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- EMDB-2048: Cryo-EM structure of the UPF-EJC complex -

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Basic information

Entry
Database: EMDB / ID: EMD-2048
TitleCryo-EM structure of the UPF-EJC complex
Map dataCryo-EM reconstruction of the nonsense-mediated mRNA decay (NMD) complex containing proteins UPF1, UPF2, UPF3 and the Exon Junction Complex (EJC).
Sample
  • Sample: Complex containing the NMD proteins UPF1, UPF2 and UPF3 bound to the exon junction complex (EJC)
  • Protein or peptide: Up-frameshift protein 1
  • Protein or peptide: Up-frameshift protein 2
  • Protein or peptide: Up-frameshift protein 3b
  • Protein or peptide: MAGO
  • Protein or peptide: Y14
  • Protein or peptide: Btz
  • Protein or peptide: eIF4AIII
  • RNA: (U)8 RNA
KeywordsNMD / EJC / UPF1 / UPF2 / UPF3 / RNA degradation / GraFix
Function / homologyWIBG, Mago-binding / UPF3 domain / Btz domain / RNA helicase UPF1, Cys/His rich zinc-binding domain / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay
Function and homology information
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 16.0 Å
AuthorsMelero R / Buchwald G / Castano R / Raabe M / Gil D / Lazaro M / Urlaub H / Conti E / Llorca O
CitationJournal: Nat Struct Mol Biol / Year: 2012
Title: The cryo-EM structure of the UPF-EJC complex shows UPF1 poised toward the RNA 3' end.
Authors: Roberto Melero / Gretel Buchwald / Raquel Castaño / Monika Raabe / David Gil / Melisa Lázaro / Henning Urlaub / Elena Conti / Oscar Llorca /
Abstract: Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that degrades aberrant mRNAs containing premature termination codons (PTCs). NMD is triggered upon the assembly of the UPF ...Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that degrades aberrant mRNAs containing premature termination codons (PTCs). NMD is triggered upon the assembly of the UPF surveillance complex near a PTC. In humans, UPF assembly is prompted by the exon junction complex (EJC). We investigated the molecular architecture of the human UPF complex bound to the EJC by cryo-EM and using positional restraints from additional EM, MS and biochemical interaction data. The heptameric assembly is built around UPF2, a scaffold protein with a ring structure that closes around the CH domain of UPF1, keeping the helicase region in an accessible and unwinding-competent state. UPF2 also positions UPF3 to interact with the EJC. The geometry is such that this transient complex poises UPF1 to elicit helicase activity toward the 3' end of the mRNP.
History
DepositionMar 13, 2012-
Header (metadata) releaseApr 10, 2012-
Map releaseApr 10, 2012-
UpdateMar 30, 2016-
Current statusMar 30, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2.4
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2048.tif

Downloads & links

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Map

FileDownload / File: emd_2048.map.gz / Format: CCP4 / Size: 9.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of the nonsense-mediated mRNA decay (NMD) complex containing proteins UPF1, UPF2, UPF3 and the Exon Junction Complex (EJC).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.2 Å/pix.
x 136 pix.
= 299.2 Å		2.2 Å/pix.
x 136 pix.
= 299.2 Å		2.2 Å/pix.
x 136 pix.
= 299.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.4 / Movie #1: 2.4
Minimum - Maximum-6.43814659 - 17.813716889999998
Average (Standard dev.)-0.02877257 (±0.96611029)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions136136136
Spacing136136136
CellA=B=C: 299.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.22.22.2
M x/y/z136136136
origin x/y/z0.0000.0000.000
length x/y/z299.200299.200299.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS136136136
D min/max/mean-6.43817.814-0.029

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Supplemental data

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Sample components

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Entire : Complex containing the NMD proteins UPF1, UPF2 and UPF3 bound to ...

EntireName: Complex containing the NMD proteins UPF1, UPF2 and UPF3 bound to the exon junction complex (EJC)
Components
  • Sample: Complex containing the NMD proteins UPF1, UPF2 and UPF3 bound to the exon junction complex (EJC)
  • Protein or peptide: Up-frameshift protein 1
  • Protein or peptide: Up-frameshift protein 2
  • Protein or peptide: Up-frameshift protein 3b
  • Protein or peptide: MAGO
  • Protein or peptide: Y14
  • Protein or peptide: Btz
  • Protein or peptide: eIF4AIII
  • RNA: (U)8 RNA

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Supramolecule #1000: Complex containing the NMD proteins UPF1, UPF2 and UPF3 bound to ...

