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- PDB-5no2: RsgA-GDPNP bound to the 30S ribosomal subunit (RsgA assembly inte... -

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Entry
Database: PDB / ID: 5no2
TitleRsgA-GDPNP bound to the 30S ribosomal subunit (RsgA assembly intermediate)
Components
  • (30S ribosomal protein ...) x 17
  • 16S ribosomal RNA
  • Small ribosomal subunit biogenesis GTPase RsgA
KeywordsRIBOSOME
Function / homology
Function and homology information


guanosine tetraphosphate binding / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / mRNA binding involved in posttranscriptional gene silencing / ornithine decarboxylase inhibitor activity / misfolded RNA binding / RNA folding / Group I intron splicing / transcription antitermination factor activity, RNA binding / transcription antitermination / ribosomal small subunit biogenesis ...guanosine tetraphosphate binding / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / mRNA binding involved in posttranscriptional gene silencing / ornithine decarboxylase inhibitor activity / misfolded RNA binding / RNA folding / Group I intron splicing / transcription antitermination factor activity, RNA binding / transcription antitermination / ribosomal small subunit biogenesis / negative regulation of translational initiation / four-way junction DNA binding / translation repressor activity, mRNA regulatory element binding / endodeoxyribonuclease activity / positive regulation of RNA splicing / DNA-templated transcription, termination / positive regulation of translational fidelity / maintenance of translational fidelity / small ribosomal subunit rRNA binding / ribosomal small subunit assembly / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / mRNA 5'-UTR binding / GDP binding / cytosolic small ribosomal subunit / regulation of translation / regulation of mRNA stability / small ribosomal subunit / negative regulation of translation / tRNA binding / rRNA binding / ribosome / structural constituent of ribosome / translation / GTPase activity / mRNA binding / GTP binding / response to antibiotic / RNA binding / membrane / metal ion binding / cytosol
RsgA GTPase / Ribosomal protein S17, conserved site / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S4/S9 / Ribosomal protein S19 conserved site / Ribosomal protein S6, conserved site / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein S7, conserved site / Ribosomal protein S16, conserved site / Ribosomal protein S9, conserved site ...RsgA GTPase / Ribosomal protein S17, conserved site / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S4/S9 / Ribosomal protein S19 conserved site / Ribosomal protein S6, conserved site / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein S7, conserved site / Ribosomal protein S16, conserved site / Ribosomal protein S9, conserved site / Ribosomal protein S5 domain 2-type fold / 30S ribosomal protein S17 / Ribosomal protein S11, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S18, conserved site / Ribosomal protein S19, superfamily / Ribosomal protein S14, conserved site / Ribosomal protein S13, conserved site / Ribosomal protein S10, conserved site / Ribosomal protein S5, N-terminal, conserved site / Ribosomal S11, conserved site / Ribosomal protein S4, conserved site / Translation elongation factor EF1B/ribosomal protein S6 / Ribosomal protein S5, N-terminal / Nucleic acid-binding, OB-fold / Ribosomal protein S13-like, H2TH / RsgA GTPase domain / S15/NS1, RNA-binding / Ribosomal protein S12/S23 / Ribosomal protein S14, bacterial/plastid / Ribosomal protein S7 domain / Ribosomal protein S7, bacterial/organellar-type / S4 domain / Ribosomal protein S20 / Ribosomal protein S17 / Ribosomal protein S16 / Ribosomal protein S15 / Ribosomal protein S14p/S29e / Ribosomal protein S13/S18 / Ribosomal protein S11 / Ribosomal protein S10p/S20e / Ribosomal protein S9/S16 / Ribosomal protein S8 / Ribosomal protein S6 / Ribosomal protein S5, C-terminal domain / Ribosomal protein S5, N-terminal domain / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S16 domain superfamily / Ribosomal protein S14/S29 / RNA-binding S4 domain superfamily / Ribosomal protein S11 superfamily / Ribosomal protein S18 superfamily / Ribosomal protein S10 domain superfamily / Ribosomal protein S7 domain superfamily / Ribosomal protein S20 superfamily / Ribosomal protein S8 superfamily / Ribosomal protein S6 superfamily / Circularly permuted (CP)-type guanine nucleotide-binding (G) domain / Ribosomal protein S10 domain / 30s ribosomal protein S13, C-terminal / P-loop containing nucleoside triphosphate hydrolase / Ribosomal protein S19, bacterial-type / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5, bacterial-type / Ribosomal protein S10 / Ribosomal protein S17/S11 / Ribosomal protein S16 / Ribosomal protein S6 / Ribosomal protein S15 / Ribosomal protein S8 / Ribosomal protein S9 / Ribosomal protein S5 / Ribosomal protein S14 / Ribosomal protein S18 / Ribosomal protein S13 / Ribosomal protein S4/S9, N-terminal / Ribosomal protein S4, bacterial-type / Ribosomal protein S11 / Ribosomal protein S19/S15 / Ribosomal protein S20 / RNA-binding S4 domain / Ribosome biogenesis GTPase RsgA / Ribosomal protein S15, bacterial-type / Ribosomal protein S5, C-terminal / Ribosomal protein S12, bacterial-type / Ribosomal protein S5/S7
