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- PDB-4d5n: Cryo-EM structures of ribosomal 80S complexes with termination fa... -

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Basic information

Entry
Database: PDB / ID: 4d5n
TitleCryo-EM structures of ribosomal 80S complexes with termination factors and cricket paralysis virus IRES reveal the IRES in the translocated state
Components
  • CRICKET PARALYSIS VIRUS IRES RNA
  • EUKARYOTIC PEPTIDE CHAIN RELEASE FACTOR SUBUNIT 1
KeywordsRIBOSOME/RNA / RIBOSOME-RNA COMPLEX / CRPV IRES / RIBOSOME / TERMINATION / RELEASE FACTORS
Function / homology
Function and homology information


translation termination factor activity / cytoplasmic translational termination / translation release factor complex / regulation of translational termination / translation release factor activity / protein methylation / translation release factor activity, codon specific / sequence-specific mRNA binding / aminoacyl-tRNA hydrolase activity / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay ...translation termination factor activity / cytoplasmic translational termination / translation release factor complex / regulation of translational termination / translation release factor activity / protein methylation / translation release factor activity, codon specific / sequence-specific mRNA binding / aminoacyl-tRNA hydrolase activity / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / Protein hydroxylation / 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) / translational termination / cytosolic ribosome / Regulation of expression of SLITs and ROBOs / ribosome binding / RNA binding / cytosol / cytoplasm
Similarity search - Function
Peptide chain release factor eRF1/aRF1 / eRF1, domain 1 / eRF1 domain 2 / eRF1 domain 2 / eRF1 domain 1 / eRF1 domain 1/Pelota-like / eRF1 domain 3 / eRF1, domain 2 superfamily / eRF1 domain 3 / eRF1_1 / 50S ribosomal protein L30e-like
Similarity search - Domain/homology
: / RNA / RNA (> 10) / RNA (> 100) / Eukaryotic peptide chain release factor subunit 1
Similarity search - Component
Biological speciesHOMO SAPIENS (human)
CRICKET PARALYSIS VIRUS
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9 Å
AuthorsMuhs, M. / Hilal, T. / Mielke, T. / Skabkin, M.A. / Sanbonmatsu, K.Y. / Pestova, T.V. / Spahn, C.M.T.
CitationJournal: Mol Cell / Year: 2015
Title: Cryo-EM of ribosomal 80S complexes with termination factors reveals the translocated cricket paralysis virus IRES.
Authors: Margarita Muhs / Tarek Hilal / Thorsten Mielke / Maxim A Skabkin / Karissa Y Sanbonmatsu / Tatyana V Pestova / Christian M T Spahn /
Abstract: The cricket paralysis virus (CrPV) uses an internal ribosomal entry site (IRES) to hijack the ribosome. In a remarkable RNA-based mechanism involving neither initiation factor nor initiator tRNA, the ...The cricket paralysis virus (CrPV) uses an internal ribosomal entry site (IRES) to hijack the ribosome. In a remarkable RNA-based mechanism involving neither initiation factor nor initiator tRNA, the CrPV IRES jumpstarts translation in the elongation phase from the ribosomal A site. Here, we present cryoelectron microscopy (cryo-EM) maps of 80S⋅CrPV-STOP ⋅ eRF1 ⋅ eRF3 ⋅ GMPPNP and 80S⋅CrPV-STOP ⋅ eRF1 complexes, revealing a previously unseen binding state of the IRES and directly rationalizing that an eEF2-dependent translocation of the IRES is required to allow the first A-site occupation. During this unusual translocation event, the IRES undergoes a pronounced conformational change to a more stretched conformation. At the same time, our structural analysis provides information about the binding modes of eRF1 ⋅ eRF3 ⋅ GMPPNP and eRF1 in a minimal system. It shows that neither eRF3 nor ABCE1 are required for the active conformation of eRF1 at the intersection between eukaryotic termination and recycling.
History
DepositionNov 6, 2014Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 4, 2015Provider: repository / Type: Initial release
Revision 1.1Mar 4, 2015Group: Database references
Revision 2.0Aug 30, 2017Group: Atomic model / Data collection / Derived calculations
Category: atom_site / em_software / struct_conn
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _em_software.fitting_id / _em_software.image_processing_id / _struct_conn.details / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

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Structure visualization

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-2810
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Assembly

Deposited unit
A: EUKARYOTIC PEPTIDE CHAIN RELEASE FACTOR SUBUNIT 1
X: CRICKET PARALYSIS VIRUS IRES RNA


Theoretical massNumber of molelcules
Total (without water)113,4052
Polymers113,4052
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS

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Components

#1: Protein EUKARYOTIC PEPTIDE CHAIN RELEASE FACTOR SUBUNIT 1 / EUKARYOTIC RELEASE FACTOR 1 / ERF1 / PROTEIN CL1 / TB3-1 / EUKARYORIC RELEASE FACTOR 1


Mass: 49040.711 Da / Num. of mol.: 1 / Fragment: RESIDUES 5-437
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HOMO SAPIENS (human) / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P62495
#2: RNA chain CRICKET PARALYSIS VIRUS IRES RNA


Mass: 64363.910 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) CRICKET PARALYSIS VIRUS / References: GenBank: 8895506
Sequence detailsFIRST CODING TRIPLET MUTATED TO STOP

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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: CRICKET PARALYSIS VIRUS IRES RNA BOUND TO MAMMALIAN 80S RIBOSOME AND ERF1
Type: RIBOSOME / Details: MICROGRAPHS SELECTED FOR ASTIGMATISM AND DRIFT
Buffer solutionName: 20 MM TRIS PH 7.5, 100 MM KCL, 1 MM DTT, 2.5 MM MGCL2, 0.5 MM GTP
pH: 7.5
Details: 20 MM TRIS PH 7.5, 100 MM KCL, 1 MM DTT, 2.5 MM MGCL2, 0.5 MM GTP
SpecimenConc.: 1.38 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Details: LIQUID ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Apr 17, 2012
Details: GOOD MICROGRAPHS SELECTED FOR ASTIGMATISM AND DRIFT
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 39000 X / Calibrated magnification: 65520 X / Nominal defocus max: 4000 nm / Nominal defocus min: 2000 nm / Cs: 2 mm
Specimen holderTemperature: 77 K
Image recordingElectron dose: 20 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 366

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Processing

EM software
IDNameCategory
1UCSF Chimeramodel fitting
2SPARX3D reconstruction
3SPIDER3D reconstruction
CTF correctionDetails: DEFOCUS GROUPS
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: MULTI-REFERENCE TEMPLATE MATCHING / Resolution: 9 Å / Num. of particles: 109596 / Nominal pixel size: 1.56 Å / Actual pixel size: 1.56 Å
Magnification calibration: CROSS- -CORRELATION DENSITIES WITH REFERENCE STRUCTURE
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2810. (DEPOSITION ID: 12907).
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Details: METHOD--RIGID BODY, FLEXIBLE FIT
RefinementHighest resolution: 9 Å
Refinement stepCycle: LAST / Highest resolution: 9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3450 4257 0 0 7707

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