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- EMDB-2810: Cryo-EM structures of ribosomal 80S complexes with termination fa... -

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Entry
Database: EMDB / ID: 2810
TitleCryo-EM structures of ribosomal 80S complexes with termination factors and cricket paralysis virus IRES reveal the IRES in the translocated state
Map dataReconstruction of mammalian termination complex with CrPV IRES-RNA and eRF1
SampleRibosomal 80S termination complex with CrPV IRES-RNA and eRF1:
ribosome-eukaryote / eukaryoric release factor 1 / nucleic-acidNucleic acid
KeywordsCrPV IRES / ribosome / Termination / release factors
Function / homologyRibosomal protein S3Ae, conserved site / WD40 repeat, conserved site / Ribosomal S24e conserved site / Ribosomal S11, conserved site / Ribosomal protein S2, conserved site / Ribosomal protein S4e, N-terminal, conserved site / Ribosomal protein S10, conserved site / Ribosomal protein S13, conserved site / Ribosomal protein S14, conserved site / Ribosomal protein S17e, conserved site ...Ribosomal protein S3Ae, conserved site / WD40 repeat, conserved site / Ribosomal S24e conserved site / Ribosomal S11, conserved site / Ribosomal protein S2, conserved site / Ribosomal protein S4e, N-terminal, conserved site / Ribosomal protein S10, conserved site / Ribosomal protein S13, conserved site / Ribosomal protein S14, conserved site / Ribosomal protein S17e, conserved site / Ribosomal protein S19e, conserved site / Ribosomal protein S21e, conserved site / Ribosomal protein S3, conserved site / Ribosomal protein S6e, conserved site / Ribosomal protein S8e, conserved site / Ubiquitin conserved site / WD40-repeat-containing domain / Ubiquitin / Ribosomal protein S17, conserved site / G-protein beta WD-40 repeat / Ribosomal protein S5 domain 2-type fold / Ribosomal protein S9, conserved site / Ribosomal protein S7, conserved site / Ribosomal protein S19 conserved site / Ribosomal protein S8e/ribosomal biogenesis NSA2 / Ribosomal Proteins L2, RNA binding domain / Ribosomal protein L2, C-terminal / Ribosomal protein L2, conserved site / Ribosomal protein S4/S9 / Ribosomal protein S15P / Ribosomal protein S19, superfamily / Ribosomal protein S4, conserved site / K homology domain-like, alpha/beta / Ribosomal protein L2, archaeal-type / Ribosomal protein S30 / eRF1 domain 3 / Ribosomal protein S5, C-terminal / Plectin/S10, N-terminal / Ribosomal protein S23, eukaryotic/archaeal / Ribosomal protein S3, eukaryotic/archaeal / Ribosomal protein S2, eukaryotic/archaeal / Ribosomal protein S4/S9, eukaryotic/archaeal / Ribosomal protein S5, eukaryotic/archaeal / Ribosomal protein S19A/S15e / Ribosomal protein S5/S7, eukaryotic/archaeal / Ribosomal protein S10, eukaryotic/archaeal / KOW / Ribosomal protein S12/S23 / Translation protein SH3-like domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / K homology domain superfamily, prokaryotic type / S15/NS1, RNA-binding / Ribosomal protein S13-like, H2TH / Zinc-binding ribosomal protein / Nucleic acid-binding, OB-fold / Ribosomal protein S13/S15, N-terminal / Ribosomal protein L23/L15e core domain superfamily / Ribosomal protein S5, N-terminal / Ribosomal protein S4e, N-terminal / Ribosomal protein S4e, central region / Ribosomal protein S6, eukaryotic / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein L2, domain 2 / Ribosomal protein L2, domain 3 / Ribosomal protein S2, flavodoxin-like domain superfamily / Ribosomal protein S7 domain / S25 ribosomal protein / Ribosomal protein S13/S18 / Ribosomal protein S3, C-terminal domain / Ribosomal protein S19 / Ubiquitin family / Ribosomal protein S14p/S29e / Ribosomal protein S15 / Ribosomal protein S2 / Ribosomal protein S5, N-terminal domain / Ribosomal protein S10p/S20e / Ribosomal protein S17 / Ribosomal protein S9/S16 / WD domain, G-beta repeat / Ribosomal protein S8 / Ribosomal protein S11 / KOW motif / Ribosomal protein S7p/S5e / Ribosomal S17 / Ribosomal family S4e / Ribosomal S3Ae family / Ribosomal protein S19e / Ribosomal protein S6e / Ribosomal protein S28e / Ribosomal protein S8e / Ribosomal protein L7Ae/L30e/S12e/Gadd45 family / Ribosomal protein S21e / Ribosomal protein S7e / Ribosomal protein S24e / Ribosomal protein S26e / S4 domain / Ribosomal protein S27a / Ribosomal Proteins L2, RNA binding domain / Ribosomal protein S12/S23 / Peptide Chain Release Factor eRF1/aRF1, N-terminal
Function and homology information
SourceOryctolagus cuniculus (rabbit) / Homo sapiens (human) / Cricket paralysis virus
Methodsingle particle reconstruction / cryo EM / 8.7 Å resolution
AuthorsMuhs M / Hilal T / Mielke T / Skabkin MA / Sanbonmatsu KY / Pestova TV / Spahn CMT
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.
Validation ReportPDB-ID: 4d5l

