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- PDB-6nqb: Role of Era in Assembly and Homeostasis of the Ribosomal Small Subunit -

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Basic information

Entry
Database: PDB / ID: 6nqb
TitleRole of Era in Assembly and Homeostasis of the Ribosomal Small Subunit
Components
  • (30S ribosomal protein ...) x 15
  • 16S RIBOSOMAL RNA
KeywordsRIBOSOME / Ribosome assembly / 30S subunit / YjeQ protein / Era protein / cryo-electron microscopy
Function / homology
Function and homology information


small ribosomal subunit / tRNA binding / ribosome / rRNA binding / structural constituent of ribosome / translation / mRNA binding
Ribosomal protein S6 superfamily / Ribosomal protein S3, C-terminal domain superfamily / Ribosomal protein S3, C-terminal domain / Ribosomal protein S12/S23 / Ribosomal protein S4/S9 N-terminal domain / RNA-binding S4 domain superfamily / Ribosomal protein S18 superfamily / Ribosomal protein S10 domain superfamily / Ribosomal protein S20 superfamily / Ribosomal protein S8 superfamily ...Ribosomal protein S6 superfamily / Ribosomal protein S3, C-terminal domain superfamily / Ribosomal protein S3, C-terminal domain / Ribosomal protein S12/S23 / Ribosomal protein S4/S9 N-terminal domain / RNA-binding S4 domain superfamily / Ribosomal protein S18 superfamily / Ribosomal protein S10 domain superfamily / Ribosomal protein S20 superfamily / Ribosomal protein S8 superfamily / Ribosomal protein S14p/S29e / Ribosomal protein S2 signature 1. / Ribosomal protein S10 domain / Ribosomal protein S16 domain superfamily / Ribosomal protein S2, flavodoxin-like domain superfamily / Ribosomal protein S19, superfamily / Ribosomal protein S14, bacterial/plastid / Ribosomal protein S4/S9 / Ribosomal protein S19 conserved site / Ribosomal protein S19 / Ribosomal protein S15 / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein S8 signature. / Ribosomal protein S3 signature. / Ribosomal protein S14 signature. / Ribosomal protein S15 signature. / Ribosomal protein S10 signature. / Ribosomal protein S19 signature. / Ribosomal protein S18 signature. / Ribosomal protein S17 signature. / Ribosomal protein S12 signature. / KH domain / Ribosomal protein S2 / Ribosomal protein S20 / S4 domain / Ribosomal protein S6 / Ribosomal protein S18 / Ribosomal protein S16 / Ribosomal protein S8 / Ribosomal protein S17 / Ribosomal protein S10p/S20e / Ribosomal protein S6, conserved site / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein S14 / Ribosomal protein S20 / Ribosomal protein S19/S15 / Ribosomal protein S4/S9, N-terminal / Ribosomal protein S2 / Ribosomal protein S10 / Ribosomal protein S18 / Ribosomal protein S3, C-terminal / Ribosomal protein S8 / K Homology domain, type 2 / Ribosomal protein S15 / Ribosomal protein S6 / Ribosomal protein S16 / Ribosomal protein S17/S11 / Ribosomal protein S2 signature 2. / Ribosomal protein S6 signature. / Type-2 KH domain profile. / S4 RNA-binding domain profile. / RNA-binding S4 domain / K Homology domain / 30S ribosomal protein S17 / Ribosomal protein S15, bacterial-type / Ribosomal protein S17, conserved site / Ribosomal protein S3, conserved site / Ribosomal protein S18, conserved site / Ribosomal protein S14, conserved site / Ribosomal protein S10, conserved site / Ribosomal protein S2, conserved site / Ribosomal protein S4, conserved site / K homology domain-like, alpha/beta / Translation elongation factor EF1B/ribosomal protein S6 / Nucleic acid-binding, OB-fold / S15/NS1, RNA-binding / K homology domain superfamily, prokaryotic type / Ribosomal protein S12/S23 / Ribosomal protein S19, bacterial-type / Ribosomal protein S4, bacterial-type / Ribosomal protein S2, bacteria/mitochondria/plastid / Ribosomal protein S3, bacterial-type / Ribosomal protein S12, bacterial-type / Ribosomal protein S4 signature.