gb:1108560344: / 30S ribosomal protein S17 / Small ribosomal subunit biogenesis GTPase RsgA / 30S ribosomal protein S18 / 30S ribosomal protein S6 / 30S ribosomal protein S7 / 30S ribosomal protein S10 / 30S ribosomal protein S11 / 30S ribosomal protein S12 / 30S ribosomal protein S13 ...gb:1108560344: / 30S ribosomal protein S17 / Small ribosomal subunit biogenesis GTPase RsgA / 30S ribosomal protein S18 / 30S ribosomal protein S6 / 30S ribosomal protein S7 / 30S ribosomal protein S10 / 30S ribosomal protein S11 / 30S ribosomal protein S12 / 30S ribosomal protein S13 / 30S ribosomal protein S19 / 30S ribosomal protein S14 / 30S ribosomal protein S20 / 30S ribosomal protein S4 / 30S ribosomal protein S5 / 30S ribosomal protein S8 / 30S ribosomal protein S9 / 30S ribosomal protein S15 / 30S ribosomal protein S16
Biological speciesEscherichia coli (E. coli)
Escherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.16 Å
AuthorsLopez-Alonso, J.P. / Kaminishi, T. / Kikuchi, T. / Hirata, Y. / Iturrioz, I. / Dhimole, N. / Schedlbauer, A. / Hase, Y. / Goto, S. / Kurita, D. ...Lopez-Alonso, J.P. / Kaminishi, T. / Kikuchi, T. / Hirata, Y. / Iturrioz, I. / Dhimole, N. / Schedlbauer, A. / Hase, Y. / Goto, S. / Kurita, D. / Muto, A. / Zhou, S. / Naoe, C. / Mills, D.J. / Gil-Carton, D. / Takemoto, C. / Himeno, H. / Fucini, P. / Connell, S.R.
CitationJournal: Nucleic Acids Res / Year: 2017
Title: RsgA couples the maturation state of the 30S ribosomal decoding center to activation of its GTPase pocket.
Authors: Jorge Pedro López-Alonso / Tatsuya Kaminishi / Takeshi Kikuchi / Yuya Hirata / Idoia Iturrioz / Neha Dhimole / Andreas Schedlbauer / Yoichi Hase / Simon Goto / Daisuke Kurita / Akira Muto / ...Authors: Jorge Pedro López-Alonso / Tatsuya Kaminishi / Takeshi Kikuchi / Yuya Hirata / Idoia Iturrioz / Neha Dhimole / Andreas Schedlbauer / Yoichi Hase / Simon Goto / Daisuke Kurita / Akira Muto / Shu Zhou / Chieko Naoe / Deryck J Mills / David Gil-Carton / Chie Takemoto / Hyouta Himeno / Paola Fucini / Sean R Connell /
Abstract: During 30S ribosomal subunit biogenesis, assembly factors are believed to prevent accumulation of misfolded intermediate states of low free energy that slowly convert into mature 30S subunits, ...During 30S ribosomal subunit biogenesis, assembly factors are believed to prevent accumulation of misfolded intermediate states of low free energy that slowly convert into mature 30S subunits, namely, kinetically trapped particles. Among the assembly factors, the circularly permuted GTPase, RsgA, plays a crucial role in the maturation of the 30S decoding center. Here, directed hydroxyl radical probing and single particle cryo-EM are employed to elucidate RsgA΄s mechanism of action. Our results show that RsgA destabilizes the 30S structure, including late binding r-proteins, providing a structural basis for avoiding kinetically trapped assembly intermediates. Moreover, RsgA exploits its distinct GTPase pocket and specific interactions with the 30S to coordinate GTPase activation with the maturation state of the 30S subunit. This coordination validates the architecture of the decoding center and facilitates the timely release of RsgA to control the progression of 30S biogenesis.
Validation Report
SummaryFull reportAbout validation report
History
DepositionApr 10, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 24, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 2, 2017Group: Data collection / Derived calculations / Category: em_software / pdbx_struct_conn_angle / Item: _em_software.name
Revision 1.2Sep 13, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: 16S ribosomal RNA
D: 30S ribosomal protein S4
E: 30S ribosomal protein S5
F: 30S ribosomal protein S6
G: 30S ribosomal protein S7
H: 30S ribosomal protein S8
I: 30S ribosomal protein S9
J: 30S ribosomal protein S10
K: 30S ribosomal protein S11
L: 30S ribosomal protein S12
M: 30S ribosomal protein S13
N: 30S ribosomal protein S14
O: 30S ribosomal protein S15
P: 30S ribosomal protein S16
Q: 30S ribosomal protein S17
R: 30S ribosomal protein S18
S: 30S ribosomal protein S19
T: 30S ribosomal protein S20
Z: Small ribosomal subunit biogenesis GTPase RsgA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)733,04292
Polymers730,72919
Non-polymers2,31373
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area87020 Å2
ΔGint-1163 kcal/mol
Surface area285460 Å2
MethodPISA

