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- EMDB-7112: Cryo-EM structure of human CPSF-160-WDR33-CPSF-30-PAS RNA complex... -

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Basic information

Entry
Database: EMDB / ID: EMD-7112
TitleCryo-EM structure of human CPSF-160-WDR33-CPSF-30-PAS RNA complex at 3.4 A resolution
Map dataHuman CPSF160-WDR33-CPSF30-PAS complex at 3.4 A resolution
Sample
  • Complex: Quaternary complex of human CPSF-160-WDR33-CPSF-30 with AAUAAA polyadenylation signal
    • Complex: CPSF-160-WDR33
      • Protein or peptide: Cleavage and polyadenylation specificity factor subunit 1
      • Protein or peptide: pre-mRNA 3' end processing protein WDR33
    • Complex: CPSF-30Cleavage and polyadenylation specificity factor
      • Protein or peptide: Cleavage and polyadenylation specificity factor subunit 4
    • Complex: AAUAAA polyadenylation signal
      • RNA: RNA (5'-R(P*AP*AP*UP*AP*AP*AP*C)-3')
  • Ligand: ZINC ION
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 / mRNA 3'-UTR AU-rich region binding / collagen trimer / mRNA 3'-end processing / Transport of Mature mRNA Derived from an Intronless Transcript / postreplication repair / tRNA processing in the nucleus ...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 / mRNA 3'-UTR AU-rich region binding / collagen trimer / mRNA 3'-end processing / Transport of Mature mRNA Derived from an Intronless Transcript / postreplication repair / tRNA processing in the nucleus / RNA Polymerase II Transcription Termination / : / Processing of Capped Intron-Containing Pre-mRNA / mRNA processing / fibrillar center / 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 / Zinc finger, CCCH-type / Zinc finger C3H1-type profile. / Collagen triple helix repeat ...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 / Zinc finger, CCCH-type / Zinc finger C3H1-type profile. / Collagen triple helix repeat / Collagen triple helix repeat (20 copies) / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / Mono-functional DNA-alkylating methyl methanesulfonate N-term / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / CPSF A subunit region / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / 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
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) / Simian virus 40
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsSun Y / Zhang Y / Hamilton K / Walz T / Tong L
CitationJournal: Proc Natl Acad Sci U S A / Year: 2018
Title: Molecular basis for the recognition of the human AAUAAA polyadenylation signal.
Authors: Yadong Sun / Yixiao Zhang / Keith Hamilton / James L Manley / Yongsheng Shi / Thomas Walz / Liang Tong /
Abstract: Nearly all eukaryotic messenger RNA precursors must undergo cleavage and polyadenylation at their 3'-end for maturation. A crucial step in this process is the recognition of the AAUAAA ...Nearly all eukaryotic messenger RNA precursors must undergo cleavage and polyadenylation at their 3'-end for maturation. A crucial step in this process is the recognition of the AAUAAA polyadenylation signal (PAS), and the molecular mechanism of this recognition has been a long-standing problem. Here, we report the cryo-electron microscopy structure of a quaternary complex of human CPSF-160, WDR33, CPSF-30, and an AAUAAA RNA at 3.4-Å resolution. Strikingly, the AAUAAA PAS assumes an unusual conformation that allows this short motif to be bound directly by both CPSF-30 and WDR33. The A1 and A2 bases are recognized specifically by zinc finger 2 (ZF2) of CPSF-30 and the A4 and A5 bases by ZF3. Interestingly, the U3 and A6 bases form an intramolecular Hoogsteen base pair and directly contact WDR33. CPSF-160 functions as an essential scaffold and preorganizes CPSF-30 and WDR33 for high-affinity binding to AAUAAA. Our findings provide an elegant molecular explanation for how PAS sequences are recognized for mRNA 3'-end formation.
History
DepositionNov 10, 2017-
Header (metadata) releaseNov 22, 2017-
Map releaseNov 22, 2017-
UpdateJun 27, 2018-
Current statusJun 27, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
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  • Surface level: 0.06
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  • Surface view with fitted model
  • Atomic models: PDB-6dnh
  • Surface level: 0.06
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_7112.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman CPSF160-WDR33-CPSF30-PAS complex at 3.4 A resolution
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.17654768 - 0.31463027
Average (Standard dev.)0.00002113147 (±0.013521084)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 205.44 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z205.440205.440205.440
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.1770.3150.000

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Supplemental data

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Sample components

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Entire : Quaternary complex of human CPSF-160-WDR33-CPSF-30 with AAUAAA po...

