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- EMDB-26553: N2 sub-domain of IF2 bound to the 30S subunit in the Pseudomonas ... -

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Basic information

Entry
Database: EMDB / ID: EMD-26553
TitleN2 sub-domain of IF2 bound to the 30S subunit in the Pseudomonas aeruginosa 70S ribosome initiation complex (focused classification and refinement)
Map dataN2 sub-domain of IF2 bound to the 30S subunit in the 70S ribosome initiation complex. Focused map.
Sample
  • Complex: Focused map of the N2 sub-domain of IF2 bound to the 30S subunit in the 70S ribosome initiation complex
    • Protein or peptide: Translation initiation factor IF-2
KeywordsInitiation Factor 2 / 70S ribosome / cryo-EM / translation initiation / initiator tRNA / conformational changes / RIBOSOME
Function / homology
Function and homology information


translational initiation / translation initiation factor activity / 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 / Translation initiation factor IF- 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 / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsBasu RS / Sherman MB / Gagnon MG
Funding support United States, 2 items
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, 2022-
Header (metadata) releaseJun 22, 2022-
Map releaseJun 22, 2022-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26553.png

Downloads & links

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Map

FileDownload / File: emd_26553.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationN2 sub-domain of IF2 bound to the 30S subunit in the 70S ribosome initiation complex. Focused map.
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.14
Minimum - Maximum-0.6647893 - 2.0286038
Average (Standard dev.)-0.0065701897 (±0.023518918)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 435.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26553_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: N2 sub-domain of IF2 bound to the 30S...

Fileemd_26553_additional_1.map
AnnotationN2 sub-domain of IF2 bound to the 30S subunit in the 70S ribosome initiation complex. Sharpened focused map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: N2 sub-domain of IF2 bound to the 30S...

Fileemd_26553_half_map_1.map
AnnotationN2 sub-domain of IF2 bound to the 30S subunit in the 70S ribosome initiation complex. Half-map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: N2 sub-domain of IF2 bound to the 30S...

Fileemd_26553_half_map_2.map
AnnotationN2 sub-domain of IF2 bound to the 30S subunit in the 70S ribosome initiation complex. Half-map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Focused map of the N2 sub-domain of IF2 bound to the 30S subunit ...

EntireName: Focused map of the N2 sub-domain of IF2 bound to the 30S subunit in the 70S ribosome initiation complex
Components
  • Complex: Focused map of the N2 sub-domain of IF2 bound to the 30S subunit in the 70S ribosome initiation complex
    • Protein or peptide: Translation initiation factor IF-2

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Supramolecule #1: Focused map of the N2 sub-domain of IF2 bound to the 30S subunit ...

SupramoleculeName: Focused map of the N2 sub-domain of IF2 bound to the 30S subunit in the 70S ribosome initiation complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)

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Macromolecule #1: Translation initiation factor IF-2

MacromoleculeName: Translation initiation factor IF-2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Molecular weightTheoretical: 91.049641 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTQVTVKELA QVVDTPVERL LLQMRDAGLP HTSAEQVVTD SEKQALLTHL KGSHGDRASE PRKITLQRKT TTTLKVGGSK TVSVEVRKK KTYVKRSPDE IEAERQRELE EQRAAEEAER LKAEEAAARQ RAEEEARKAE EAARAKAAQE AAATAGAEPA V VADVAVAE ...String:
MTQVTVKELA QVVDTPVERL LLQMRDAGLP HTSAEQVVTD SEKQALLTHL KGSHGDRASE PRKITLQRKT TTTLKVGGSK TVSVEVRKK KTYVKRSPDE IEAERQRELE EQRAAEEAER LKAEEAAARQ RAEEEARKAE EAARAKAAQE AAATAGAEPA V VADVAVAE PVAKPAAVEE RKKEEPRRVP KRDEDDDRRD RKHTQHRPSV KEKEKVPAPR VAPRSTDEES DGYRRGGRGG KS KLKKRNQ HGFQNPTGPI VREVNIGETI TVAELAAQMS VKGAEVVKFM FKMGSPVTIN QVLDQETAQL VAEELGHKVK LVS ENALEE QLAESLKFEG EAVTRAPVVT VMGHVDHGKT SLLDYIRRAK VAAGEAGGIT QHIGAYHVET ERGMVTFLDT PGHA AFTAM RARGAQATDI VILVVAADDG VMPQTQEAVQ HAKAAGVPIV VAVNKIDKPE ANPDNIKNGL AALDVIPEEW GGDAP FVPV SAKLGTGVDE LLEAVLLQAE VLELKATPSA PGRGVVVESR LDKGRGPVAT VLVQDGTLRQ GDMVLVGINY GRVRAM LDE NGKPIKEAGP SIPVEILGLD GTPDAGDEMT VVADEKKARE VALFRQGKFR EVKLARAHAG KLENIFENMG QEEKKTL NI VLKADVRGSL EALQGSLSGL GNDEVQVRVV GGGVGGITES DANLALASNA VLFGFNVRAD AGARKIVEAE GLDMRYYN V IYDIIEDVKK ALTGMLGSDL RENILGIAEV RDVFRSPKFG AIAGCMVTEG MVHRNRPIRV LRDDVVIFEG ELESLRRFK DDVAEVRAGM ECGIGVKSYN DVKVGDKIEV FEKVEVARSL

UniProtKB: Translation initiation factor IF-2

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
GridModel: Quantifoil R2/1 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 295 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.2 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 8056 / Average exposure time: 1.0 sec. / Average electron dose: 31.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 878576
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1.0)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.1.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1.0) / Number images used: 123146

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7uiu:
N2 sub-domain of IF2 bound to the 30S subunit in the Pseudomonas aeruginosa 70S ribosome initiation complex (focused classification and refinement)

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