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- PDB-6eoj: PolyA polymerase module of the cleavage and polyadenylation facto... -

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Basic information

Entry
Database: PDB / ID: 6eoj
TitlePolyA polymerase module of the cleavage and polyadenylation factor (CPF) from Saccharomyces cerevisiae
Components
  • Polyadenylation factor subunit 2,Polyadenylation factor subunit 2
  • Protein CFT1
  • mRNA 3'-end-processing protein YTH1
KeywordsRNA BINDING PROTEIN / WD40 / Beta-propeller / Zinc finger / 3'end processing
Function / homology
Function and homology information


Processing of Intronless Pre-mRNAs / termination of RNA polymerase II transcription, poly(A)-coupled / mRNA cleavage and polyadenylation specificity factor complex / mRNA 3'-end processing / termination of RNA polymerase II transcription / mRNA processing / mitochondrion / RNA binding / nucleus / metal ion binding / cytosol
Similarity search - Function
CPSF complex subunit CPSF4-like / RNA-binding, Nab2-type zinc finger / 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 / Zinc finger, CCCH-type / Zinc finger C3H1-type profile. / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / : ...CPSF complex subunit CPSF4-like / RNA-binding, Nab2-type zinc finger / 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 / Zinc finger, CCCH-type / Zinc finger C3H1-type profile. / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / : / CPSF A subunit region / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Polyadenylation factor subunit 2 / mRNA 3'-end-processing protein YTH1 / Protein CFT1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Saccharomyces cerevisiae S288c (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.55 Å
AuthorsCasanal, A. / Kumar, A. / Hill, C.H. / Emsley, P. / Passmore, L.A.
Funding support United Kingdom, 5items
OrganizationGrant numberCountry
H2020 European Research Council725685 United Kingdom
Medical Research Council (United Kingdom)MC_U105192715 United Kingdom
European Research Council261151 United Kingdom
European Molecular Biology OrganizationALTF66-2015 United Kingdom
Bill & Melinda Gates Foundation United Kingdom
CitationJournal: Science / Year: 2017
Title: Architecture of eukaryotic mRNA 3'-end processing machinery.
Authors: Ana Casañal / Ananthanarayanan Kumar / Chris H Hill / Ashley D Easter / Paul Emsley / Gianluca Degliesposti / Yuliya Gordiyenko / Balaji Santhanam / Jana Wolf / Katrin Wiederhold / Gillian ...Authors: Ana Casañal / Ananthanarayanan Kumar / Chris H Hill / Ashley D Easter / Paul Emsley / Gianluca Degliesposti / Yuliya Gordiyenko / Balaji Santhanam / Jana Wolf / Katrin Wiederhold / Gillian L Dornan / Mark Skehel / Carol V Robinson / Lori A Passmore /
Abstract: Newly transcribed eukaryotic precursor messenger RNAs (pre-mRNAs) are processed at their 3' ends by the ~1-megadalton multiprotein cleavage and polyadenylation factor (CPF). CPF cleaves pre-mRNAs, ...Newly transcribed eukaryotic precursor messenger RNAs (pre-mRNAs) are processed at their 3' ends by the ~1-megadalton multiprotein cleavage and polyadenylation factor (CPF). CPF cleaves pre-mRNAs, adds a polyadenylate tail, and triggers transcription termination, but it is unclear how its various enzymes are coordinated and assembled. Here, we show that the nuclease, polymerase, and phosphatase activities of yeast CPF are organized into three modules. Using electron cryomicroscopy, we determined a 3.5-angstrom-resolution structure of the ~200-kilodalton polymerase module. This revealed four β propellers, in an assembly markedly similar to those of other protein complexes that bind nucleic acid. Combined with in vitro reconstitution experiments, our data show that the polymerase module brings together factors required for specific and efficient polyadenylation, to help coordinate mRNA 3'-end processing.
History
DepositionOct 9, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 15, 2017Provider: repository / Type: Initial release
Revision 1.1Dec 6, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jan 31, 2018Group: Author supporting evidence / Experimental preparation
Category: em_sample_support / pdbx_audit_support
Item: _em_sample_support.grid_type / _pdbx_audit_support.funding_organization
Revision 1.3May 15, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: audit_author / chem_comp_atom ...audit_author / chem_comp_atom / chem_comp_bond / database_2 / struct_ref_seq / struct_sheet
Item: _audit_author.name / _database_2.pdbx_DOI ..._audit_author.name / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq.db_align_beg / _struct_ref_seq.db_align_end / _struct_sheet.number_strands

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Structure visualization

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Assembly

Deposited unit
A: Protein CFT1
B: mRNA 3'-end-processing protein YTH1
D: Polyadenylation factor subunit 2,Polyadenylation factor subunit 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)231,9055
Polymers231,7743
Non-polymers1312
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13370 Å2
ΔGint-63 kcal/mol
Surface area56480 Å2
MethodPISA

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Components

#1: Protein Protein CFT1 / Cleavage factor two protein 1


Mass: 153577.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: CFT1, YHH1, YDR301W / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q06632
#2: Protein mRNA 3'-end-processing protein YTH1 / Yeast 30 kDa homolog 1


Mass: 24560.416 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: YTH1, YPR107C / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q06102
#3: Protein Polyadenylation factor subunit 2,Polyadenylation factor subunit 2


Mass: 53636.645 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast), (gene. exp.) Saccharomyces cerevisiae S288c (yeast)
Strain: ATCC 204508 / S288c / Gene: PFS2, YNL317W, N0348 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P42841
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / 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

ComponentName: Complex of Cft1, Yth1, Pfs2 and Fip1 / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.27 MDa / Experimental value: NO
Source (natural)Organism: SaccharSaccharomyces cerevisiae (strain ATCC 204508 / S288c)omyces (yeast)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.9
Buffer component
IDConc.NameBuffer-ID
110 mMHepes1
2150 mMSodium Chloride1
31 mMTCEP1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 16 sec. / Electron dose: 45 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 5 / Num. of real images: 4227
EM imaging opticsEnergyfilter name: GIF Quantum LS
Image scansMovie frames/image: 20

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Processing

EM software
IDNameVersionCategoryDetails
1RELION2particle selection
2RELION2image acquisition
4RELION2CTF correctionGCTF
7Cootmodel fitting
9RELION2initial Euler assignment
10RELION2final Euler assignment
11RELION2classification
12RELION23D reconstruction
13REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 460167
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.55 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 77197 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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