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- EMDB-4728: Cryo-EM structure of a poly(A) RNP bound to the Pan2-Pan3 deadenylase -

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Basic information

Entry
Database: EMDB / ID: EMD-4728
TitleCryo-EM structure of a poly(A) RNP bound to the Pan2-Pan3 deadenylase
Map data
SampleTernary Complex of Pan2-Pan3, Pab1 and poly(A) RNA
  • Pan2-Pan3
  • (Polyadenylate-binding protein, cytoplasmic and ...) x 2
  • RNA
  • PAN2-PAN3 deadenylation complex catalytic subunit PAN2
  • nucleic-acidNucleic acid
  • (PAN2-PAN3 deadenylation complex subunit ...) x 2
  • ligand
Function / homology
Function and homology information


Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / L13a-mediated translational silencing of Ceruloplasmin expression / regulation of nuclear-transcribed mRNA poly(A) tail shortening / ribonuclease inhibitor activity / PAN complex / positive regulation of cytoplasmic mRNA processing body assembly / ec:3.1.13.4: / poly(A)-specific ribonuclease activity / nuclear-transcribed mRNA poly(A) tail shortening ...Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / L13a-mediated translational silencing of Ceruloplasmin expression / regulation of nuclear-transcribed mRNA poly(A) tail shortening / ribonuclease inhibitor activity / PAN complex / positive regulation of cytoplasmic mRNA processing body assembly / ec:3.1.13.4: / poly(A)-specific ribonuclease activity / nuclear-transcribed mRNA poly(A) tail shortening / deadenylation-dependent decapping of nuclear-transcribed mRNA / regulation of translational initiation / RNA phosphodiester bond hydrolysis, exonucleolytic / poly(A) binding / postreplication repair / poly(U) RNA binding / mRNA transport / protein kinase A catalytic subunit binding / promoter-specific chromatin binding / mRNA 3'-UTR binding / P-body / mRNA processing / cytoplasmic stress granule / ribonucleoprotein complex / ribosome / nucleic acid binding / mRNA binding / protein kinase activity / DNA repair / RNA binding / ATP binding / metal ion binding / nucleus / cytosol / cytoplasm
Protein kinase domain / PABC (PABP) domain / RNA recognition motif domain, eukaryote / Polyadenylate-binding protein/Hyperplastic disc protein / Quinoprotein alcohol dehydrogenase-like superfamily / Zinc finger, CCCH-type / RNA recognition motif domain / Ribonuclease H-like superfamily / Nucleotide-binding alpha-beta plait domain superfamily / Exonuclease, RNase T/DNA polymerase III ...Protein kinase domain / PABC (PABP) domain / RNA recognition motif domain, eukaryote / Polyadenylate-binding protein/Hyperplastic disc protein / Quinoprotein alcohol dehydrogenase-like superfamily / Zinc finger, CCCH-type / RNA recognition motif domain / Ribonuclease H-like superfamily / Nucleotide-binding alpha-beta plait domain superfamily / Exonuclease, RNase T/DNA polymerase III / WD40/YVTN repeat-like-containing domain superfamily / PAN2 domain / Ubiquitin specific protease domain / PAN2-PAN3 deadenylation complex catalytic subunit PAN2 / PAN2-PAN3 deadenylation complex subunit PAN3 / Protein kinase-like domain superfamily / RNA-binding domain superfamily / Ribonuclease H superfamily / Protein kinase domain profile. / Poly(A)-binding protein C-terminal (PABC) domain profile. / Ubiquitin specific protease (USP) domain profile. / Zinc finger C3H1-type profile. / Eukaryotic RNA Recognition Motif (RRM) profile. / Pan3 Pseudokinase domain / Papain-like cysteine peptidase superfamily / Ubiquitin carboxyl-terminal hydrolase / Exonuclease / Poly-adenylate binding protein, unique domain / RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) / Pan3 pseudokinase domain / Polyadenylate binding protein, human types 1, 2, 3, 4
Polyadenylate-binding protein, cytoplasmic and nuclear / PAN2-PAN3 deadenylation complex subunit PAN3 / PAN2-PAN3 deadenylation complex catalytic subunit PAN2
Biological speciesSaccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) / Saccharomyces cerevisiae S288C (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsSchaefer IB / Conti E
CitationJournal: Cell / Year: 2019
Title: Molecular Basis for poly(A) RNP Architecture and Recognition by the Pan2-Pan3 Deadenylase.
Authors: Ingmar B Schäfer / Masami Yamashita / Jan Michael Schuller / Steffen Schüssler / Peter Reichelt / Mike Strauss / Elena Conti /
Abstract: The stability of eukaryotic mRNAs is dependent on a ribonucleoprotein (RNP) complex of poly(A)-binding proteins (PABPC1/Pab1) organized on the poly(A) tail. This poly(A) RNP not only protects mRNAs ...The stability of eukaryotic mRNAs is dependent on a ribonucleoprotein (RNP) complex of poly(A)-binding proteins (PABPC1/Pab1) organized on the poly(A) tail. This poly(A) RNP not only protects mRNAs from premature degradation but also stimulates the Pan2-Pan3 deadenylase complex to catalyze the first step of poly(A) tail shortening. We reconstituted this process in vitro using recombinant proteins and show that Pan2-Pan3 associates with and degrades poly(A) RNPs containing two or more Pab1 molecules. The cryo-EM structure of Pan2-Pan3 in complex with a poly(A) RNP composed of 90 adenosines and three Pab1 protomers shows how the oligomerization interfaces of Pab1 are recognized by conserved features of the deadenylase and thread the poly(A) RNA substrate into the nuclease active site. The structure reveals the basis for the periodic repeating architecture at the 3' end of cytoplasmic mRNAs. This illustrates mechanistically how RNA-bound Pab1 oligomers act as rulers for poly(A) tail length over the mRNAs' lifetime.
Validation ReportPDB-ID: 6r5k

