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- PDB-6fuw: Cryo-EM structure of the human CPSF160-WDR33-CPSF30 complex bound... -

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Basic information

Entry
Database: PDB / ID: 6fuw
TitleCryo-EM structure of the human CPSF160-WDR33-CPSF30 complex bound to the PAS AAUAAA motif at 3.1 Angstrom resolution
Components
  • Cleavage and polyadenylation specificity factor subunit 1
  • Cleavage and polyadenylation specificity factor subunit 4
  • RNA (5'-R(P*AP*AP*UP*AP*AP*AP*GP*G)-3')
  • pre-mRNA 3' end processing protein WDR33
KeywordsRNA BINDING PROTEIN / 3' pre-mRNA processing / polyadenylation / CPSF / beta propeller / AAUAAA
Function / homology
Function and homology information


co-transcriptional RNA 3'-end processing, cleavage and polyadenylation pathway / Inhibition of Host mRNA Processing and RNA Silencing / Processing of Intronless Pre-mRNAs / mRNA cleavage and polyadenylation specificity factor complex / collagen trimer / mRNA 3'-end processing / mRNA 3'-UTR AU-rich region binding / Transport of Mature mRNA Derived from an Intronless Transcript / mRNA 3'-end processing / DNA damage tolerance ...co-transcriptional RNA 3'-end processing, cleavage and polyadenylation pathway / Inhibition of Host mRNA Processing and RNA Silencing / Processing of Intronless Pre-mRNAs / mRNA cleavage and polyadenylation specificity factor complex / collagen trimer / mRNA 3'-end processing / mRNA 3'-UTR AU-rich region binding / Transport of Mature mRNA Derived from an Intronless Transcript / mRNA 3'-end processing / DNA damage tolerance / tRNA processing in the nucleus / RNA Polymerase II Transcription Termination / Processing of Capped Intron-Containing Pre-mRNA / fibrillar center / mRNA processing / sequence-specific double-stranded DNA binding / spermatogenesis / intracellular membrane-bounded organelle / enzyme binding / RNA binding / zinc ion binding / nucleoplasm / nucleus
Similarity search - Function
Zinc-finger CCCH domain / Zinc-finger containing family / CPSF complex subunit CPSF4-like / Pre-mRNA 3' end processing protein Pfs2-like / Zinc finger C-x8-C-x5-C-x3-H type (and similar) / Zinc finger, CCCH-type superfamily / zinc finger / Collagen triple helix repeat / Collagen triple helix repeat (20 copies) / Zinc finger, CCCH-type ...Zinc-finger CCCH domain / Zinc-finger containing family / CPSF complex subunit CPSF4-like / Pre-mRNA 3' end processing protein Pfs2-like / Zinc finger C-x8-C-x5-C-x3-H type (and similar) / Zinc finger, CCCH-type superfamily / zinc finger / Collagen triple helix repeat / Collagen triple helix repeat (20 copies) / Zinc finger, CCCH-type / Zinc finger C3H1-type profile. / RSE1/DDB1/CPSF1 second beta-propeller / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / : / CPSF A subunit region / RSE1/DDB1/CPSF1 first beta-propeller / YVTN repeat-like/Quinoprotein amine dehydrogenase / 7 Propeller / Methylamine Dehydrogenase; Chain H / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / WD domain, G-beta repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Mainly Beta
Similarity search - Domain/homology
RNA / Cleavage and polyadenylation specificity factor subunit 4 / Cleavage and polyadenylation specificity factor subunit 1 / pre-mRNA 3' end processing protein WDR33
Similarity search - Component
Biological speciesHomo sapiens (human)
unidentified adenovirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.07 Å
AuthorsClerici, M. / Faini, M. / Jinek, M.
Funding support Belgium, Germany, 4items
OrganizationGrant numberCountry
European Research CouncilERC-StG-337284 Belgium
European Molecular Biology OrganizationEMBO ALTF-343-2013 Germany
European Research CouncilERC Advanced Grants no. 233226 and no. 670821 Belgium
European UnionPROSPECTS, HEALTH-F4-2008-201648 Belgium
CitationJournal: Nat Struct Mol Biol / Year: 2018
Title: Structural basis of AAUAAA polyadenylation signal recognition by the human CPSF complex.
Authors: Marcello Clerici / Marco Faini / Lena M Muckenfuss / Ruedi Aebersold / Martin Jinek /
Abstract: Mammalian mRNA biogenesis requires specific recognition of a hexanucleotide AAUAAA motif in the polyadenylation signals (PAS) of precursor mRNA (pre-mRNA) transcripts by the cleavage and ...Mammalian mRNA biogenesis requires specific recognition of a hexanucleotide AAUAAA motif in the polyadenylation signals (PAS) of precursor mRNA (pre-mRNA) transcripts by the cleavage and polyadenylation specificity factor (CPSF) complex. Here we present a 3.1-Å-resolution cryo-EM structure of a core CPSF module bound to the PAS hexamer motif. The structure reveals the molecular interactions responsible for base-specific recognition, providing a rationale for mechanistic differences between mammalian and yeast 3' polyadenylation.
History
DepositionFeb 28, 2018Deposition site: PDBE / Processing site: PDBE
SupersessionMar 21, 2018ID: 6FBS
Revision 1.0Mar 21, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 11, 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
Revision 1.2Feb 22, 2023Group: Database references / Refinement description / Category: database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.3Jul 10, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Cleavage and polyadenylation specificity factor subunit 1
B: pre-mRNA 3' end processing protein WDR33
C: Cleavage and polyadenylation specificity factor subunit 4
D: RNA (5'-R(P*AP*AP*UP*AP*AP*AP*GP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)232,6477
Polymers232,4504
Non-polymers1963
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14140 Å2
ΔGint-59 kcal/mol
Surface area66850 Å2
MethodPISA

