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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: EMD-3908
TitlePolyA polymerase module of the cleavage and polyadenylation factor (CPF) from Saccharomyces cerevisiae
Map data
Sample
  • Complex: Complex of Cft1, Yth1, Pfs2 and Fip1
    • Protein or peptide: Protein CFT1
    • Protein or peptide: mRNA 3'-end-processing protein YTH1
    • Protein or peptide: Polyadenylation factor subunit 2,Polyadenylation factor subunit 2
  • Ligand: ZINC ION
KeywordsWD40 / Beta-propeller / Zinc finger / 3'end processing / RNA BINDING PROTEIN
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 speciesSaccharSaccharomyces cerevisiae (strain ATCC 204508 / S288c)omyces (yeast) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) / Saccharomyces cerevisiae S288c (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.55 Å
AuthorsCasanal A / Kumar A
Funding support United Kingdom, 5 items
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, 2017-
Header (metadata) releaseNov 15, 2017-
Map releaseNov 15, 2017-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.15
  • Imaged by UCSF Chimera
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  • 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
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3908.png

Downloads & links

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Map

FileDownload / File: emd_3908.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15 / Movie #1: 0.15
Minimum - Maximum-0.55501544 - 0.8373203
Average (Standard dev.)0.00017478284 (±0.031612962)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 224.0 Å
α=β=γ: 90.0 °

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
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
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_3908_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_3908_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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
Components
  • Complex: Complex of Cft1, Yth1, Pfs2 and Fip1
    • Protein or peptide: Protein CFT1
    • Protein or peptide: mRNA 3'-end-processing protein YTH1
    • Protein or peptide: Polyadenylation factor subunit 2,Polyadenylation factor subunit 2
  • Ligand: ZINC ION

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Supramolecule #1: Complex of Cft1, Yth1, Pfs2 and Fip1

SupramoleculeName: Complex of Cft1, Yth1, Pfs2 and Fip1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: SaccharSaccharomyces cerevisiae (strain ATCC 204508 / S288c)omyces (yeast)
Molecular weightTheoretical: 270 KDa

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Macromolecule #1: Protein CFT1

MacromoleculeName: Protein CFT1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 153.577156 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MNVYDDVLDA TVVSHSLATH FTTSDYEELL VVRTNILSVY RPTRDGKLYL TDEFKFHGLI TDIGLIPQKD SPLSCLLLCT GVAKISILK FNTLTNSIDT LSLHYYEGKF KGKSLVELAK ISTLRMDPGS SCALLFNNDI IAFLPFHVNK NDDDEEEEDE D ENIDDSEL ...String:
MNVYDDVLDA TVVSHSLATH FTTSDYEELL VVRTNILSVY RPTRDGKLYL TDEFKFHGLI TDIGLIPQKD SPLSCLLLCT GVAKISILK FNTLTNSIDT LSLHYYEGKF KGKSLVELAK ISTLRMDPGS SCALLFNNDI IAFLPFHVNK NDDDEEEEDE D ENIDDSEL IHSMNQKSQG TNTFNKRKRT KLGDKFTAPS VVLVASELYE GAKNIIDIQF LKNFTKPTIA LLYQPKLVWA GN TTISKLP TQYVILTLNI QPAESATKIE STTIAFVKEL PWDLHTIVPV SNGAIIVGTN ELAFLDNTGV LQSTVLLNSF ADK ELQKTK IINNSSLEIM FREKNTTSIW IPSSKSKNGG SNNDETLLLM DLKSNIYYIQ MEAEGRLLIK FDIFKLPIVN DLLK ENSNP KCITRLNATN SNKNMDLFIG FGSGNALVLR LNNLKSTIET REAHNPSSGT NSLMDINDDD DEEMDDLYAD EAPEN GLTT NDSKGTVETV QPFDIELLSS LRNVGPITSL TVGKVSSIDD VVKGLPNPNK NEYSLVATSG NGSGSHLTVI QTSVQP EIE LALKFISITQ IWNLKIKGRD RYLITTDSTK SRSDIYESDN NFKLHKGGRL RRDATTVYIS MFGEEKRIIQ VTTNHLY LY DTHFRRLTTI KFDYEVIHVS VMDPYILVTV SRGDIKIFEL EEKNKRKLLK VDLPEILNEM VITSGLILKS NMCNEFLI G LSKSQEEQLL FTFVTADNQI IFFTKDHNDR IFQLNGVDQL NESLYISTYQ LGDEIVPDPS IKQVMINKLG HDNKEEYLT ILTFGGEIYQ YRKLPQRRSR FYRNVTRNDL AITGAPDNAY AKGVSSIERI MHYFPDYNGY SVIFVTGSVP YILIKEDDST PKIFKFGNI PLVSVTPWSE RSVMCVDDIK NARVYTLTTD NMYYGNKLPL KQIKISNVLD DYKTLQKLVY HERAQLFLVS Y CKRVPYEA LGEDGEKVIG YDENVPHAEG FQSGILLINP KSWKVIDKID FPKNSVVNEM RSSMIQINSK TKRKREYIIA GV ANATTED TPPTGAFHIY DVIEVVPEPG KPDTNYKLKE IFQEEVSGTV STVCEVSGRF MISQSQKVLV RDIQEDNSVI PVA FLDIPV FVTDSKSFGN LLIIGDAMQG FQFIGFDAEP YRMISLGRSM SKFQTMSLEF LVNGGDMYFA ATDADRNVHV LKYA PDEPN SLSGQRLVHC SSFTLHSTNS CMMLLPRNEE FGSPQVPSFQ NVGGQVDGSV FKIVPLSEEK YRRLYVIQQQ IIDRE LQLG GLNPRMERLA NDFYQMGHSM RPMLDFNVIR RFCGLAIDRR KSIAQKAGRH AHFEAWRDII NIEFSMRSLC QGK

