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- PDB-6r5k: Cryo-EM structure of a poly(A) RNP bound to the Pan2-Pan3 deadenylase -

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Basic information

Entry
Database: PDB / ID: 6r5k
TitleCryo-EM structure of a poly(A) RNP bound to the Pan2-Pan3 deadenylase
Components
  • (PAN2-PAN3 deadenylation complex subunit ...) x 2
  • PAN2-PAN3 deadenylation complex catalytic subunit PAN2
  • Polyadenylate-binding protein, cytoplasmic and nuclear
  • poly(A) RNA
KeywordsRNA BINDING PROTEIN / poly(A)-tail / mRNA / RNP / PABP / Pab1 / Pan2-Pan3 / Ccr4-Not / deadenylase / RRM / cryoEM
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 (baker's yeast)
Saccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsSchaefer, I.B. / 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 Report
SummaryFull reportAbout validation report
DateDeposition: Mar 25, 2019 / Release: May 29, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0May 29, 2019Structure modelrepositoryInitial release
1.1Jun 12, 2019Structure modelData collection / Database referencescitation / em_admin / pdbx_database_proc / pdbx_seq_map_depositor_info_citation.journal_volume / _citation.page_first / _em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code / _pdbx_seq_map_depositor_info.one_letter_code_mod

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Structure visualization

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Assembly

Deposited unit
A: PAN2-PAN3 deadenylation complex catalytic subunit PAN2
D: Polyadenylate-binding protein, cytoplasmic and nuclear
E: poly(A) RNA
F: Polyadenylate-binding protein, cytoplasmic and nuclear
H: Polyadenylate-binding protein, cytoplasmic and nuclear
N: PAN2-PAN3 deadenylation complex subunit PAN3
O: PAN2-PAN3 deadenylation complex subunit PAN3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)482,2799
Polymers482,2317
Non-polymers492
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, cross-linking, assay for oligomerization, immunoprecipitation
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TypeNameSymmetry operationNumber
identity operation1_5551
Buried area44360 Å2
ΔGint-134 kcal/mol
Surface area139370 Å2
MethodPISA

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Components

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Protein/peptide , 2 types, 4 molecules ADFH

#1: Protein/peptide PAN2-PAN3 deadenylation complex catalytic subunit PAN2 / PAB1P-dependent poly(A)-specific ribonuclease / Poly(A)-nuclease deadenylation complex subunit 2 / PAN deadenylation complex subunit 2


Mass: 127186.602 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: PAN2, YGL094C / Cell line (production host): Hi5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P53010, EC: 3.1.13.4
#2: Protein/peptide Polyadenylate-binding protein, cytoplasmic and nuclear / Poly(A)-binding protein / ARS consensus-binding protein ACBP-67 / Polyadenylate tail-binding protein


Mass: 64778.363 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: PAB1, YER165W / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P04147

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PAN2-PAN3 deadenylation complex subunit ... , 2 types, 2 molecules NO

#4: Protein/peptide PAN2-PAN3 deadenylation complex subunit PAN3 / PAB1P-dependent poly(A)-specific ribonuclease / Poly(A)-nuclease deadenylation complex subunit 3 / PAN deadenylation complex subunit 3


Mass: 53218.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: PAN3, ECM35, YKL025C / Cell line (production host): Hi5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P36102
#5: Protein/peptide PAN2-PAN3 deadenylation complex subunit PAN3 / PAB1P-dependent poly(A)-specific ribonuclease / Poly(A)-nuclease deadenylation complex subunit 3 / PAN deadenylation complex subunit 3


Mass: 77906.688 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: PAN3, ECM35, YKL025C / Cell line (production host): Hi5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P36102

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RNA chain / Non-polymers , 2 types, 3 molecules E

#3: RNA chain poly(A) RNA


Mass: 29583.547 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288C (yeast)
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Magnesium

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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

Component

Type: COMPLEX

IDNameEntity IDParent-IDSource
1Ternary Complex of Pan2-Pan3, Pab1 and poly(A) RNA1, 2, 3, 4, 50MULTIPLE SOURCES
2Pan2-Pan31,4,51RECOMBINANT
3Polyadenylate-binding protein, cytoplasmic and nuclear21RECOMBINANT
4RNA31RECOMBINANT
Molecular weightValue: 0.498 MDa / Experimental value: NO
Source (natural)

Ncbi tax-ID: 559292

IDEntity assembly-IDOrganism
12Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
23Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
34Saccharomyces cerevisiae S288C (yeast)
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Trichoplusia ni (cabbage looper)7111
23Escherichia coli BL21(DE3) (bacteria)469008
34synthetic construct (others)32630
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 8 sec. / Electron dose: 51 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6463
Image scansSampling size: 5 µns / Movie frames/image: 40 / Used frames/image: 2-40

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Processing

Software
NameVersionClassification
phenix.real_space_refinedev_3026refinement
PHENIXdev_3026refinement
EM software
IDNameVersionCategory
2SerialEM3.5image acquisition
4Gctf1.06CTF correction
7UCSF Chimera1.13.1model fitting
9RELION3.0-beta-1initial Euler assignment
10RELION3.0-beta-1final Euler assignment
11RELION3.0-beta-1classification
12RELION3.0-beta-13D reconstruction
13Coot0.8model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 2141230
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29165 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 45.7 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient
RefinementHighest resolution: 4.8 Å / Stereochemistry target values: GeoStd + Monomer Library
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.005224156
f_angle_d1.109433112
f_chiral_restr0.06053767
f_plane_restr0.00714042
f_dihedral_angle_d11.088114290

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