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- PDB-7zki: Cryo-EM structure of aIF1A:aIF5B:Met-tRNAiMet complex from a Pyro... -

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Basic information

Entry
Database: PDB / ID: 7zki
TitleCryo-EM structure of aIF1A:aIF5B:Met-tRNAiMet complex from a Pyrococcus abyssi 30S initiation complex
Components
  • (Translation initiation factor ...) x 2
  • Met-tRNAiMet
KeywordsTRANSLATION / Initiation complex / translation initiation / small ribosomal subunit / aIF5b
Function / homology
Function and homology information


UDP phosphatase activity / GDP phosphatase activity / proteoglycan biosynthetic process / intein-mediated protein splicing / intron homing / translation initiation factor activity / endonuclease activity / GTPase activity / calcium ion binding / GTP binding / RNA binding
Similarity search - Function
Apyrase / Apyrase superfamily / Apyrase / Translation initiation factor aIF-2, archaea / Elongation factor Tu-type domain / Elongation factor Tu domain 4 / Translation initiation factor IF- 2, domain 3 / Translation-initiation factor 2 / Translation initiation factor IF- 2 / Translation initiation factor IF-2, domain 3 superfamily ...Apyrase / Apyrase superfamily / Apyrase / Translation initiation factor aIF-2, archaea / Elongation factor Tu-type domain / Elongation factor Tu domain 4 / Translation initiation factor IF- 2, domain 3 / Translation-initiation factor 2 / Translation initiation factor IF- 2 / Translation initiation factor IF-2, domain 3 superfamily / Translation initiation factor 1A (eIF-1A), conserved site / Eukaryotic initiation factor 1A signature. / Intein splicing domain / eukaryotic translation initiation factor 1A / Translation initiation factor 1A (eIF-1A) / Intein DOD homing endonuclease / Intein DOD-type homing endonuclease domain profile. / RNA-binding domain, S1, IF1 type / Translation initiation factor 1A / IF-1 / S1 domain IF1 type 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
PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / METHIONINE / : / RNA / RNA (> 10) / Apyrase / Probable translation initiation factor IF-2 / Translation initiation factor 1A
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Pyrococcus abyssi GE5 (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsCoureux, P.D. / Bourgeois, G. / Mechulam, Y. / Schmitt, E. / Kazan, R.
Funding support France, 1items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 20, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 31, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
4: Met-tRNAiMet
6: Translation initiation factor 1A
7: Translation initiation factor 5B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)109,9896
Polymers109,2943
Non-polymers6963
Water00
1


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

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Components

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RNA chain , 1 types, 1 molecules 4

#1: RNA chain Met-tRNAiMet


Mass: 24833.904 Da / Num. of mol.: 1 / Mutation: C1A, A72U
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / References: GenBank: 1334604293

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Translation initiation factor ... , 2 types, 2 molecules 67

#2: Protein Translation initiation factor 1A / aIF-1A


Mass: 15336.709 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi GE5 (archaea) / Gene: eIF1A, aif1A, PYRAB05910, PAB2441 / Strain: GE5 / Orsay / Production host: Escherichia coli (E. coli) / References: UniProt: Q9V138
#3: Protein Translation initiation factor 5B


Mass: 69122.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi GE5 (archaea) / Gene: WBA_LOCUS12468, infB, PYRAB11390, PAB0755 / Strain: GE5 / Orsay / Production host: Escherichia coli (E. coli) / References: UniProt: A0A3P7EHT1, UniProt: Q9UZK7

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Non-polymers , 3 types, 3 molecules

#4: Chemical ChemComp-MET / METHIONINE


Type: L-peptide linking / Mass: 149.211 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H11NO2S
#5: Chemical ChemComp-GNP / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER


Mass: 522.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O13P3 / Feature type: SUBJECT OF INVESTIGATION
Comment: GppNHp, GMPPNP, energy-carrying molecule analogue*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

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

Component
IDNameTypeEntity IDParent-IDSource
1tRNA-aIF5B-aIF1A moiety of a Pyrococcus abyssi translation initiation complex with 30S ribosomal subunit,tRNA, mRNA and initiation factors 1A and 5B.COMPLEX#1-#30MULTIPLE SOURCES
2tRNA-MetCOMPLEX#11RECOMBINANT
3Translation initiation factor 1A and Probable translation initiation factor IF-2COMPLEX#2-#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Pyrococcus abyssi GE5 (archaea)272844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Escherichia coli (E. coli)562
Buffer solutionpH: 6.7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationCryogen name: ETHANE

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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 defocus max: 2500 nm / Nominal defocus min: 800 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 39 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2000000
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 37000
Details: Resolution calculated by the post-processing procedure in Relion.
Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0027403
ELECTRON MICROSCOPYf_angle_d0.47410385
ELECTRON MICROSCOPYf_dihedral_angle_d12.2491684
ELECTRON MICROSCOPYf_chiral_restr0.041247
ELECTRON MICROSCOPYf_plane_restr0.0041051

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