SupramoleculeName: Complex containing the NMD proteins UPF1, UPF2 and UPF3 bound to the exon junction complex (EJC)
type: sample / ID: 1000 / Oligomeric state: Hetero-octameric / Number unique components: 8
Molecular weightExperimental: 390 KDa / Theoretical: 390 KDa

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Macromolecule #1: Up-frameshift protein 1

MacromoleculeName: Up-frameshift protein 1 / type: protein_or_peptide / ID: 1 / Name.synonym: UPF1 / Details: 115-914 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 90 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceGO: nuclear-transcribed mRNA catabolic process, nonsense-mediated decay
InterPro: RNA helicase UPF1, Cys/His rich zinc-binding domain

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Macromolecule #2: Up-frameshift protein 2

MacromoleculeName: Up-frameshift protein 2 / type: protein_or_peptide / ID: 2 / Name.synonym: UPF2 / Details: full length / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 148 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceGO: nuclear-transcribed mRNA catabolic process, nonsense-mediated decay
InterPro: RNA helicase UPF1, Cys/His rich zinc-binding domain

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Macromolecule #3: Up-frameshift protein 3b

MacromoleculeName: Up-frameshift protein 3b / type: protein_or_peptide / ID: 3 / Name.synonym: UPF3b / Details: full length / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 56 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceGO: nuclear-transcribed mRNA catabolic process, nonsense-mediated decay
InterPro: UPF3 domain

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Macromolecule #4: MAGO

MacromoleculeName: MAGO / type: protein_or_peptide / ID: 4 / Name.synonym: MAGO / Details: full length / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 16 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceGO: nuclear-transcribed mRNA catabolic process, nonsense-mediated decay
InterPro: WIBG, Mago-binding

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Macromolecule #5: Y14

MacromoleculeName: Y14 / type: protein_or_peptide / ID: 5 / Name.synonym: Y14 / Details: residues 66-154 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 10 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceGO: nuclear-transcribed mRNA catabolic process, nonsense-mediated decay
InterPro: WIBG, Mago-binding

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Macromolecule #6: Btz

MacromoleculeName: Btz / type: protein_or_peptide / ID: 6 / Name.synonym: Btz / Details: residues 136-286 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 17 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceGO: nuclear-transcribed mRNA catabolic process, nonsense-mediated decay
InterPro: Btz domain

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Macromolecule #7: eIF4AIII

MacromoleculeName: eIF4AIII / type: protein_or_peptide / ID: 7 / Name.synonym: eIF4AIII / Details: full length / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 45 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceGO: nuclear-transcribed mRNA catabolic process, nonsense-mediated decay
InterPro: Btz domain

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Macromolecule #8: (U)8 RNA

MacromoleculeName: (U)8 RNA / type: rna / ID: 8 / Name.synonym: RNA / Details: Oligo(U) 8nt / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 3 KDa
SequenceString:
UUUUUUUU

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.175 mg/mL
BufferpH: 7.5
Details: 50 mM K-phosphate,150 mM NaCl, 3 mM MgCl2, 20% sucrose, 0.1% glutaraldehyde
GridDetails: Quantifoil grids (R2/2) with thin carbon film on top
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK III
Method: Manual Application (3.5 microliters) Humidifier Off During Process Blot offset: -2 mm Blot Total: 2 Blot Time: 2 s Wait Time: 30 s Drain Time: 1 s

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Electron microscopy

MicroscopeJEOL 2200FS
TemperatureMin: 83 K / Max: 100 K / Average: 91.5 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected calculating the Fourier Transform of the CCD frames. Phase flipping
Specialist opticsEnergy filter - Name: FEI / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 10.0 eV
DateOct 20, 2010
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Average electron dose: 15 e/Å2 / Details: 0.22 nm per pixel, final sampling / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 69494 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.112 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Eucentric. 626 cryo-holder (Gatan Inc., Warrendale, PA, USA)
Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: Each CCD Frame using BSOFT
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN, XMIPP, SPIDER, BSOFT
Details: Initial reference obtained by random conical tilt of negative-stained images
Number images used: 85000

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Atomic model buiding 1

Initial modelPDB ID:

1uw4

SoftwareName: Chimera
DetailsProtocol: Rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: R-factor

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Atomic model buiding 2

Initial modelPDB ID:

2xb2

SoftwareName: Chimera
DetailsProtocol: Rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: R-factor

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Atomic model buiding 3

Initial modelPDB ID:

2wjv

SoftwareName: Chimera
DetailsProtocol: Rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: R-factor

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