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- PDB-4crn: Cryo-EM of a pretermination complex with eRF1 and eRF3 -

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Basic information

Entry
Database: PDB / ID: 4crn
TitleCryo-EM of a pretermination complex with eRF1 and eRF3
Components
  • ERF1 IN RIBOSOME-BOUND ERF1-ERF3-GDPNP COMPLEXEukaryotic release factors
  • ERF3 IN RIBOSOME BOUND ERF1-ERF3-GDPNP COMPLEXGSPT1
KeywordsTRANSLATION / TERMINATION / CRYO-EM
Function / homologyTranslation protein, beta-barrel domain superfamily / eRF1 domain 2 / Elongation factor Tu domain 2 / Elongation factor Tu C-terminal domain / Elongation factor Tu GTP binding domain / Tr-type G domain, conserved site / 50S ribosomal protein L30e-like / P-loop containing nucleoside triphosphate hydrolase / Peptide Chain Release Factor eRF1/aRF1, N-terminal / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal ...Translation protein, beta-barrel domain superfamily / eRF1 domain 2 / Elongation factor Tu domain 2 / Elongation factor Tu C-terminal domain / Elongation factor Tu GTP binding domain / Tr-type G domain, conserved site / 50S ribosomal protein L30e-like / P-loop containing nucleoside triphosphate hydrolase / Peptide Chain Release Factor eRF1/aRF1, N-terminal / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / eRF1 domain 3 / eRF1 domain 2 / eRF1 domain 1/Pelota-like / Peptide chain release factor eRF1/aRF1 / Translation elongation factor EFTu-like, domain 2 / Translation elongation factor EFTu/EF1A, C-terminal / Eukaryotic peptide chain release factor GTP-binding subunit / Transcription factor, GTP-binding domain / eRF1 domain 3 / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / mRNA decay by 3' to 5' exoribonuclease / Eukaryotic Translation Termination / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / eRF1 domain 1 / translation release factor complex / cytoplasmic translational termination / translation release factor activity, codon specific / translation release factor activity / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / translational termination / DNA-templated transcription, termination / cytoplasmic stress granule / GTPase activity / GTP binding / identical protein binding / cytosol / Eukaryotic peptide chain release factor GTP-binding subunit / Eukaryotic peptide chain release factor subunit 1
Function and homology information
Specimen sourceSACCHAROMYCES CEREVISIAE (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 9.1 Å resolution
AuthorsPreis, A. / Heuer, A. / Barrio-Garcia, C. / Hauser, A. / Eyler, D. / Berninghausen, O. / Green, R. / Becker, T. / Beckmann, R.
CitationJournal: Cell Rep / Year: 2014
Title: Cryoelectron microscopic structures of eukaryotic translation termination complexes containing eRF1-eRF3 or eRF1-ABCE1.
Authors: Anne Preis / Andre Heuer / Clara Barrio-Garcia / Andreas Hauser / Daniel E Eyler / Otto Berninghausen / Rachel Green / Thomas Becker / Roland Beckmann
Abstract: Termination and ribosome recycling are essential processes in translation. In eukaryotes, a stop codon in the ribosomal A site is decoded by a ternary complex consisting of release factors eRF1 and ...Termination and ribosome recycling are essential processes in translation. In eukaryotes, a stop codon in the ribosomal A site is decoded by a ternary complex consisting of release factors eRF1 and guanosine triphosphate (GTP)-bound eRF3. After GTP hydrolysis, eRF3 dissociates, and ABCE1 can bind to eRF1-loaded ribosomes to stimulate peptide release and ribosomal subunit dissociation. Here, we present cryoelectron microscopic (cryo-EM) structures of a pretermination complex containing eRF1-eRF3 and a termination/prerecycling complex containing eRF1-ABCE1. eRF1 undergoes drastic conformational changes: its central domain harboring the catalytically important GGQ loop is either packed against eRF3 or swung toward the peptidyl transferase center when bound to ABCE1. Additionally, in complex with eRF3, the N-terminal domain of eRF1 positions the conserved NIKS motif proximal to the stop codon, supporting its suggested role in decoding, yet it appears to be delocalized in the presence of ABCE1. These results suggest that stop codon decoding and peptide release can be uncoupled during termination.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 28, 2014 / Release: Jul 23, 2014
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jul 23, 2014Structure modelrepositoryInitial release
1.1May 11, 2016Structure modelStructure summary
1.2Aug 23, 2017Structure modelData collectionem_image_scans / em_software_em_software.image_processing_id
Remark 700 SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "PB" IN EACH CHAIN ON SHEET RECORDS BELOW ... SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "PB" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 6-STRANDED BARREL THIS IS REPRESENTED BY A 7-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "PC" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 6-STRANDED BARREL THIS IS REPRESENTED BY A 7-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL.

