a domain-wise fashion, while late 50S assembly proceeds incrementally. Furthermore, we find that both ribosomal proteins and folded rRNA helices, occupying surface exposed regions on pre-50S particles, induce, or stabilize rRNA folds within adjacent regions, thereby creating cooperativity.
History
DepositionJan 21, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 5, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
2: 50S ribosomal protein L34
A: 23S rRNA
B: 5S rRNA
E: 50S ribosomal protein L4
F: 50S ribosomal protein L5
J: 50S ribosomal protein L13
L: 50S ribosomal protein L15
O: 50S ribosomal protein L18
Q: 50S ribosomal protein L20
R: 50S ribosomal protein L21
S: 50S ribosomal protein L22
U: 50S ribosomal protein L24
V: 50S ribosomal protein L25
W: 50S ribosomal protein L27
Y: 50S ribosomal protein L29
Z: 50S ribosomal protein L30


Theoretical massNumber of molelcules
Total (without water)1,154,21116
Polymers1,154,21116
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area56710 Å2
ΔGint-449 kcal/mol
Surface area247270 Å2
MethodPISA

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Components

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50S ribosomal protein ... , 14 types, 14 molecules 2EFJLOQRSUVWYZ

#1: Protein/peptide 50S ribosomal protein L34 / ribosomal protein bL34


Mass: 5397.463 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: Can20-12E / References: UniProt: P0A7P5
#4: Protein 50S ribosomal protein L4 / ribosomal protein uL4


Mass: 22121.566 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: Can20-12E / References: UniProt: P60723
#5: Protein 50S ribosomal protein L5 / ribosomal protein uL5


Mass: 20333.611 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: Can20-12E / References: UniProt: P62399
#6: Protein 50S ribosomal protein L13 / ribosomal protein uL13


Mass: 16050.606 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: Can20-12E / References: UniProt: P0AA10
#7: Protein 50S ribosomal protein L15 / ribosomal protein uL15


Mass: 15008.471 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: Can20-12E / References: UniProt: P02413
#8: Protein 50S ribosomal protein L18 / ribosomal protein uL18


Mass: 12794.668 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: Can20-12E / References: UniProt: P0C018
#9: Protein 50S ribosomal protein L20 / ribosomal protein bL20


Mass: 13528.024 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: Can20-12E / References: UniProt: P0A7L3
#10: Protein 50S ribosomal protein L21 / ribosomal protein bL21


Mass: 11586.374 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: Can20-12E / References: UniProt: P0AG48
#11: Protein 50S ribosomal protein L22 / ribosomal protein uL22


Mass: 12253.359 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: Can20-12E / References: UniProt: P61175
#12: Protein 50S ribosomal protein L24 / ribosomal protein uL24


Mass: 11339.250 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: Can20-12E / References: UniProt: P60624
#13: Protein 50S ribosomal protein L25 / ribosomal protein bL25


Mass: 10713.465 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: Can20-12E / References: UniProt: P68919
#14: Protein 50S ribosomal protein L27 / Large ribosomal subunit protein bL27


Mass: 9146.540 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: AKR was replaced by TKR / Source: (natural) Escherichia coli (E. coli) / Strain: Can20-12E / References: UniProt: P0A7L8
#15: Protein 50S ribosomal protein L29 / ribosomal protein uL29


Mass: 7286.464 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: Can20-12E / References: UniProt: P0A7M6
#16: Protein 50S ribosomal protein L30 / ribosomal protein uL30


Mass: 6554.820 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: Can20-12E / References: UniProt: P0AG51

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RNA chain , 2 types, 2 molecules AB

#2: RNA chain 23S rRNA


Mass: 941612.375 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: errorneous sequence alignment. Modelled residues: 1-684,794-1023,1135-1270,2003-2040,2259-2423,2893-2903
Source: (natural) Escherichia coli (E. coli) / Strain: Can20-12E / References: GenBank: 1109114233
#3: RNA chain 5S rRNA


Mass: 38483.926 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: Can20-12E / References: GenBank: 1435226024

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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: large ribosomal subunit precursor d12-CP / Type: RIBOSOME / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: Can20-12E
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: OTHER
Electron lensMode: OTHER / Nominal magnification: 31000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Temperature (max): 83 K / Temperature (min): 82 K
Image recordingAverage exposure time: 10 sec. / Electron dose: 62 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 50

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Processing

EM software
IDNameVersionCategory
1Gautomatch0.53particle selection
2Leginonimage acquisition
4Gctf1.06CTF correction
7Coot0.9.6.2model fitting
9cryoSPARC3.3initial Euler assignment
11cryoSPARC3.3classification
12RELION3.1classification
13cryoSPARC3.33D reconstruction
14PHENIX1.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 653029
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 12740 / Symmetry type: POINT
Atomic model buildingSpace: REAL

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