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

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Basic information

Entry
Database: PDB / ID: 6uro
TitleCryo-EM structure of human CPSF160-WDR33-CPSF30-PAS RNA-CstF77 complex
Components
  • (Cleavage and polyadenylation specificity factor subunit ...) x 2
  • Cleavage stimulation factor subunit 3
  • PAS RNA
  • pre-mRNA 3' end processing protein WDR33
KeywordsRNA BINDING PROTEIN/RNA / pre-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 / fibrillar center / mRNA processing / 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 / Tetratricopeptide repeat domain / Serine Threonine Protein Phosphatase 5, Tetratricopeptide repeat / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / Alpha Horseshoe / 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 / Mainly Alpha
Similarity search - Domain/homology
RNA / RNA (> 10) / 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
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsSun, Y. / Zhang, Y. / Walz, T. / Tong, L.
Funding support United States, 1items
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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 27, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Mar 4, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.year
Revision 1.3Mar 20, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Cleavage and polyadenylation specificity factor subunit 1
B: pre-mRNA 3' end processing protein WDR33
C: Cleavage and polyadenylation specificity factor subunit 4
D: PAS RNA
E: Cleavage stimulation factor subunit 3
F: Cleavage stimulation factor subunit 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)438,1389
Polymers437,9426
Non-polymers1963
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Cleavage and polyadenylation specificity factor subunit ... , 2 types, 2 molecules AC

#1: Protein Cleavage and polyadenylation specificity factor subunit 1 / / Cleavage and polyadenylation specificity factor 160 kDa subunit / CPSF 160 kDa subunit


Mass: 161074.234 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CPSF1, CPSF160 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q10570
#3: Protein Cleavage and polyadenylation specificity factor subunit 4 / / Cleavage and polyadenylation specificity factor 30 kDa subunit / CPSF 30 kDa subunit / NS1 effector ...Cleavage and polyadenylation specificity factor 30 kDa subunit / CPSF 30 kDa subunit / NS1 effector domain-binding protein 1 / Neb-1 / No arches homolog


Mass: 28417.883 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CPSF4, CPSF30, NAR, NEB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O95639

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Protein , 2 types, 3 molecules BEF

#2: Protein pre-mRNA 3' end processing protein WDR33 / WD repeat-containing protein 33 / WD repeat-containing protein WDC146


Mass: 67546.812 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: WDR33, WDC146 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9C0J8
#5: Protein Cleavage stimulation factor subunit 3 / / CF-1 77 kDa subunit / Cleavage stimulation factor 77 kDa subunit / CstF-77


Mass: 83039.625 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CSTF3 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q12996

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RNA chain / Non-polymers , 2 types, 4 molecules D

#4: RNA chain PAS RNA


Mass: 14823.655 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Macaca mulatta polyomavirus 1
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: The complex of human mPSF-PAS RNA-CstF / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8 / Details: 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM DTT
Buffer componentConc.: 150 mM / Name: sodium chloride / Formula: NaClSodium chloride
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 225000 X / Calibrated magnification: 46729 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm / Calibrated defocus min: 900 nm / Calibrated defocus max: 2800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 70 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameCategory
4CTFFINDCTF correction
12RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50092 / Symmetry type: POINT
Atomic model building
IDDetails
1
2The model went through phenix.refine from the crystal structure(2OOE), but was not manually rebuilt due to lack of sufficient side chain density.
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16DNH

6dnh

16DNH1PDBexperimental model
22OOE

2ooe

12OOE2PDBexperimental model
36DNH

6dnh

26DNH1PDBexperimental model
42OOE

2ooe

22OOE2PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00922961
ELECTRON MICROSCOPYf_angle_d0.99131079
ELECTRON MICROSCOPYf_dihedral_angle_d7.29513791
ELECTRON MICROSCOPYf_chiral_restr0.0563379
ELECTRON MICROSCOPYf_plane_restr0.0073933

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