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Structure viewerMolecule:
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Assembly

Deposited unit
P: ERF3 IN RIBOSOME BOUND ERF1-ERF3-GDPNP COMPLEX
X: ERF1 IN RIBOSOME-BOUND ERF1-ERF3-GDPNP COMPLEX
hetero molecules


Theoretical massNumber of molelcules
Total (without water)97,3963
Polyers96,8732
Non-polymers5221
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

#1: Protein/peptide ERF3 IN RIBOSOME BOUND ERF1-ERF3-GDPNP COMPLEX / GSPT1


Mass: 47840.965 Da / Num. of mol.: 1
Details: FOR CHAIN P (ERF3) THE FIRST 254 AMINO ACIDS ARE MISSING.
Source: (gene. exp.) SACCHAROMYCES CEREVISIAE (baker's yeast)
Plasmid name: PTYB2 / Production host: ESCHERICHIA COLI (E. coli) / References: UniProt: P05453
#2: Protein/peptide ERF1 IN RIBOSOME-BOUND ERF1-ERF3-GDPNP COMPLEX / Eukaryotic release factors


Mass: 49032.375 Da / Num. of mol.: 1
Details: FOR CHAIN X THE LAST 25 AMINO ACIDS (416-440) ARE MISSING
Source: (gene. exp.) SACCHAROMYCES CEREVISIAE (baker's yeast)
Plasmid name: PTYB2 / Production host: ESCHERICHIA COLI (E. coli) / References: UniProt: P12385
#3: Chemical ChemComp-GNP / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER


Mass: 522.196 Da / Num. of mol.: 1 / Formula: C10H17N6O13P3
Comment: GppNHp (GMPPNP, energy-carrying molecule analogue) *YM
Sequence detailsTHE FIRST 254 AMINO ACIDS FOR THE N-TERMINAL DOMAIN ARE MISSING IN THE MODEL THE LAST AMINO ACIDS ...THE FIRST 254 AMINO ACIDS FOR THE N-TERMINAL DOMAIN ARE MISSING IN THE MODEL THE LAST AMINO ACIDS (RESIDUES 416-440) ARE MISSING IN THE MODEL

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: CMV-STALLED WHEAT GERM 80S-RNC BOUND TO ERF1 AND ABCE1-ADPNP
Type: RIBOSOME
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: CARBON
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Details: LIQUID ETHANE, VITROBOT MARK 4

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS / Date: Feb 20, 2013
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 147136 / Nominal defocus max: 3500 nm / Nominal defocus min: 1300 nm / Cs: 2.7 mm
Image recordingElectron dose: 0.02 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k)

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Processing

EM software
IDNameCategory
1Cootmodel fitting
2UCSF Chimeramodel fitting
3STARFISHparticle selection
4SPIDER3D reconstruction
SymmetryPoint symmetry: C1
3D reconstructionMethod: PROJECTION MATCHING / Resolution: 9.1 Å / Number of particles: 39309
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2597 (DEPOSITION ID: 12368).
Symmetry type: POINT
Least-squares processHighest resolution: 9.1 Å
Refine hist #LASTHighest resolution: 9.1 Å
Number of atoms included #LASTProtein: 6661 / Nucleic acid: 0 / Ligand: 32 / Solvent: 0 / Total: 6693

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