EntireName: Quaternary complex of human CPSF-160-WDR33-CPSF-30 with AAUAAA polyadenylation signal
Components
  • Complex: Quaternary complex of human CPSF-160-WDR33-CPSF-30 with AAUAAA polyadenylation signal
    • Complex: CPSF-160-WDR33
      • Protein or peptide: Cleavage and polyadenylation specificity factor subunit 1
      • Protein or peptide: pre-mRNA 3' end processing protein WDR33
    • Complex: CPSF-30Cleavage and polyadenylation specificity factor
      • Protein or peptide: Cleavage and polyadenylation specificity factor subunit 4
    • Complex: AAUAAA polyadenylation signal
      • RNA: RNA (5'-R(P*AP*AP*UP*AP*AP*AP*C)-3')
  • Ligand: ZINC ION

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Supramolecule #1: Quaternary complex of human CPSF-160-WDR33-CPSF-30 with AAUAAA po...

SupramoleculeName: Quaternary complex of human CPSF-160-WDR33-CPSF-30 with AAUAAA polyadenylation signal
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Molecular weightExperimental: 255 KDa

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Supramolecule #2: CPSF-160-WDR33

SupramoleculeName: CPSF-160-WDR33 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
Molecular weightTheoretical: 225 KDa

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Supramolecule #3: CPSF-30

SupramoleculeName: CPSF-30 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 27 KDa

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Supramolecule #4: AAUAAA polyadenylation signal

SupramoleculeName: AAUAAA polyadenylation signal / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Simian virus 40
Molecular weightTheoretical: 5 KDa

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Macromolecule #1: Cleavage and polyadenylation specificity factor subunit 1

MacromoleculeName: Cleavage and polyadenylation specificity factor subunit 1
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 161.074234 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MYAVYKQAHP PTGLEFSMYC NFFNNSERNL VVAGTSQLYV YRLNRDAEAL TKNDRSTEGK AHREKLELAA SFSFFGNVMS MASVQLAGA KRDALLLSFK DAKLSVVEYD PGTHDLKTLS LHYFEEPELR DGFVQNVHTP RVRVDPDGRC AAMLVYGTRL V VLPFRRES ...String:
MYAVYKQAHP PTGLEFSMYC NFFNNSERNL VVAGTSQLYV YRLNRDAEAL TKNDRSTEGK AHREKLELAA SFSFFGNVMS MASVQLAGA KRDALLLSFK DAKLSVVEYD PGTHDLKTLS LHYFEEPELR DGFVQNVHTP RVRVDPDGRC AAMLVYGTRL V VLPFRRES LAEEHEGLVG EGQRSSFLPS YIIDVRALDE KLLNIIDLQF LHGYYEPTLL ILFEPNQTWP GRVAVRQDTC SI VAISLNI TQKVHPVIWS LTSLPFDCTQ ALAVPKPIGG VVVFAVNSLL YLNQSVPPYG VALNSLTTGT TAFPLRTQEG VRI TLDCAQ ATFISYDKMV ISLKGGEIYV LTLITDGMRS VRAFHFDKAA ASVLTTSMVT MEPGYLFLGS RLGNSLLLKY TEKL QEPPA SAVREAADKE EPPSKKKRVD ATAGWSAAGK SVPQDEVDEI EVYGSEAQSG TQLATYSFEV CDSILNIGPC ANAAV GEPA FLSEEFQNSP EPDLEIVVCS GHGKNGALSV LQKSIRPQVV TTFELPGCYD MWTVIAPVRK EEEDNPKGEG TEQEPS TTP EADDDGRRHG FLILSREDST MILQTGQEIM ELDTSGFATQ GPTVFAGNIG DNRYIVQVSP LGIRLLEGVN QLHFIPV DL GAPIVQCAVA DPYVVIMSAE GHVTMFLLKS DSYGGRHHRL ALHKPPLHHQ SKVITLCLYR DLSGMFTTES RLGGARDE L GGRSGPEAEG LGSETSPTVD DEEEMLYGDS GSLFSPSKEE ARRSSQPPAD RDPAPFRAEP THWCLLVREN GTMEIYQLP DWRLVFLVKN FPVGQRVLVD SSFGQPTTQG EARREEATRQ GELPLVKEVL LVALGSRQSR PYLLVHVDQE LLIYEAFPHD SQLGQGNLK VRFKKVPHNI NFREKKPKPS KKKAEGGGAE EGAGARGRVA RFRYFEDIYG YSGVFICGPS PHWLLVTGRG A LRLHPMAI DGPVDSFAPF HNVNCPRGFL YFNRQGELRI SVLPAYLSYD APWPVRKIPL RCTAHYVAYH VESKVYAVAT ST NTPCARI PRMTGEEKEF ETIERDERYI HPQQEAFSIQ LISPVSWEAI PNARIELQEW EHVTCMKTVS LRSEETVSGL KGY VAAGTC LMQGEEVTCR GRILIMDVIE VVPEPGQPLT KNKFKVLYEK EQKGPVTALC HCNGHLVSAI GQKIFLWSLR ASEL TGMAF IDTQLYIHQM ISVKNFILAA DVMKSISLLR YQEESKTLSL VSRDAKPLEV YSVDFMVDNA QLGFLVSDRD RNLMV YMYL PEAKESFGGM RLLRRADFHV GAHVNTFWRT PCRGATEGLS KKSVVWENKH ITWFATLDGG IGLLLPMQEK TYRRLL MLQ NALTTMLPHH AGLNPRAFRM LHVDRRTLQN AVRNVLDGEL LNRYLYLSTM ERSELAKKIG TTPDIILDDL LETDRVT AH F

