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- PDB-6fuw: Cryo-EM structure of the human CPSF160-WDR33-CPSF30 complex bound... -

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Basic information

Entry
Database: PDB / ID: 6fuw
TitleCryo-EM structure of the human CPSF160-WDR33-CPSF30 complex bound to the PAS AAUAAA motif at 3.1 Angstrom resolution
Components
  • Cleavage and polyadenylation specificity factor subunit 1
  • Cleavage and polyadenylation specificity factor subunit 4
  • RNA (5'-R(P*AP*AP*UP*AP*AP*AP*GP*G)-3')
  • pre-mRNA 3' end processing protein WDR33
KeywordsRNA BINDING PROTEIN / 3' pre-mRNA processing / polyadenylation / CPSF / beta propeller / AAUAAA
Function / homologyZinc-finger containing family / Zinc finger C3H1-type profile. / WD40-repeat-containing domain superfamily / Zinc finger, CCCH-type superfamily / Zinc finger, CCHC-type superfamily / Zinc knuckle / WD domain, G-beta repeat / Zinc finger C-x8-C-x5-C-x3-H type (and similar) / CPSF A subunit region / Trp-Asp (WD) repeats profile. ...Zinc-finger containing family / Zinc finger C3H1-type profile. / WD40-repeat-containing domain superfamily / Zinc finger, CCCH-type superfamily / Zinc finger, CCHC-type superfamily / Zinc knuckle / WD domain, G-beta repeat / Zinc finger C-x8-C-x5-C-x3-H type (and similar) / CPSF A subunit region / Trp-Asp (WD) repeats profile. / Zinc finger CCHC-type profile. / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / Trp-Asp (WD) repeats circular profile. / Cleavage of Growing Transcript in the Termination Region / Transport of Mature mRNA Derived from an Intronless Transcript / Inhibition of Host mRNA Processing and RNA Silencing / tRNA processing in the nucleus / mRNA Splicing - Major Pathway / mRNA 3'-end processing / Processing of Intronless Pre-mRNAs / Mono-functional DNA-alkylating methyl methanesulfonate N-term / WD40-repeat-containing domain / WD40/YVTN repeat-like-containing domain superfamily / Zinc finger, CCHC-type / WD40 repeat / Zinc finger, CCCH-type / modification by virus of host mRNA processing / mRNA 3'-UTR AU-rich region binding / mRNA cleavage and polyadenylation specificity factor complex / pre-mRNA cleavage required for polyadenylation / postreplication repair / mRNA 3'-end processing / collagen trimer / mRNA cleavage / termination of RNA polymerase II transcription / mRNA export from nucleus / endoribonuclease activity / mRNA polyadenylation / fibrillar center / mRNA splicing, via spliceosome / spermatogenesis / intracellular membrane-bounded organelle / enzyme binding / RNA binding / zinc ion binding / nucleoplasm / nucleus / Cleavage and polyadenylation specificity factor subunit 4 / Cleavage and polyadenylation specificity factor subunit 1 / pre-mRNA 3' end processing protein WDR33
Function and homology information
Specimen sourceHomo sapiens (human)
unidentified adenovirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.07 Å resolution
AuthorsClerici, M. / Faini, M. / Jinek, M.
CitationJournal: Nat. Struct. Mol. Biol. / Year: 2018
Title: Structural basis of AAUAAA polyadenylation signal recognition by the human CPSF complex.
Authors: Marcello Clerici / Marco Faini / Lena M Muckenfuss / Ruedi Aebersold / Martin Jinek
DateDeposition: Feb 28, 2018 / Release: Mar 21, 2018

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Structure visualization

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Structure viewerMolecule:
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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: RNA (5'-R(P*AP*AP*UP*AP*AP*AP*GP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)232,6477
Polyers232,4504
Non-polymers1963
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)14140
ΔGint (kcal/M)-59
Surface area (Å2)66850
MethodPISA

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Components

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


Mass: 161346.484 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Gene: CPSF1, CPSF160
Production host: Spodoptera aff. frugiperda 2 RZ-2014 (insect)
References: UniProt: Q10570
#2: Protein/peptide pre-mRNA 3' end processing protein WDR33 / WD repeat-containing protein 33 / WD repeat-containing protein WDC146


Mass: 47448.910 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Gene: WDR33, WDC146
Production host: Spodoptera aff. frugiperda 2 RZ-2014 (insect)
References: UniProt: Q9C0J8
#3: Protein/peptide Cleavage and polyadenylation specificity factor subunit 4 / / Cleavage and polyadenylation specificity factor 30 kDa subunit / CPSF 30 kDa subunit / NS1 effector domain-binding protein 1 / Neb-1 / No arches homolog


Mass: 20422.855 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Gene: CPSF4, CPSF30, NAR, NEB1
Production host: Spodoptera aff. frugiperda 2 RZ-2014 (insect)
References: UniProt: O95639
#4: RNA chain RNA (5'-R(P*AP*AP*UP*AP*AP*AP*GP*G)-3')


Mass: 3232.036 Da / Num. of mol.: 1 / Source: (synth.) unidentified adenovirus
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Formula: Zn / Zinc

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Experimental details

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Experiment

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

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Sample preparation

Component
IDNameTypeEntity IDParent IDSource
1Human CPSF160-WDR33-CPSF30 heterotrimer bound to the AAUAAA motif of the Polyadenylation SignalCOMPLEX1, 2, 3, 40MULTIPLE SOURCES
2Human CPSF160-WDR33-CPSF30 heterotrimerCOMPLEX1, 2, 31RECOMBINANT
3AAUAAA motif of the Polyadenylation SignalCOMPLEX41RECOMBINANT
Molecular weightValue: 0.22 MDa / Experimental value: NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
229606Homo sapiens (human)
3310535unidentified adenovirus
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganism
221491790Spodoptera aff. frugiperda 2 RZ-2014 (insect)
3332630synthetic construct (others)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer ID
1140 mMPotassium ChlorideKCl1
220 mMHEPESHEPES1
31 mMDithiothreitolDTT1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 293 kelvins / Details: 15 seconds wait time prior to blotting

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 47259 / Nominal defocus max: 2200 nm / Nominal defocus min: 1100 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 80 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1 / Number of real images: 1070
EM imaging opticsEnergyfilter name: GIF Quantum LS
Image scansMovie frames/image: 50

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2.1b1_cu8.0particle selection
2SerialEMimage acquisition
4RELION2.1b1_cu8.0CTF correction
7Coot0.8.8model fitting
9PHENIX1.12-2829model refinement
10RELION2.1b1_cu8.0initial Euler assignment
11RELION2.1b1_cu8.0final Euler assignment
12RELION2.1b1_cu8.0classification
13RELION2.1b1_cu8.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 263000
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 137000 / Symmetry type: POINT
Atomic model buildingDetails: phenix.real_space_refine / Ref protocol: RIGID BODY FIT / Ref space: REAL
Atomic model buildingPDB-ID: 6F9N
Details: fitted manually in Coot
Least-squares processHighest resolution: 3.07 Å
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01013762
ELECTRON MICROSCOPYf_angle_d0.96818688
ELECTRON MICROSCOPYf_dihedral_angle_d11.8268173
ELECTRON MICROSCOPYf_chiral_restr0.0582081
ELECTRON MICROSCOPYf_plane_restr0.0062352

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