SummaryFull reportAbout validation report
DateDeposition: Mar 25, 2019 / Header (metadata) release: May 29, 2019 / Map release: May 29, 2019 / Update: Jun 12, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6r5k
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_4728.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 288 pix.
= 304.128 Å
1.06 Å/pix.
x 288 pix.
= 304.128 Å
1.06 Å/pix.
x 288 pix.
= 304.128 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.056 Å
Density
Contour LevelBy AUTHOR: 0.014 / Movie #1: 0.014
Minimum - Maximum-0.012846572 - 0.051474016
Average (Standard dev.)0.00014905825 (±0.0026519466)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 304.128 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0561.0561.056
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z304.128304.128304.128
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.0130.0510.000

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Supplemental data

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Mask #1

Fileemd_4728_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Ternary Complex of Pan2-Pan3, Pab1 and poly(A) RNA

EntireName: Ternary Complex of Pan2-Pan3, Pab1 and poly(A) RNA / Number of components: 10

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Component #1: protein, Ternary Complex of Pan2-Pan3, Pab1 and poly(A) RNA

ProteinName: Ternary Complex of Pan2-Pan3, Pab1 and poly(A) RNA / Recombinant expression: No
MassTheoretical: 498 kDa

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Component #2: protein, Pan2-Pan3

ProteinName: Pan2-Pan3 / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #3: protein, Polyadenylate-binding protein, cytoplasmic and nuclear

ProteinName: Polyadenylate-binding protein, cytoplasmic and nuclear
Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #4: protein, RNA

ProteinName: RNA / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae S288C (yeast)
Source (engineered)Expression System: synthetic construct (others)

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Component #5: protein, PAN2-PAN3 deadenylation complex catalytic subunit PAN2

ProteinName: PAN2-PAN3 deadenylation complex catalytic subunit PAN2
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 127.186602 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #6: protein, Polyadenylate-binding protein, cytoplasmic and nuclear

ProteinName: Polyadenylate-binding protein, cytoplasmic and nuclear
Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 64.778363 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #7: nucleic-acid, poly(A) RNA

nucleic acidName: poly(A) RNA / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA
MassTheoretical: 29.583547 kDa
SourceSpecies: Saccharomyces cerevisiae S288C (yeast)

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Component #8: protein, PAN2-PAN3 deadenylation complex subunit PAN3

ProteinName: PAN2-PAN3 deadenylation complex subunit PAN3 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 53.218609 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #9: protein, PAN2-PAN3 deadenylation complex subunit PAN3

ProteinName: PAN2-PAN3 deadenylation complex subunit PAN3 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 77.906688 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #10: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.5
VitrificationCryogen name: OTHER

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 51 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 6463 / Sampling size: 5 µm

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 29165
3D reconstructionSoftware: RELION / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Target criteria: Correlation coefficient / Refinement space: REAL / Overall bvalue: 45.7
Output model

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