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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 / homologyCleavage/polyadenylation specificity factor, A subunit, C-terminal / Trp-Asp (WD) repeats profile. / WD domain, G-beta repeat / Zinc finger, CCCH-type superfamily / WD40-repeat-containing domain superfamily / WD40-repeat-containing domain / WD40/YVTN repeat-like-containing domain superfamily / WD40 repeat / Zinc finger, CCCH-type / CPSF A subunit region ...Cleavage/polyadenylation specificity factor, A subunit, C-terminal / Trp-Asp (WD) repeats profile. / WD domain, G-beta repeat / Zinc finger, CCCH-type superfamily / WD40-repeat-containing domain superfamily / WD40-repeat-containing domain / WD40/YVTN repeat-like-containing domain superfamily / WD40 repeat / Zinc finger, CCCH-type / CPSF A subunit region / Trp-Asp (WD) repeats circular profile. / Zinc finger C3H1-type profile. / Processing of Intronless Pre-mRNAs / mRNA cleavage factor complex / mRNA cleavage and polyadenylation specificity factor complex / pre-mRNA cleavage required for polyadenylation / termination of RNA polymerase II transcription / endoribonuclease activity / mRNA polyadenylation / mRNA binding / RNA binding / nucleus / metal ion binding / cytosol / Polyadenylation factor subunit 2 / mRNA 3'-end-processing protein YTH1 / Protein CFT1
Function and homology information
Specimen sourceSaccharomyces cerevisiae (baker's yeast)
Saccharomyces cerevisiae S288c (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.55 Å resolution
AuthorsCasanal, A. / Kumar, A. / Hill, C.H. / Emsley, P. / Passmore, L.
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 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 9, 2017 / Release: Nov 15, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Nov 15, 2017Structure modelrepositoryInitial release
1.1Dec 6, 2017Structure modelDatabase referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last
1.2Jan 31, 2018Structure modelAuthor supporting evidence / Experimental preparationem_sample_support / pdbx_audit_support_em_sample_support.grid_type / _pdbx_audit_support.funding_organization

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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
Polyers231,7743
Non-polymers1312
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)13370
ΔGint (kcal/M)-63
Surface area (Å2)56480
MethodPISA

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Components

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


Mass: 153577.156 Da / Num. of mol.: 1
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/peptide mRNA 3'-end-processing protein YTH1 / Yeast 30 kDa homolog 1


Mass: 24560.416 Da / Num. of mol.: 1
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/peptide Polyadenylation factor subunit 2,Polyadenylation factor subunit 2


Mass: 53636.645 Da / Num. of mol.: 1
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 / Formula: Zn / Zinc

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Complex of Cft1, Yth1, Pfs2 and Fip1 / Type: COMPLEX / Entity ID: 1, 2, 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 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 / Cs: 2.7 mm / C2 aperture diameter: 70 mm / 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) / Number of grids imaged: 5 / Number of real images: 4227
EM imaging opticsEnergyfilter name: GIF Quantum LS
Image scansMovie frames/image: 20

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Processing

EM software
IDNameVersionCategoryDetails
1RELION2.0particle selection
2RELION2.0image acquisition
4RELION2.0CTF correctionGCTF
7Cootmodel fitting
9RELION2.0initial Euler assignment
10RELION2.0final Euler assignment
11RELION2.0classification
12RELION2.03D reconstruction
13REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 460167
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.55 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 77197 / Algorithm: FOURIER SPACE / Number of class averages: 1 / Symmetry type: POINT
Atomic model buildingRef protocol: AB INITIO MODEL / Ref space: REAL

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