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Macromolecule #2: pre-mRNA 3' end processing protein WDR33

MacromoleculeName: pre-mRNA 3' end processing protein WDR33 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 67.546812 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGSSHHHHHH SSGLVMATEI GSPPRFFHMP RFQHQAPRQL FYKRPDFAQQ QAMQQLTFDG KRMRKAVNRK TIDYNPSVIK YLENRIWQR DQRDMRAIQP DAGYYNDLVP PIGMLNNPMN AVTTKFVRTS TNKVKCPVFV VRWTPEGRRL VTGASSGEFT L WNGLTFNF ...String:
MGSSHHHHHH SSGLVMATEI GSPPRFFHMP RFQHQAPRQL FYKRPDFAQQ QAMQQLTFDG KRMRKAVNRK TIDYNPSVIK YLENRIWQR DQRDMRAIQP DAGYYNDLVP PIGMLNNPMN AVTTKFVRTS TNKVKCPVFV VRWTPEGRRL VTGASSGEFT L WNGLTFNF ETILQAHDSP VRAMTWSHND MWMLTADHGG YVKYWQSNMN NVKMFQAHKE AIREASFSPT DNKFATCSDD GT VRIWDFL RCHEERILRG HGADVKCVDW HPTKGLVVSG SKDSQQPIKF WDPKTGQSLA TLHAHKNTVM EVKLNLNGNW LLT ASRDHL CKLFDIRNLK EELQVFRGHK KEATAVAWHP VHEGLFASGG SDGSLLFWHV GVEKEVGGME MAHEGMIWSL AWHP LGHIL CSGSNDHTSK FWTRNRPGDK MRDRYNLNLL PGMSEDGVEY DDLEPNSLAV IPGMGIPEQL KLAMEQEQMG KDESN EIEM TIPGLDWGME EVMQKDQKKV PQKKVPYAKP IPAQFQQAWM QNKVPIPAPN EVLNDRKEDI KLEEKKKTQA EIEQEM ATL QYTNPQLLEQ LKIERLAQKQ VEQI

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Macromolecule #3: Cleavage and polyadenylation specificity factor subunit 4

MacromoleculeName: Cleavage and polyadenylation specificity factor subunit 4
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 27.646055 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GMQEIIASVD HIKFDLEIAV EQQLGAQPLP FPGMDKSGAA VCEFFLKAAC GKGGMCPFRH ISGEKTVVCK HWLRGLCKKG DQCEFLHEY DMTKMPECYF YSKFGECSNK ECPFLHIDPE SKIKDCPWYD RGFCKHGPLC RHRHTRRVIC VNYLVGFCPE G PSCKFMHP ...String:
GMQEIIASVD HIKFDLEIAV EQQLGAQPLP FPGMDKSGAA VCEFFLKAAC GKGGMCPFRH ISGEKTVVCK HWLRGLCKKG DQCEFLHEY DMTKMPECYF YSKFGECSNK ECPFLHIDPE SKIKDCPWYD RGFCKHGPLC RHRHTRRVIC VNYLVGFCPE G PSCKFMHP RFELPMGTTE QPPLPQQTQP PAKQRTPQVI GVMQSQNSSA GNRGPRPLEQ VTCYKCGEKG HYANRCTKGH LA FLSGQ

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Macromolecule #4: RNA (5'-R(P*AP*AP*UP*AP*AP*AP*C)-3')

MacromoleculeName: RNA (5'-R(P*AP*AP*UP*AP*AP*AP*C)-3') / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Simian virus 40
Molecular weightTheoretical: 5.361317 KDa
SequenceString:
AACCUCCAAU AAACAAC

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 3 / 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

Concentration0.18 mg/mL
BufferpH: 7.9 / Component - Concentration: 0.38 mM / Component - Formula: NaClSodium chloride / Component - Name: sodium chloride / Details: 25 mM Tris-HCl, pH 7.9, 380 mM NaCl, 5 mM DTT
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 2.8 µm / Calibrated defocus min: 0.9 µm / Calibrated magnification: 46729 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 225000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 2468 / Average exposure time: 10.0 sec. / Average electron dose: 70.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1144122
CTF correctionSoftware - Name: CTFFIND
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 173632

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Atomic model buiding 1

Initial modelPDB ID:

Chain - Chain ID: C / Chain - Residue range: 56-116
Output model

PDB-6dnh:
Cryo-EM structure of human CPSF-160-WDR33-CPSF-30-PAS RNA complex at 3.4 A resolution

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