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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


regulation of nuclear-transcribed mRNA poly(A) tail shortening / ribonuclease inhibitor activity / PAN complex / poly(A)-specific ribonuclease / poly(A)-specific ribonuclease activity / nuclear-transcribed mRNA poly(A) tail shortening / poly(A) binding / postreplication repair / poly(U) RNA binding / regulation of translational initiation ...regulation of nuclear-transcribed mRNA poly(A) tail shortening / ribonuclease inhibitor activity / PAN complex / poly(A)-specific ribonuclease / poly(A)-specific ribonuclease activity / nuclear-transcribed mRNA poly(A) tail shortening / poly(A) binding / postreplication repair / poly(U) RNA binding / regulation of translational initiation / Translation initiation complex formation / intracellular non-membrane-bounded organelle / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / L13a-mediated translational silencing of Ceruloplasmin expression / mRNA transport / molecular condensate scaffold activity / mRNA 3'-UTR binding / promoter-specific chromatin binding / P-body / mRNA processing / cytoplasmic stress granule / nucleic acid binding / ribosome / protein kinase activity / ribonucleoprotein complex / DNA repair / mRNA binding / RNA binding / ATP binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
PAN2-PAN3 deadenylation complex subunit PAN3 / Pan3 pseudokinase domain / Pan3 Pseudokinase domain / PAN2, UCH domain / PAN2-PAN3 deadenylation complex catalytic subunit PAN2 / : / Ubiquitin carboxyl-terminal hydrolase / PAN2 N-terminal / : / PABP, RNA recognition motif 2 ...PAN2-PAN3 deadenylation complex subunit PAN3 / Pan3 pseudokinase domain / Pan3 Pseudokinase domain / PAN2, UCH domain / PAN2-PAN3 deadenylation complex catalytic subunit PAN2 / : / Ubiquitin carboxyl-terminal hydrolase / PAN2 N-terminal / : / PABP, RNA recognition motif 2 / Polyadenylate binding protein, human types 1, 2, 3, 4 / Polyadenylate-binding protein/Hyperplastic disc protein / Poly-adenylate binding protein, unique domain / Poly(A)-binding protein C-terminal (PABC) domain profile. / C-terminal domain of Poly(A)-binding protein. Present also in Drosophila hyperplastics discs protein. / PABC (PABP) domain / Exonuclease / RNA recognition motif domain, eukaryote / RNA recognition motif / Exonuclease, RNase T/DNA polymerase III / EXOIII / Zinc finger, CCCH-type / Zinc finger C3H1-type profile. / Ubiquitin specific protease domain / Ubiquitin specific protease (USP) domain profile. / Quinoprotein alcohol dehydrogenase-like superfamily / Papain-like cysteine peptidase superfamily / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / RNA-binding domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Nucleotide-binding alpha-beta plait domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / Polyadenylate-binding protein, cytoplasmic and nuclear / PAN2-PAN3 deadenylation complex subunit PAN3 / PAN2-PAN3 deadenylation complex catalytic subunit PAN2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer'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.
History
DepositionMar 25, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 29, 2019Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2019Group: Data collection / Database references
Category: citation / em_admin ...citation / em_admin / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _citation.journal_volume / _citation.page_first ..._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
Revision 1.2Nov 6, 2019Group: Data collection / Refinement description / Category: em_3d_fitting / software / Item: _em_3d_fitting.target_criteria / _software.name
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Structure visualization

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  • Deposited structure unit
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  • EMDB-4728
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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
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 , 2 types, 4 molecules ADFH

#1: Protein PAN2-PAN3 deadenylation complex catalytic subunit PAN2 / PAB1P-dependent poly(A)-specific ribonuclease / Poly(A)-nuclease deadenylation complex subunit 2 / ...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, poly(A)-specific ribonuclease
#2: Protein 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 PAN2-PAN3 deadenylation complex subunit PAN3 / PAB1P-dependent poly(A)-specific ribonuclease / Poly(A)-nuclease deadenylation complex subunit 3 / ...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 PAN2-PAN3 deadenylation complex subunit PAN3 / PAB1P-dependent poly(A)-specific ribonuclease / Poly(A)-nuclease deadenylation complex subunit 3 / ...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

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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
IDNameTypeEntity IDParent-IDSource
1Ternary Complex of Pan2-Pan3, Pab1 and poly(A) RNACOMPLEX#1-#50MULTIPLE SOURCES
2Pan2-Pan3COMPLEX#1, #4-#51RECOMBINANT
3Polyadenylate-binding protein, cytoplasmic and nuclearCOMPLEX#21RECOMBINANT
4RNACOMPLEX#31RECOMBINANT
Molecular weightValue: 0.498 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)559292
23Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)559292
34Saccharomyces cerevisiae S288C (yeast)559292
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 µm / Movie frames/image: 40 / Used frames/image: 2-40

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Processing

Software
NameVersionClassification
PHENIXdev_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: Cross-correlation coefficient
RefinementHighest resolution: 4.8 Å / Stereochemistry target values: GeoStd + Monomer Library
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005224156
ELECTRON MICROSCOPYf_angle_d1.109433112
ELECTRON MICROSCOPYf_chiral_restr0.06053767
ELECTRON MICROSCOPYf_plane_restr0.00714042
ELECTRON MICROSCOPYf_dihedral_angle_d11.088114290

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