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- PDB-7unt: Compact IF2-GDP bound to the Pseudomonas aeruginosa 70S ribosome ... -

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Basic information

Entry
Database: PDB / ID: 7unt
TitleCompact IF2-GDP bound to the Pseudomonas aeruginosa 70S ribosome initiation complex, from focused classification and refinement (I-B)
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
GUANOSINE-5'-DIPHOSPHATE / Translation initiation factor IF-2
Similarity search - Component
Biological speciesPseudomonas aeruginosa PAO1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 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: Basu, R.S. / Sherman, M.B. / Gagnon, M.G.
History
DepositionApr 11, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 22, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 14, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
x: Translation initiation factor IF-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)91,4932
Polymers91,0501
Non-polymers4431
Water00
1


  • Idetical with deposited unit
  • defined by author
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
#2: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: GDP, energy-carrying molecule*YM
Has ligand of interestY

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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: Compact IF2-GDP bound to the Pseudomonas aeruginosa 70S ribosome initiation complex, from focused classification and refinement (I-B)
Type: RIBOSOME / Entity ID: #1 / Source: NATURAL
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 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
8UCSF Chimera1.14model fitting
10PHENIX1.19.2model refinement
11cryoSPARC3.1.0initial Euler assignment
12cryoSPARC3.1.0final Euler assignment
13cryoSPARC3.1.0classification
14cryoSPARC3.1.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 878576
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 24614 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 3JCJ

3jcj


Accession code: 3JCJ / Source name: PDB / Type: experimental model

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