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- EMDB-14763: Cryo-EM structure of aIF1A:aIF5B:Met-tRNAiMet complex from a Pyro... -

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Basic information

Entry
Database: EMDB / ID: EMD-14763
TitleCryo-EM structure of aIF1A:aIF5B:Met-tRNAiMet complex from a Pyrococcus abyssi 30S initiation complex
Map data
Sample
  • Complex: tRNA-aIF5B-aIF1A moiety of a Pyrococcus abyssi translation initiation complex with 30S ribosomal subunit,tRNA, mRNA and initiation factors 1A and 5B.
    • Complex: tRNA-Met
      • RNA: Met-tRNAiMet
    • Complex: Translation initiation factor 1A and Probable translation initiation factor IF-2
      • Protein or peptide: Translation initiation factor 1A
      • Protein or peptide: Translation initiation factor 5B
  • Ligand: METHIONINE
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
Function / homology
Function and homology information


nucleoside diphosphate phosphatase activity / intron homing / intein-mediated protein splicing / translation initiation factor activity / endonuclease activity / GTPase activity / calcium ion binding / GTP binding / RNA binding / cytoplasm
Similarity search - Function
Apyrase / Apyrase superfamily / Translation initiation factor aIF-2, archaea / Elongation factor Tu-type domain / Elongation factor Tu domain 4 / Translation initiation factor 1A (eIF-1A), conserved site / Eukaryotic initiation factor 1A signature. / eukaryotic translation initiation factor 1A / Translation initiation factor 1A (eIF-1A) / Translation initiation factor IF- 2, domain 3 ...Apyrase / Apyrase superfamily / Translation initiation factor aIF-2, archaea / Elongation factor Tu-type domain / Elongation factor Tu domain 4 / Translation initiation factor 1A (eIF-1A), conserved site / Eukaryotic initiation factor 1A signature. / eukaryotic translation initiation factor 1A / Translation initiation factor 1A (eIF-1A) / Translation initiation factor IF- 2, domain 3 / Translation-initiation factor 2 / Translation initiation factor IF- 2 / Translation initiation factor IF-2, domain 3 superfamily / Intein splicing domain / RNA-binding domain, S1, IF1 type / Translation initiation factor 1A / IF-1 / S1 domain IF1 type profile. / Intein DOD homing endonuclease / Intein DOD-type homing endonuclease domain profile. / Intein C-terminal splicing region / Intein C-terminal splicing motif profile. / Hint domain C-terminal / Hint (Hedgehog/Intein) domain C-terminal region / Intein N-terminal splicing region / Intein N-terminal splicing motif profile. / Hint domain N-terminal / Hint (Hedgehog/Intein) domain N-terminal region / Homing endonuclease / Hint domain superfamily / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Rossmann-like alpha/beta/alpha sandwich fold / Small GTP-binding protein domain / Translation protein, beta-barrel domain superfamily / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Uncharacterized protein / Probable translation initiation factor IF-2 / Translation initiation factor 1A
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Pyrococcus abyssi GE5 (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsCoureux PD / Bourgeois G / Mechulam Y / Schmitt E / Kazan R
Funding support France, 1 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR) France
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Role of aIF5B in archaeal translation initiation.
Authors: Ramy Kazan / Gabrielle Bourgeois / Christine Lazennec-Schurdevin / Eric Larquet / Yves Mechulam / Pierre-Damien Coureux / Emmanuelle Schmitt /
Abstract: In eukaryotes and in archaea late steps of translation initiation involve the two initiation factors e/aIF5B and e/aIF1A. In eukaryotes, the role of eIF5B in ribosomal subunit joining is established ...In eukaryotes and in archaea late steps of translation initiation involve the two initiation factors e/aIF5B and e/aIF1A. In eukaryotes, the role of eIF5B in ribosomal subunit joining is established and structural data showing eIF5B bound to the full ribosome were obtained. To achieve its function, eIF5B collaborates with eIF1A. However, structural data illustrating how these two factors interact on the small ribosomal subunit have long been awaited. The role of the archaeal counterparts, aIF5B and aIF1A, remains to be extensively addressed. Here, we study the late steps of Pyrococcus abyssi translation initiation. Using in vitro reconstituted initiation complexes and light scattering, we show that aIF5B bound to GTP accelerates subunit joining without the need for GTP hydrolysis. We report the crystallographic structures of aIF5B bound to GDP and GTP and analyze domain movements associated to these two nucleotide states. Finally, we present the cryo-EM structure of an initiation complex containing 30S bound to mRNA, Met-tRNAiMet, aIF5B and aIF1A at 2.7 Å resolution. Structural data shows how archaeal 5B and 1A factors cooperate to induce a conformation of the initiator tRNA favorable to subunit joining. Archaeal and eukaryotic features of late steps of translation initiation are discussed.
History
DepositionApr 13, 2022-
Header (metadata) releaseJul 20, 2022-
Map releaseJul 20, 2022-
UpdateAug 31, 2022-
Current statusAug 31, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14763.png

Downloads & links

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Map

FileDownload / File: emd_14763.map.gz / Format: CCP4 / Size: 307.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 432 pix.
= 371.52 Å		0.86 Å/pix.
x 432 pix.
= 371.52 Å		0.86 Å/pix.
x 432 pix.
= 371.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.012
Minimum - Maximum-0.02421813 - 0.07090903
Average (Standard dev.)-9.843442e-05 (±0.0018164722)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions432432432
Spacing432432432
CellA=B=C: 371.52002 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Not sharpened map

Fileemd_14763_additional_1.map
AnnotationNot sharpened map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_14763_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_14763_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : tRNA-aIF5B-aIF1A moiety of a Pyrococcus abyssi translation initia...

