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- PDB-8b6z: CryoEM Structure of Extended eEF1A bound to the Ribosome in the C... -

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Basic information

Entry
Database: PDB / ID: 8b6z
TitleCryoEM Structure of Extended eEF1A bound to the Ribosome in the Classical Pre State
Components
  • 28S ribosomal RNA
  • Elongation factor 1-alpha 2
KeywordsRIBOSOME / translation / protein biogenesis / elongation factor
Function / homology
Function and homology information


cytoplasmic side of lysosomal membrane / Eukaryotic Translation Elongation / eukaryotic translation elongation factor 1 complex / regulation of chaperone-mediated autophagy / translation factor activity, RNA binding / translational elongation / positive regulation of lipid kinase activity / translation elongation factor activity / translation / positive regulation of apoptotic process ...cytoplasmic side of lysosomal membrane / Eukaryotic Translation Elongation / eukaryotic translation elongation factor 1 complex / regulation of chaperone-mediated autophagy / translation factor activity, RNA binding / translational elongation / positive regulation of lipid kinase activity / translation elongation factor activity / translation / positive regulation of apoptotic process / GTPase activity / synapse / GTP binding / protein kinase binding / cytoplasm
Similarity search - Function
Translation elongation factor EF1A, eukaryotic/archaeal / Translation elongation factor EFTu/EF1A, C-terminal / Elongation factor Tu C-terminal domain / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain ...Translation elongation factor EF1A, eukaryotic/archaeal / Translation elongation factor EFTu/EF1A, C-terminal / Elongation factor Tu C-terminal domain / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / 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. / Translation protein, beta-barrel domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
: / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Elongation factor 1-alpha 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsGemmer, M. / Fedry, J.M.M. / Forster, F.G.
Funding supportEuropean Union, Netherlands, 3items
OrganizationGrant numberCountry
European Research Council (ERC)724425European Union
Netherlands Organisation for Scientific Research (NWO)724.016.001 Netherlands
Netherlands Organisation for Scientific Research (NWO)212.152 Netherlands
CitationJournal: Nature / Year: 2023
Title: Visualization of translation and protein biogenesis at the ER membrane.
Authors: Max Gemmer / Marten L Chaillet / Joyce van Loenhout / Rodrigo Cuevas Arenas / Dimitrios Vismpas / Mariska Gröllers-Mulderij / Fujiet A Koh / Pascal Albanese / Richard A Scheltema / Stuart C ...Authors: Max Gemmer / Marten L Chaillet / Joyce van Loenhout / Rodrigo Cuevas Arenas / Dimitrios Vismpas / Mariska Gröllers-Mulderij / Fujiet A Koh / Pascal Albanese / Richard A Scheltema / Stuart C Howes / Abhay Kotecha / Juliette Fedry / Friedrich Förster /
Abstract: The dynamic ribosome-translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome. It governs the synthesis, translocation, membrane ...The dynamic ribosome-translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome. It governs the synthesis, translocation, membrane insertion, N-glycosylation, folding and disulfide-bond formation of nascent proteins. Although individual components of this machinery have been studied at high resolution in isolation, insights into their interplay in the native membrane remain limited. Here we use cryo-electron tomography, extensive classification and molecular modelling to capture snapshots of mRNA translation and protein maturation at the ER membrane at molecular resolution. We identify a highly abundant classical pre-translocation intermediate with eukaryotic elongation factor 1a (eEF1a) in an extended conformation, suggesting that eEF1a may remain associated with the ribosome after GTP hydrolysis during proofreading. At the ER membrane, distinct polysomes bind to different ER translocons specialized in the synthesis of proteins with signal peptides or multipass transmembrane proteins with the translocon-associated protein complex (TRAP) present in both. The near-complete atomic model of the most abundant ER translocon variant comprising the protein-conducting channel SEC61, TRAP and the oligosaccharyltransferase complex A (OSTA) reveals specific interactions of TRAP with other translocon components. We observe stoichiometric and sub-stoichiometric cofactors associated with OSTA, which are likely to include protein isomerases. In sum, we visualize ER-bound polysomes with their coordinated downstream machinery.
History
DepositionSep 27, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 7, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 25, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year
Revision 1.2Feb 8, 2023Group: 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 / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Elongation factor 1-alpha 2
B: 28S ribosomal RNA


Theoretical massNumber of molelcules
Total (without water)1,690,7672
Polymers1,690,7672
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area720 Å2
ΔGint0 kcal/mol
Surface area22310 Å2

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Components

#1: Protein Elongation factor 1-alpha 2 / EF-1-alpha-2 / Eukaryotic elongation factor 1 A-2 / eEF1A-2 / Statin-S1


Mass: 50545.102 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q05639
#2: RNA chain 28S ribosomal RNA /


Mass: 1640222.125 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: GenBank: 86475748

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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: Ribosome in the Classical Pre+ state / Type: RIBOSOME / Entity ID: all / Source: NATURAL
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R3.5/1
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 200 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameCategory
1RELIONparticle selection
4CTFFINDCTF correction
10RELIONinitial Euler assignment
11RELIONclassification
12RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 19046 / Symmetry type: POINT

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