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- EMDB-20861: Cryo-EM structure of human CPSF160-WDR33-CPSF30-PAS RNA-CstF77 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-20861
TitleCryo-EM structure of human CPSF160-WDR33-CPSF30-PAS RNA-CstF77 complex
Map datahuman CPSF160-WDR33-CPSF30-PAS RNA-CstF77 complex
Sample
  • Complex: The complex of human mPSF-PAS RNA-CstF
    • Protein or peptide: Cleavage and polyadenylation specificity factor subunit 1
    • Protein or peptide: pre-mRNA 3' end processing protein WDR33
    • Protein or peptide: Cleavage and polyadenylation specificity factor subunit 4
    • RNA: PAS RNA
    • Protein or peptide: Cleavage stimulation factor subunit 3
  • Ligand: ZINC ION
Keywordspre-mRNA 3'-end processing / RNA binding / AAUAAA polyadenylation signal / RNA BINDING PROTEIN-RNA complex / mPSF
Function / homology
Function and homology information


mRNA cleavage stimulating factor complex / co-transcriptional RNA 3'-end processing, cleavage and polyadenylation pathway / Inhibition of Host mRNA Processing and RNA Silencing / co-transcriptional mRNA 3'-end processing, cleavage and polyadenylation pathway / RNA 3'-end processing / 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 ...mRNA cleavage stimulating factor complex / co-transcriptional RNA 3'-end processing, cleavage and polyadenylation pathway / Inhibition of Host mRNA Processing and RNA Silencing / co-transcriptional mRNA 3'-end processing, cleavage and polyadenylation pathway / RNA 3'-end processing / 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 / mRNA binding / intracellular membrane-bounded organelle / enzyme binding / RNA binding / zinc ion binding / nucleoplasm / nucleus
Similarity search - Function
mRNA 3'-end-processing protein Rna14-like / Zinc-finger CCCH domain / Zinc-finger containing family / Suppressor of forked / Suppressor of forked protein (Suf) / 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 ...mRNA 3'-end-processing protein Rna14-like / Zinc-finger CCCH domain / Zinc-finger containing family / Suppressor of forked / Suppressor of forked protein (Suf) / 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) / HAT (Half-A-TPR) repeat / HAT (Half-A-TPR) repeats / 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. / Tetratricopeptide-like helical domain superfamily / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / 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 / Cleavage stimulation factor subunit 3 / pre-mRNA 3' end processing protein WDR33
Similarity search - Component
Biological speciesHomo sapiens (human) / Macaca mulatta polyomavirus 1
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsSun Y / Zhang Y / Walz T / Tong L
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118093 United States
CitationJournal: Mol Cell / Year: 2020
Title: Structural Insights into the Human Pre-mRNA 3'-End Processing Machinery.
Authors: Yixiao Zhang / Yadong Sun / Yongsheng Shi / Thomas Walz / Liang Tong /
Abstract: The mammalian pre-mRNA 3'-end-processing machinery consists of cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and other proteins, but the overall ...The mammalian pre-mRNA 3'-end-processing machinery consists of cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and other proteins, but the overall architecture of this machinery remains unclear. CPSF contains two functionally distinct modules: a cleavage factor (mCF) and a polyadenylation specificity factor (mPSF). Here, we have produced recombinant human CPSF and CstF and examined these factors by electron microscopy (EM). We find that mPSF is the organizational core of the machinery, while the conformations of mCF and CstF and the position of mCF relative to mPSF are highly variable. We have identified by cryo-EM a segment in CPSF100 that tethers mCF to mPSF, and we have named it the PSF interaction motif (PIM). Mutations in the PIM can abolish CPSF formation, indicating that it is a crucial contact in CPSF. We have also obtained reconstructions of mCF and CstF77 by cryo-EM, assembled around the mPSF core.
History
DepositionOct 23, 2019-
Header (metadata) releaseNov 13, 2019-
Map releaseNov 27, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
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  • Surface view colored by radius
  • Surface level: 0.02
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  • Surface view with fitted model
  • Atomic models: PDB-6uro
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20861.png

Downloads & links

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Map

FileDownload / File: emd_20861.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhuman CPSF160-WDR33-CPSF30-PAS RNA-CstF77 complex
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.06084295 - 0.1047449
Average (Standard dev.)0.00000038817905 (±0.0034893255)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 299.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z299.600299.600299.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.0610.1050.000

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Supplemental data

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Sample components

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Entire : The complex of human mPSF-PAS RNA-CstF

EntireName: The complex of human mPSF-PAS RNA-CstF
Components
  • Complex: The complex of human mPSF-PAS RNA-CstF
    • Protein or peptide: Cleavage and polyadenylation specificity factor subunit 1
    • Protein or peptide: pre-mRNA 3' end processing protein WDR33
    • Protein or peptide: Cleavage and polyadenylation specificity factor subunit 4
    • RNA: PAS RNA
    • Protein or peptide: Cleavage stimulation factor subunit 3
  • Ligand: ZINC ION

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Supramolecule #1: The complex of human mPSF-PAS RNA-CstF

SupramoleculeName: The complex of human mPSF-PAS RNA-CstF / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Homo sapiens (human)