EntireName: tRNA-aIF5B-aIF1A moiety of a Pyrococcus abyssi translation initiation complex with 30S ribosomal subunit,tRNA, mRNA and initiation factors 1A and 5B.
Components
  • Complex: tRNA-aIF5B-aIF1A moiety of a Pyrococcus abyssi translation initiation complex with 30S ribosomal subunit,tRNA, mRNA and initiation factors 1A and 5B.
    • Complex: tRNA-Met
      • RNA: Met-tRNAiMet
    • Complex: Translation initiation factor 1A and Probable translation initiation factor IF-2
      • Protein or peptide: Translation initiation factor 1A
      • Protein or peptide: Translation initiation factor 5B
  • Ligand: METHIONINE
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: tRNA-aIF5B-aIF1A moiety of a Pyrococcus abyssi translation initia...

SupramoleculeName: tRNA-aIF5B-aIF1A moiety of a Pyrococcus abyssi translation initiation complex with 30S ribosomal subunit,tRNA, mRNA and initiation factors 1A and 5B.
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: tRNA-Met

SupramoleculeName: tRNA-Met / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: Translation initiation factor 1A and Probable translation initiat...

SupramoleculeName: Translation initiation factor 1A and Probable translation initiation factor IF-2
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Met-tRNAiMet

MacromoleculeName: Met-tRNAiMet / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 24.833904 KDa
SequenceString:
AGCGGGG(4SU)GG AGCAGCCUGG (H2U)AGCUCGUCG GG(OMC)UCAUAAC CCGAAGAUCG UCGG(5MU)(PSU)CAAA UCCGGCCCC CGCUACCA

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Macromolecule #2: Translation initiation factor 1A

MacromoleculeName: Translation initiation factor 1A / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea) / Strain: GE5 / Orsay
Molecular weightTheoretical: 15.336709 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MGSSSHHHHH HSSGLVPRGS HMPKKERKVE GDEVIRVPLP EGNQLFGVVE QALGAGWMDV RCEDGKIRRC RIPGKLRRRV WIRVGDLVI VQPWPVQSDK RGDIVYRYTQ TQVDWLLRKG KITQEFLTGG SLLVE

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Macromolecule #3: Translation initiation factor 5B

MacromoleculeName: Translation initiation factor 5B / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea) / Strain: GE5 / Orsay
Molecular weightTheoretical: 69.122945 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MMTKRIRQPI IAVLGHVDHG KTTLLDRIRK TNVAAKEAGG ITQHIGATEV PIEVVKKIAG PLIKLWKAE IKLPGLLFID TPGHEAFTSL RARGGSLADL AVLVVDINEG FQPQTIESIE ILRKYRTPFV VAANKIDRIK G WVIEEDEP ...String:
MGSSHHHHHH SSGLVPRGSH MMTKRIRQPI IAVLGHVDHG KTTLLDRIRK TNVAAKEAGG ITQHIGATEV PIEVVKKIAG PLIKLWKAE IKLPGLLFID TPGHEAFTSL RARGGSLADL AVLVVDINEG FQPQTIESIE ILRKYRTPFV VAANKIDRIK G WVIEEDEP FLMNIKKQDQ RAVQELETKL WELIGKFYEF GFQANRFDRV QNFTRELAIV PISAKYGIGI AELLVLIAGL SQ RYLEEKL KIEVEGPARG TILEVREEPG LGHTIDVIIY DGTLHKDDTI VVGGKDKAIV TKIRALLKPK PLDEIRDPRF RFD YVDEVT AAAGVKIAAP GLEEALAGSP VIAAPTPEDV EKAKQEILEQ IERVVISTDK VGVIVKADTL GSLEALSKEL QEKE IPIRK ADVGNVSKTD VMEALSVKEE EPKYGVILGF NVKVNEDAEE VAKAKDVKIF VGNVIYKLIE DYEEWVKEEE EKKKR ELLS KVTFPGVIRL YPDERYVFRR SNPAIVGIEV IEGRIKPGVT LIKQNGQKVG VIRSIKSRDE FLQEAKKGQA VAIAIE GAI VGRHIHPGET LYVDLSRDDA ITLLKHLRDT LEDTDIKALK MIAKVKAKED PFWRAI

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Macromolecule #4: METHIONINE

MacromoleculeName: METHIONINE / type: ligand / ID: 4 / Number of copies: 1 / Formula: MET
Molecular weightTheoretical: 149.211 Da
Chemical component information

ChemComp-MET:
METHIONINE

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Macromolecule #5: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / type: ligand / ID: 5 / Number of copies: 1 / Formula: GNP
Molecular weightTheoretical: 522.196 Da
Chemical component information

ChemComp-GNP:
PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / GppNHp, GMPPNP, energy-carrying molecule analogue*YM

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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: 6.7
GridModel: Quantifoil R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2.0 nm
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 39.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2000000
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7yzn

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Resolution calculated by the post-processing procedure in Relion.
Number images used: 37000
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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