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Components

#1: Protein Cleavage and polyadenylation specificity factor subunit 1 / Cleavage and polyadenylation specificity factor 160 kDa subunit / CPSF 160 kDa subunit


Mass: 161346.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CPSF1, CPSF160
Production host: Spodoptera aff. frugiperda 2 RZ-2014 (butterflies/moths)
References: UniProt: Q10570
#2: Protein pre-mRNA 3' end processing protein WDR33 / WD repeat-containing protein 33 / WD repeat-containing protein WDC146


Mass: 47448.910 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: WDR33, WDC146
Production host: Spodoptera aff. frugiperda 2 RZ-2014 (butterflies/moths)
References: UniProt: Q9C0J8
#3: Protein Cleavage and polyadenylation specificity factor subunit 4 / Cleavage and polyadenylation specificity factor 30 kDa subunit / CPSF 30 kDa subunit / NS1 effector ...Cleavage and polyadenylation specificity factor 30 kDa subunit / CPSF 30 kDa subunit / NS1 effector domain-binding protein 1 / Neb-1 / No arches homolog


Mass: 20422.855 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CPSF4, CPSF30, NAR, NEB1
Production host: Spodoptera aff. frugiperda 2 RZ-2014 (butterflies/moths)
References: UniProt: O95639
#4: RNA chain RNA (5'-R(P*AP*AP*UP*AP*AP*AP*GP*G)-3')


Mass: 3232.036 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) unidentified adenovirus
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn

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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
1Human CPSF160-WDR33-CPSF30 heterotrimer bound to the AAUAAA motif of the Polyadenylation SignalCOMPLEX#1-#40MULTIPLE SOURCES
2Human CPSF160-WDR33-CPSF30 heterotrimerCOMPLEX#1-#31RECOMBINANT
3AAUAAA motif of the Polyadenylation SignalCOMPLEX#41RECOMBINANT
Molecular weightValue: 0.22 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33unidentified adenovirus10535
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Spodoptera aff. frugiperda 2 RZ-2014 (butterflies/moths)1491790
33synthetic construct (others)32630
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1140 mMPotassium ChlorideKCl1
220 mMHEPESHEPES1
31 mMDithiothreitolDTT1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 293 K / Details: 15 seconds wait time prior to blotting

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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 FIELD / Calibrated magnification: 47259 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1100 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 80 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1070
EM imaging opticsEnergyfilter name: GIF Quantum LS
Image scansMovie frames/image: 50

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2.1b1_cu8.0particle selection
2SerialEMimage acquisition
4RELION2.1b1_cu8.0CTF correction
7Coot0.8.8model fitting
9PHENIX1.12-2829model refinement
10RELION2.1b1_cu8.0initial Euler assignment
11RELION2.1b1_cu8.0final Euler assignment
12RELION2.1b1_cu8.0classification
13RELION2.1b1_cu8.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 263000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 137000 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Details: phenix.real_space_refine
Atomic model buildingPDB-ID: 6F9N

6f9n


Accession code: 6F9N / Details: fitted manually in Coot / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.07 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0113762
ELECTRON MICROSCOPYf_angle_d0.96818688
ELECTRON MICROSCOPYf_dihedral_angle_d11.8268173
ELECTRON MICROSCOPYf_chiral_restr0.0582081
ELECTRON MICROSCOPYf_plane_restr0.0062352

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