UniProtKB: Protein CFT1

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Macromolecule #2: mRNA 3'-end-processing protein YTH1

MacromoleculeName: mRNA 3'-end-processing protein YTH1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 24.560416 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: PSLIHPDTAK YPFKFEPFLR QEYSFSLDPD RPICEFYNSR EGPKSCPRGP LCPKKHVLPI FQNKIVCRHW LRGLCKKNDQ CEYLHEYNL RKMPECVFFS KNGYCTQSPD CQYLHIDPAS KIPKCENYEM GFCPLGSSCP RRHIKKVFCQ RYMTGFCPLG K DECDMEHP ...String:
PSLIHPDTAK YPFKFEPFLR QEYSFSLDPD RPICEFYNSR EGPKSCPRGP LCPKKHVLPI FQNKIVCRHW LRGLCKKNDQ CEYLHEYNL RKMPECVFFS KNGYCTQSPD CQYLHIDPAS KIPKCENYEM GFCPLGSSCP RRHIKKVFCQ RYMTGFCPLG K DECDMEHP QFIIPDEGSK LRIKRDDEIN TRKMDEEKER RLNAIINGEV

UniProtKB: mRNA 3'-end-processing protein YTH1

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Macromolecule #3: Polyadenylation factor subunit 2,Polyadenylation factor subunit 2

MacromoleculeName: Polyadenylation factor subunit 2,Polyadenylation factor subunit 2
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 53.636645 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDGHNQNQYQ NQNQIQQSQQ PPLKKYVTQR RSVDVSSPYI NLYYNRRHGL PNLVVEPETS YTIDIMPPNA YRGRDRVINL PSKFTHLSS NKVKHVIPAI QWTPEGRRLV VATYSGEFSL WNASSFTFET LMQAHDSAVT TMKYSHDSDW MISGDADGMI K IWQPNFSM ...String:
MDGHNQNQYQ NQNQIQQSQQ PPLKKYVTQR RSVDVSSPYI NLYYNRRHGL PNLVVEPETS YTIDIMPPNA YRGRDRVINL PSKFTHLSS NKVKHVIPAI QWTPEGRRLV VATYSGEFSL WNASSFTFET LMQAHDSAVT TMKYSHDSDW MISGDADGMI K IWQPNFSM VKEIDAAHTE SIRDMAFSSN DSKFVTCSDD NILKIWNFSN GKQERVLSGH HWDVKSCDWH PEMGLIASAS KD NLVKLWD PRSGNCISSI LKFKHTVLKT RFQPTKGNLL MAISKDKSCR VFDIRYSMKE LMCVRDETDY MTLEWHPINE SMF TLACYD GSLKHFDLLQ NLNEPILTIP YAHDKCITSL SYNPVGHIFA TAAKDRTIRF WTRARPIDPN AYDDPTYNNK KING WFFGI NNDINAVREK SEFGAAPPPP ATLEPHALPN MNGFINKKPR QEIPGIDSNI KSSTLPGLSI (UNK)(UNK)(UNK) (UNK)(UNK)

UniProtKB: Polyadenylation factor subunit 2

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.9
Component:
ConcentrationName
10.0 mMHepes
150.0 mMSodium Chloride
1.0 mMTCEP
GridModel: Quantifoil UltrAuFoil / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 5 / Number real images: 4227 / Average exposure time: 16.0 sec. / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 460167
Startup modelType of model: OTHER
Details: 3D model generated from a previous data set collected from a CPF native sample in a Titan Krios equipped with a Falcon II detector.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.55 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 77197
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.0)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 2.0)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6eoj:
PolyA polymerase module of the cleavage and polyadenylation factor (CPF) from Saccharomyces cerevisiae

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