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- PDB-7uiu: N2 sub-domain of IF2 bound to the 30S subunit in the Pseudomonas ... -

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Basic information

Entry
Database: PDB / ID: 7uiu
TitleN2 sub-domain of IF2 bound to the 30S subunit in the Pseudomonas aeruginosa 70S ribosome initiation complex (focused classification and refinement)
ComponentsTranslation initiation factor IF-2
KeywordsRIBOSOME / Initiation Factor 2 / 70S ribosome / cryo-EM / translation initiation / initiator tRNA / conformational changes
Function / homology
Function and homology information


translation initiation factor activity / translational initiation / GTPase activity / GTP binding / cytoplasm
Similarity search - Function
Initiation factor 2 associated domain, bacterial / Bacterial translation initiation factor IF-2 associated region / Translation initiation factor IF-2, N-terminal / Translation initiation factor IF-2, N-terminal region / Translation initiation factor IF-2, domain II / Initiation factor 2 signature. / Translation initiation factor IF-2, bacterial-like / Translation initiation factor IF- 2, domain 3 / Translation-initiation factor 2 / : ...Initiation factor 2 associated domain, bacterial / Bacterial translation initiation factor IF-2 associated region / Translation initiation factor IF-2, N-terminal / Translation initiation factor IF-2, N-terminal region / Translation initiation factor IF-2, domain II / Initiation factor 2 signature. / Translation initiation factor IF-2, bacterial-like / Translation initiation factor IF- 2, domain 3 / Translation-initiation factor 2 / : / Elongation factor G domain 2 / Translation initiation factor IF- 2 / Translation initiation factor IF-2, domain 3 superfamily / Putative DNA-binding domain superfamily / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Small GTP-binding protein domain / Translation protein, beta-barrel domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Translation initiation factor IF-2
Similarity search - Component
Biological speciesPseudomonas aeruginosa PAO1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsBasu, R.S. / Sherman, M.B. / Gagnon, M.G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM136936 United States
Robert A. Welch FoundationH-2032-20200401 United States
CitationJournal: Nat Commun / Year: 2022
Title: Compact IF2 allows initiator tRNA accommodation into the P site and gates the ribosome to elongation.
Authors: Ritwika S Basu / Michael B Sherman / Matthieu G Gagnon /
Abstract: During translation initiation, initiation factor 2 (IF2) holds initiator transfer RNA (fMet-tRNA) in a specific orientation in the peptidyl (P) site of the ribosome. Upon subunit joining IF2 ...During translation initiation, initiation factor 2 (IF2) holds initiator transfer RNA (fMet-tRNA) in a specific orientation in the peptidyl (P) site of the ribosome. Upon subunit joining IF2 hydrolyzes GTP and, concomitant with inorganic phosphate (P) release, changes conformation facilitating fMet-tRNA accommodation into the P site and transition of the 70 S ribosome initiation complex (70S-IC) to an elongation-competent ribosome. The mechanism by which IF2 separates from initiator tRNA at the end of translation initiation remains elusive. Here, we report cryo-electron microscopy (cryo-EM) structures of the 70S-IC from Pseudomonas aeruginosa bound to compact IF2-GDP and initiator tRNA. Relative to GTP-bound IF2, rotation of the switch 2 α-helix in the G-domain bound to GDP unlocks a cascade of large-domain movements in IF2 that propagate to the distal tRNA-binding domain C2. The C2-domain relocates 35 angstroms away from tRNA, explaining how IF2 makes way for fMet-tRNA accommodation into the P site. Our findings provide the basis by which IF2 gates the ribosome to the elongation phase.
History
DepositionMar 29, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 22, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 29, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Feb 14, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
x: Translation initiation factor IF-2


Theoretical massNumber of molelcules
Total (without water)91,0501
Polymers91,0501
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Translation initiation factor IF-2


Mass: 91049.641 Da / Num. of mol.: 1 / Fragment: N2 sub-domain of IF2
Source method: isolated from a genetically manipulated source
Details: Initiation Factor 2 / Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria) / Gene: infB, PA4744 / Plasmid: pET28a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Star(DE3) / References: UniProt: Q9HV55

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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: Focused map of the N2 sub-domain of IF2 bound to the 30S subunit in the 70S ribosome initiation complex
Type: RIBOSOME / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pET28a
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R2/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2100 nm / Nominal defocus min: 200 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 1 sec. / Electron dose: 31 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8056

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.1.0particle selection
2EPU2.11.1image acquisition
4cryoSPARC3.1.0CTF correction
7Coot0.8.9.1model fitting
9cryoSPARC3.1.0initial Euler assignment
10cryoSPARC3.1.0final Euler assignment
11cryoSPARC3.1.0classification
12cryoSPARC3.1.03D reconstruction
13PHENIX1.19.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 878576
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 123146 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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