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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

UniProtKB: Cleavage and polyadenylation specificity factor subunit 1

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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

UniProtKB: pre-mRNA 3' end processing protein WDR33

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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: 28.417883 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MQEIIASVDH IKFDLEIAVE QQLGAQPLPF PGMDKSGAAV CEFFLKAACG KGGMCPFRHI SGEKTVVCKH WLRGLCKKGD QCEFLHEYD MTKMPECYFY SKFGECSNKE CPFLHIDPES KIKDCPWYDR GFCKHGPLCR HRHTRRVICV NYLVGFCPEG P SCKFMHPR ...String:
MQEIIASVDH IKFDLEIAVE QQLGAQPLPF PGMDKSGAAV CEFFLKAACG KGGMCPFRHI SGEKTVVCKH WLRGLCKKGD QCEFLHEYD MTKMPECYFY SKFGECSNKE CPFLHIDPES KIKDCPWYDR GFCKHGPLCR HRHTRRVICV NYLVGFCPEG P SCKFMHPR FELPMGTTEQ PPLPQQTQPP AKQRTPQVIG VMQSQNSSAG NRGPRPLEQV TCYKCGEKGH YANRCTKGHL AF LSGQHHH HHH

UniProtKB: Cleavage and polyadenylation specificity factor subunit 4

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Macromolecule #5: Cleavage stimulation factor subunit 3

MacromoleculeName: Cleavage stimulation factor subunit 3 / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 83.039625 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSGDGATEQA AEYVPEKVKK AEKKLEENPY DLDAWSILIR EAQNQPIDKA RKTYERLVAQ FPSSGRFWKL YIEAEIKAKN YDKVEKLFQ RCLMKVLHID LWKCYLSYVR ETKGKLPSYK EKMAQAYDFA LDKIGMEIMS YQIWVDYINF LKGVEAVGSY A ENQRITAV ...String:
MSGDGATEQA AEYVPEKVKK AEKKLEENPY DLDAWSILIR EAQNQPIDKA RKTYERLVAQ FPSSGRFWKL YIEAEIKAKN YDKVEKLFQ RCLMKVLHID LWKCYLSYVR ETKGKLPSYK EKMAQAYDFA LDKIGMEIMS YQIWVDYINF LKGVEAVGSY A ENQRITAV RRVYQRGCVN PMINIEQLWR DYNKYEEGIN IHLAKKMIED RSRDYMNARR VAKEYETVMK GLDRNAPSVP PQ NTPQEAQ QVDMWKKYIQ WEKSNPLRTE DQTLITKRVM FAYEQCLLVL GHHPDIWYEA AQYLEQSSKL LAEKGDMNNA KLF SDEAAN IYERAISTLL KKNMLLYFAY ADYEESRMKY EKVHSIYNRL LAIEDIDPTL VYIQYMKFAR RAEGIKSGRM IFKK AREDT RTRHHVYVTA ALMEYYCSKD KSVAFKIFEL GLKKYGDIPE YVLAYIDYLS HLNEDNNTRV LFERVLTSGS LPPEK SGEI WARFLAFESN IGDLASILKV EKRRFTAFKE EYEGKETALL VDRYKFMDLY PCSASELKAL GYKDVSRAKL AAIIPD PVV APSIVPVLKD EVDRKPEYPK PDTQQMIPFQ PRHLAPPGLH PVPGGVFPVP PAAVVLMKLL PPPICFQGPF VQVDELM EI FRRCKIPNTV EEAVRIITGG APELAVEGNG PVESNAVLTK AVKRPNEDSD EDEEKGAVVP PVHDIYRARQ QKRIR

UniProtKB: Cleavage stimulation factor subunit 3

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Macromolecule #4: PAS RNA

MacromoleculeName: PAS RNA / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Macaca mulatta polyomavirus 1
Molecular weightTheoretical: 14.823655 KDa
SequenceString:
UUCACAAAUA AACAUUUUUU UCACUGCAUU CUAGUUGUGG UUUGUCC

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / 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

Concentration0.25 mg/mL
BufferpH: 8 / Component - Concentration: 150.0 mM / Component - Formula: NaClSodium chloride / Component - Name: sodium chloride / Details: 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM DTT
GridSupport film - Material: CARBON / Support film - topology: HOLEY / Details: unspecified
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: 2.8000000000000003 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 / 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

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 50092

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Atomic model buiding 1

Initial model
PDB IDChain

6dnh

source_name: PDB, initial_model_type: experimental model

2ooe

source_name: PDB, initial_model_type: experimental model
Output model

PDB-6uro:
Cryo-EM structure of human CPSF160-WDR33-CPSF30-PAS RNA-CstF77 complex

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Atomic model buiding 2

Initial model
PDB IDChain

6dnh

source_name: PDB, initial_model_type: experimental model

2ooe

source_name: PDB, initial_model_type: experimental model
DetailsThe model went through phenix.refine from the crystal structure(2OOE), but was not manually rebuilt due to lack of sufficient side chain density.
Output model

PDB-6uro:
Cryo-EM structure of human CPSF160-WDR33-CPSF30-PAS RNA-CstF77 complex

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