30S ribosomal protein S19 / 30S ribosomal protein S2 / 30S ribosomal protein S20 / 30S ribosomal protein S16 / 30S ribosomal protein S15 / 30S ribosomal protein S4 / 30S ribosomal protein S10 / 30S ribosomal protein S8 / 30S ribosomal protein S18 / 30S ribosomal protein S3 ...30S ribosomal protein S19 / 30S ribosomal protein S2 / 30S ribosomal protein S20 / 30S ribosomal protein S16 / 30S ribosomal protein S15 / 30S ribosomal protein S4 / 30S ribosomal protein S10 / 30S ribosomal protein S8 / 30S ribosomal protein S18 / 30S ribosomal protein S3 / un:a0a0t5xim4: / 30S ribosomal protein S17 / 30S ribosomal protein S14 / 30S ribosomal protein S6 / 30S ribosomal protein S12
Biological speciesEscherichia coli (E. coli)
Escherichia coli H736 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsOrtega, J.
Funding supportCanada , 1件
OrganizationGrant numberCountry
Canadian Institutes of Health ResearchPJT-153044Canada
CitationJournal: Nucleic Acids Res. / Year: 2019
Title: Role of Era in assembly and homeostasis of the ribosomal small subunit.
Authors: Aida Razi / Joseph H Davis / Yumeng Hao / Dushyant Jahagirdar / Brett Thurlow / Kaustuv Basu / Nikhil Jain / Josue Gomez-Blanco / Robert A Britton / Javier Vargas / Alba Guarné / Sarah A Woodson / James R Williamson / Joaquin Ortega /
Abstract: Assembly factors provide speed and directionality to the maturation process of the 30S subunit in bacteria. To gain a more precise understanding of how these proteins mediate 30S maturation, it is ...Assembly factors provide speed and directionality to the maturation process of the 30S subunit in bacteria. To gain a more precise understanding of how these proteins mediate 30S maturation, it is important to expand on studies of 30S assembly intermediates purified from bacterial strains lacking particular maturation factors. To reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, high resolution cryo-electron microscopy and in-cell footprinting. Our combined approach allowed for visualization of the small subunit as it assembled and revealed that with the exception of key helices in the platform domain, all other 16S rRNA domains fold even in the absence of Era. Notably, the maturing particles did not stall while waiting for the platform domain to mature and instead re-routed their folding pathway to enable concerted maturation of other structural motifs spanning multiple rRNA domains. We also found that binding of Era to the mature 30S subunit destabilized helix 44 and the decoding center preventing binding of YjeQ, another assembly factor. This work establishes Era's role in ribosome assembly and suggests new roles in maintaining ribosome homeostasis.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jan 20, 2019 / Release: Jun 26, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jun 26, 2019Structure modelrepositoryInitial release
1.1Jul 17, 2019Structure modelData collection / Database referencescitation_citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
C: 30S ribosomal protein S3
J: 30S ribosomal protein S10
N: 30S ribosomal protein S14
S: 30S ribosomal protein S19
A: 16S RIBOSOMAL RNA
D: 30S ribosomal protein S4
E: 30S ribosomal protein S5
F: 30S ribosomal protein S6
H: 30S ribosomal protein S8
L: 30S ribosomal protein S12
O: 30S ribosomal protein S15
P: 30S RIBOSOMAL PROTEIN bS16
Q: 30S ribosomal protein S17
R: 30S ribosomal protein S18
T: 30S ribosomal protein S20
B: 30S ribosomal protein S2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)699,25917
Polymers699,23516
Non-polymers241
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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30S ribosomal protein ... , 15 types, 15 molecules CJNSDEFHLOPQRTB

