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- EMDB-3908: PolyA polymerase module of the cleavage and polyadenylation facto... -

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Basic information

Entry
Database: EMDB / ID: 3908
TitlePolyA polymerase module of the cleavage and polyadenylation factor (CPF) from Saccharomyces cerevisiae
Map data
SampleComplex of Cft1, Yth1, Pfs2 and Fip1
  • Protein CFT1
  • mRNA 3'-end-processing protein YTH1
  • Polyadenylation factor subunit 2,Polyadenylation factor subunit 2
  • ligand
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
SourceSaccharSaccharomyces cerevisiae (strain ATCC 204508 / S288c)omyces (yeast)
Methodsingle particle reconstruction / cryo EM / 3.55 Å resolution
AuthorsCasanal A / Kumar A
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 ReportPDB-ID: 6eoj

SummaryFull reportAbout validation report
DateDeposition: Oct 9, 2017 / Header (metadata) release: Nov 15, 2017 / Map release: Nov 15, 2017 / Last update: Jan 31, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.15
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.15
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6eoj
  • Surface level: 0.15
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_3908.map.gz (map file in CCP4 format, 16385 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
160 pix
1.4 Å/pix.
= 224. Å
160 pix
1.4 Å/pix.
= 224. Å
160 pix
1.4 Å/pix.
= 224. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.4 Å
Density
Contour Level:0.15 (by author), 0.15 (movie #1):
Minimum - Maximum-0.55501544 - 0.8373203
Average (Standard dev.)0.00017478284 (0.031612962)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions160160160
Origin000
Limit159159159
Spacing160160160
CellA=B=C: 224 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.41.41.4
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z224.000224.000224.000
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.5550.8370.000

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Supplemental data

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Mask #1

Fileemd_3908_msk_1.map ( map file in CCP4 format, 16385 KB )
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms
Data typeImage stored as Reals
Space group number1

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Mask #1~

Fileemd_3908_msk_1.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms

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Sample components

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Entire Complex of Cft1, Yth1, Pfs2 and Fip1

EntireName: Complex of Cft1, Yth1, Pfs2 and Fip1 / Number of components: 5
MassTheoretical: 270 kDa

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Component #1: protein, Complex of Cft1, Yth1, Pfs2 and Fip1

ProteinName: Complex of Cft1, Yth1, Pfs2 and Fip1 / Recombinant expression: No
MassTheoretical: 270 kDa
SourceSpecies: SaccharSaccharomyces cerevisiae (strain ATCC 204508 / S288c)omyces (yeast)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #2: protein, Protein CFT1

ProteinName: Protein CFT1 / Recombinant expression: No
MassTheoretical: 153.577156 kDa
Source (engineered)Expression System: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c

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Component #3: protein, mRNA 3'-end-processing protein YTH1

ProteinName: mRNA 3'-end-processing protein YTH1 / Recombinant expression: No
MassTheoretical: 24.560416 kDa
Source (engineered)Expression System: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c

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Component #4: protein, Polyadenylation factor subunit 2,Polyadenylation factor ...

ProteinName: Polyadenylation factor subunit 2,Polyadenylation factor subunit 2
Recombinant expression: No
MassTheoretical: 53.636645 kDa
Source (engineered)Expression System: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c

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Component #5: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.1 mg/ml / pH: 7.9
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 45 e/Å2 / Illumination mode: OTHER
LensMagnification: 81000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 4227

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 77197
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 3.55 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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