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- EMDB-27870: CRYO-EM STRUCTURE OF the human MPSF -

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Basic information

Entry
Database: EMDB / ID: EMD-27870
TitleCRYO-EM STRUCTURE OF the human MPSF
Map datamPSF mixed with a FAM labeled 17-mer RNA containing the AAGAAA PAS (No binding observed)
Sample
  • Complex: human mPSF
    • Protein or peptide: Cleavage and polyadenylation specificity factor subunit 1
    • Protein or peptide: Cleavage and polyadenylation specificity factor subunit 4
    • Protein or peptide: pre-mRNA 3' end processing protein WDR33
  • Ligand: ZINC ION
KeywordsmRNA / 3'processing / polyadenylation / CPSF / RNA BINDING PROTEIN
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 / collagen trimer / mRNA 3'-UTR AU-rich region binding / mRNA 3'-end processing / Transport of Mature mRNA Derived from an Intronless Transcript / mRNA 3'-end processing / 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 / collagen trimer / mRNA 3'-UTR AU-rich region binding / mRNA 3'-end processing / Transport of Mature mRNA Derived from an Intronless Transcript / mRNA 3'-end processing / tRNA processing in the nucleus / postreplication repair / RNA Polymerase II Transcription Termination / Processing of Capped Intron-Containing Pre-mRNA / fibrillar center / mRNA processing / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.68 Å
AuthorsGutierrez PA / Wei J / Sun Y / 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: RNA / Year: 2022
Title: Molecular basis for the recognition of the AUUAAA polyadenylation signal by mPSF.
Authors: Pedro A Gutierrez / Jia Wei / Yadong Sun / Liang Tong /
Abstract: The polyadenylation signal (PAS) is a key sequence element for 3'-end cleavage and polyadenylation of messenger RNA precursors (pre-mRNAs). This hexanucleotide motif is recognized by the mammalian ...The polyadenylation signal (PAS) is a key sequence element for 3'-end cleavage and polyadenylation of messenger RNA precursors (pre-mRNAs). This hexanucleotide motif is recognized by the mammalian polyadenylation specificity factor (mPSF), consisting of CPSF160, WDR33, CPSF30, and Fip1 subunits. Recent studies have revealed how the AAUAAA PAS, the most frequently observed PAS, is recognized by mPSF. We report here the structure of human mPSF in complex with the AUUAAA PAS, the second most frequently identified PAS. Conformational differences are observed for the A1 and U2 nucleotides in AUUAAA compared to the A1 and A2 nucleotides in AAUAAA, while the binding modes of the remaining 4 nt are essentially identical. The 5' phosphate of U2 moves by 2.6 Å and the U2 base is placed near the six-membered ring of A2 in AAUAAA, where it makes two hydrogen bonds with zinc finger 2 (ZF2) of CPSF30, which undergoes conformational changes as well. We also attempted to determine the binding modes of two rare PAS hexamers, AAGAAA and GAUAAA, but did not observe the RNA in the cryo-electron microscopy density. The residues in CPSF30 (ZF2 and ZF3) and WDR33 that recognize PAS are disordered in these two structures.
History
DepositionAug 17, 2022-
Header (metadata) releaseJan 18, 2023-
Map releaseJan 18, 2023-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27870.png

Downloads & links

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Map

FileDownload / File: emd_27870.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationmPSF mixed with a FAM labeled 17-mer RNA containing the AAGAAA PAS (No binding observed)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 212.48 Å		0.83 Å/pix.
x 256 pix.
= 212.48 Å		0.83 Å/pix.
x 256 pix.
= 212.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.35
Minimum - Maximum-2.5599027 - 3.4161632
Average (Standard dev.)0.0003617861 (±0.124242604)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A

Fileemd_27870_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_27870_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : human mPSF

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

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Supramolecule #1: human mPSF

SupramoleculeName: human mPSF / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: mPSF with the AAGAAA poly(A) signal, but the RNA was not observed
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: Cleavage and polyadenylation specificity factor subunit 4

MacromoleculeName: Cleavage and polyadenylation specificity factor subunit 4
type: protein_or_peptide / ID: 2 / 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

UniProtKB: Cleavage and polyadenylation specificity factor subunit 4

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

MacromoleculeName: pre-mRNA 3' end processing protein WDR33 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 65.912039 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MATEIGSPPR FFHMPRFQHQ APRQLFYKRP DFAQQQAMQQ LTFDGKRMRK AVNRKTIDYN PSVIKYLENR IWQRDQRDMR AIQPDAGYY NDLVPPIGML NNPMNAVTTK FVRTSTNKVK CPVFVVRWTP EGRRLVTGAS SGEFTLWNGL TFNFETILQA H DSPVRAMT ...String:
MATEIGSPPR FFHMPRFQHQ APRQLFYKRP DFAQQQAMQQ LTFDGKRMRK AVNRKTIDYN PSVIKYLENR IWQRDQRDMR AIQPDAGYY NDLVPPIGML NNPMNAVTTK FVRTSTNKVK CPVFVVRWTP EGRRLVTGAS SGEFTLWNGL TFNFETILQA H DSPVRAMT WSHNDMWMLT ADHGGYVKYW QSNMNNVKMF QAHKEAIREA SFSPTDNKFA TCSDDGTVRI WDFLRCHEER IL RGHGADV KCVDWHPTKG LVVSGSKDSQ QPIKFWDPKT GQSLATLHAH KNTVMEVKLN LNGNWLLTAS RDHLCKLFDI RNL KEELQV FRGHKKEATA VAWHPVHEGL FASGGSDGSL LFWHVGVEKE VGGMEMAHEG MIWSLAWHPL GHILCSGSND HTSK FWTRN RPGDKMRDRY NLNLLPGMSE DGVEYDDLEP NSLAVIPGMG IPEQLKLAME QEQMGKDESN EIEMTIPGLD WGMEE VMQK DQKKVPQKKV PYAKPIPAQF QQAWMQNKVP IPAPNEVLND RKEDIKLEEK KKTQAEIEQE MATLQYTNPQ LLEQLK IER LAQKQVEQI

UniProtKB: pre-mRNA 3' end processing protein WDR33

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 1 / 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

Concentration0.2 mg/mL
BufferpH: 8 / Details: 25 mM Tris (pH 8.0), 150 mM NaCl, and 5 mM DTT
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 4607 / Average electron dose: 58.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -2.0 µm / Nominal defocus min: -1.0 µm
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: 4998847
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.68 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 614782
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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Atomic model buiding 1

Initial modelPDB ID:

6dnh


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-8e3q:
CRYO-EM STRUCTURE OF the human MPSF

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