#1: Protein/peptide 30S ribosomal protein S3 /


Mass: 23078.785 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpsC, NCTC9094_00425 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A376HTV6
#2: Protein/peptide 30S ribosomal protein S10 /


Mass: 11196.988 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpsJ, HMPREF9552_00980 / Production host: Escherichia coli (E. coli) / References: UniProt: D7X302
#3: Protein/peptide 30S ribosomal protein S14 /


Mass: 11360.276 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: rpsN, C0R28_24180, D9J44_12955, ERS085374_04715, SAMEA3485113_04711
Production host: Escherichia coli (E. coli) / References: UniProt: A0A090BZT4
#4: Protein/peptide 30S ribosomal protein S19 /


Mass: 8441.836 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli H736 (bacteria) / Gene: rpsS, ECHG_03182 / Production host: Escherichia coli (E. coli) / References: UniProt: F4SQ43
#6: Protein/peptide 30S ribosomal protein S4 /


Mass: 23383.002 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpsD, A1UM_03988 / Production host: Escherichia coli (E. coli) / References: UniProt: L3PZ69
#7: Protein/peptide 30S ribosomal protein S5 /


Mass: 15675.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpsE, AOX65_22485 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0T5XIM4
#8: Protein/peptide 30S ribosomal protein S6 /


Mass: 10972.643 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: rpsF, B9M99_08080, BE963_12515, BMT53_12235, CIJ94_19015, D2188_21030, DTM45_20815, DU321_10545, Eco118UI_24760, ECONIH1_24970, EFV14_11625, EIA08_19855, HMPREF3040_03088, NCTC9077_05624
Production host: Escherichia coli (E. coli) / References: UniProt: A0A074QGH8
#9: Protein/peptide 30S ribosomal protein S8 /


Mass: 14015.361 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpsH, HMPREF9530_00381 / Production host: Escherichia coli (E. coli) / References: UniProt: D8A1L7
#10: Protein/peptide 30S ribosomal protein S12 /


Mass: 13636.961 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpsL, EC970259_3994 / Production host: Escherichia coli (E. coli) / References: UniProt: V6FZ95
#11: Protein/peptide 30S ribosomal protein S15 /


Mass: 9944.415 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / References: UniProt: Q8X9M2
#12: Protein/peptide 30S RIBOSOMAL PROTEIN bS16 /


Mass: 8936.237 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / References: UniProt: S0UHH0
#13: Protein/peptide 30S ribosomal protein S17 /


Mass: 9164.815 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0G3QJD8
#14: Protein/peptide 30S ribosomal protein S18 /


Mass: 6109.118 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpsR, HMPREF9534_03906 / Production host: Escherichia coli (E. coli) / References: UniProt: D7ZI16
#15: Protein/peptide 30S ribosomal protein S20 /


Mass: 9506.190 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpsT, G749_00187 / Production host: Escherichia coli (E. coli) / References: UniProt: T6N332
#16: Protein/peptide 30S ribosomal protein S2 /


Mass: 24122.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpsB, HMPREF1620_04659 / Production host: Escherichia coli (E. coli) / References: UniProt: U9ZNW8

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RNA chain / Non-polymers , 2 types, 2 molecules A

#17: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Magnesium
#5: RNA chain 16S RIBOSOMAL RNA /


Mass: 499690.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)

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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: 30S assembly intermediate (class P) / Type: RIBOSOME
Details: 30S assembly intermediate (class P) purified from Era-depleted E.coli cells
Entity ID: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16
Source: NATURAL
Molecular weightValue: 0.9 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 5 mA / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: 2750 nm / Nominal defocus min: 1250 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 35 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2.1.0.particle selection
2EPUimage acquisition
4GctfCTF correction
10RELION2.1.0.initial Euler assignment
11RELION2.1.0.final Euler assignment
12RELION2.1.0.classification
13RELION2.1.0.3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 847135
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 423567 / Symmetry type: POINT
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.00646213
f_angle_d0.95569052
f_dihedral_angle_d16.2224755
f_chiral_restr0.0478824
f_plane_restr0.0063762

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