SummaryFull report
PDB-ID: 4d5n

SummaryFull report
PDB-ID: 4d5y

SummaryFull report
About validation report
DateDeposition: Nov 3, 2014 / Header (metadata) release: Dec 24, 2014 / Map release: Feb 4, 2015 / Last update: Mar 11, 2015

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.5
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  • Surface view colored by height
  • Surface level: 2.5
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  • Surface view with fitted model
  • Atomic models: : PDB-4d5l, PDB-4d5n, PDB-4d5y
  • Surface level: 2.5
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-4d5l
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-4d5n
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-4d5y
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_2810.map.gz (map file in CCP4 format, 105470 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
300 pix
1.56 Å/pix.
= 468. Å
300 pix
1.56 Å/pix.
= 468. Å
300 pix
1.56 Å/pix.
= 468. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.56 Å
Density
Contour Level:2.5 (by author), 2.5 (movie #1):
Minimum - Maximum-3.84100437 - 12.03551006
Average (Standard dev.)0.20275222 (0.88032973)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions300300300
Origin-150-150-149
Limit149149150
Spacing300300300
CellA=B=C: 467.99997 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.561.561.56
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z468.000468.000468.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-32-96
NX/NY/NZ8165193
MAP C/R/S123
start NC/NR/NS-150-150-149
NC/NR/NS300300300
D min/max/mean-3.84112.0360.203

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Supplemental data

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Sample components

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Entire Ribosomal 80S termination complex with CrPV IRES-RNA and eRF1

EntireName: Ribosomal 80S termination complex with CrPV IRES-RNA and eRF1
Number of components: 3
Oligomeric State: CrPV IRES and eRF1 bound to one 80S ribosome
MassTheoretical: 4.5 MDa

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Component #1: ribosome-eukaryote, 80S ribosome

Ribosome-eukaryoteName: 80S ribosome / Eukaryote: ALL / Recombinant expression: No
MassTheoretical: 4.5 MDa
SourceSpecies: Oryctolagus cuniculus (rabbit)

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Component #2: protein, eukaryoric release factor 1

ProteinName: eukaryoric release factor 1 / a.k.a: eRF1Eukaryotic release factors / Oligomeric Details: Monomer / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 50 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21 (bacteria)
External referencesUniProt: Eukaryotic peptide chain release factor subunit 1

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Component #3: nucleic-acid, Cricket paralysis virus IRES RNA

Nucleic-acidName: Cricket paralysis virus IRES RNA / a.k.a: CrPV IRES RNA / Class: RNA / Structure: OTHER
Sequence:
AAAAAUGUGA UCUUGCUUGU AAAUACAAUU UUGAGAGGUU AAUAAAUUAC AAGUAGUGCU AUUUUUGUAU UUAGGUUAGC UAUUUAGCUU UACGUUCCAG GAUGCCUAGU GGCAGCCCCA CAAUAUCCAG GAAGCCCUCU CUGCGGUUUU UCAGAUUAGG UAGUCGAAAA ACCUAAGAAA UUUACCUUAA GGCUUCCUCG A

Synthetic: No
SourceSpecies: Cricket paralysis virus

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 1.38 mg/ml
Buffer solution: 20 mM Tris pH 7.5, 100 mM KCl, 1 mM DTT, 2.5 mM MgCl2, 0.5 mM GTP
pH: 7.5
Support filmQuantifoil grids with additional continuous carbon support
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 85 % / Method: blot for 2/4 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20 / Date: Apr 17, 2012 / Details: minimal dose system
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 39000 X (nominal), 65520 X (calibrated)
Astigmatism: Objective lens astigmatism was corrected at 200,000 times magnification
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 2000 - 4000 nm
Specimen HolderModel: GATAN LIQUID NITROGEN / Temperature: K ( 77 - K)
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of digital images: 366 / Scanner: OTHER / Bit depth: 16

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 109596
Details: The particles were selected using SIGNATURE and processed by using SPIDER and SPARX
3D reconstructionAlgorithm: Multi-reference template matching / Software: SPIDER, SPARX / CTF correction: Defocus group
Details: The particles were selected using SIGNATURE and processed by using SPIDER and SPARX.
Resolution: 8.7 Å / Resolution method: FSC 0.5, semi-independent

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Atomic model buiding

Modeling #1Software: CHIMERA / Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 4CXC
Chain ID: A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, Z, a, b, c, d, e, f, g, 1
Modeling #2Software: CHIMERA / Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 4CXD
Chain ID: A, B, C, D, E, F, G, H, I, J, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, Z, a, b, c, d, e, f, g, h, i, j, k, l, m, n, o, p, t, u
Output model

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