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Components

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30S ribosomal protein ... , 17 types, 17 molecules DEFGHIJKLMNOPQRST

#2: Protein 30S ribosomal protein S4 / / Small ribosomal subunit protein uS4


Mass: 23383.002 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / References: UniProt: P0A7V8
#3: Protein 30S ribosomal protein S5 / / Small ribosomal subunit protein uS5


Mass: 16361.878 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / References: UniProt: P0A7W1
#4: Protein 30S ribosomal protein S6 / / Small ribosomal subunit protein bS6


Mass: 12326.251 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / References: UniProt: P02358
#5: Protein 30S ribosomal protein S7 / / Small ribosomal subunit protein uS7


Mass: 14543.850 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / References: UniProt: P02359
#6: Protein 30S ribosomal protein S8 / / Small ribosomal subunit protein uS8


Mass: 14015.361 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / References: UniProt: P0A7W7
#7: Protein 30S ribosomal protein S9 / / Small ribosomal subunit protein uS9


Mass: 14554.882 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / References: UniProt: P0A7X3
#8: Protein 30S ribosomal protein S10 / / Small ribosomal subunit protein uS10


Mass: 11325.117 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / References: UniProt: P0A7R5
#9: Protein 30S ribosomal protein S11 / / Small ribosomal subunit protein uS11


Mass: 12487.200 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / References: UniProt: P0A7R9
#10: Protein/peptide 30S ribosomal protein S12 / / Small ribosomal subunit protein uS12


Mass: 1641.981 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / References: UniProt: P0A7S3
#11: Protein 30S ribosomal protein S13 / / Small ribosomal subunit protein uS13


Mass: 12625.753 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / References: UniProt: P0A7S9
#12: Protein 30S ribosomal protein S14 / / Small ribosomal subunit protein uS14


Mass: 11475.364 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / References: UniProt: P0AG59
#13: Protein 30S ribosomal protein S15 / / Small ribosomal subunit protein uS15


Mass: 10159.621 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / References: UniProt: P0ADZ4
#14: Protein 30S ribosomal protein S16 / / Small ribosomal subunit protein bS16


Mass: 9207.572 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / References: UniProt: P0A7T3
#15: Protein 30S ribosomal protein S17 / / Small ribosomal subunit protein uS17


Mass: 9263.946 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / References: UniProt: P0AG63
#16: Protein 30S ribosomal protein S18 / / Small ribosomal subunit protein bS18


Mass: 6466.477 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / References: UniProt: P0A7T7
#17: Protein 30S ribosomal protein S19 / / Small ribosomal subunit protein uS19


Mass: 9057.626 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / References: UniProt: P0A7U3
#18: Protein 30S ribosomal protein S20 / / Small ribosomal subunit protein bS20


Mass: 9577.268 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / References: UniProt: P0A7U7

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RNA chain / Protein , 2 types, 2 molecules AZ

#19: Protein Small ribosomal subunit biogenesis GTPase RsgA


Mass: 34850.457 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rsgA, engC, yjeQ, b4161, JW4122 / Plasmid: p7XNH3 / Production host: Escherichia coli BL21(DE3)
References: UniProt: P39286, Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides
#1: RNA chain 16S ribosomal RNA /


Mass: 497404.969 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: GenBank: 1108560344

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Non-polymers , 3 types, 73 molecules

#20: Chemical...
ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 71 / Source method: obtained synthetically / Formula: Mg
#21: Chemical ChemComp-ZN / ZINC ION / Zinc


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#22: Chemical ChemComp-GNP / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / 5'-Guanylyl imidodiphosphate


Mass: 522.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O13P3
Comment: GppNHp, GMPPNP, energy-carrying molecule analogue*YM

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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 ribosomal subunit bound by RsgA / Type: RIBOSOME / Entity ID: #1-#19 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli K12 (bacteria)
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPESC8H18N2O4S1
210 mMMagnesium chlorideMgCl21
3150 mMAmmonium acetateC2H7NO21
46 mM2-mercaptoethanolC2H6OS1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 278 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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Calibrated magnification: 101000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 2.3 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of real images: 3408
Image scansWidth: 2048 / Height: 2048 / Movie frames/image: 13

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Processing

SoftwareName: PHENIX / Classification: refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fittingrigid docking
8Cootmodel fittingmanual fitting
10RELION1.4initial Euler assignment
11RELION2final Euler assignment
12RELION2classification
13RELION23D reconstruction
14PHENIXdev-2621-000model refinement
15REFMAC5.8.0155model refinementinitial jelly body
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 878976
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 5.16 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61908 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE
Displacement parametersBiso max: 348.57 Å2 / Biso mean: 157.4923 Å2 / Biso min: 29.92 Å2
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00253327
ELECTRON MICROSCOPYf_angle_d0.52279630
ELECTRON MICROSCOPYf_chiral_restr0.02910134
ELECTRON MICROSCOPYf_plane_restr0.0054402
ELECTRON MICROSCOPYf_dihedral_angle_d11.8628598

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