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- PDB-6dnh: Cryo-EM structure of human CPSF-160-WDR33-CPSF-30-PAS RNA complex... -

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Basic information

Entry
Database: PDB / ID: 6dnh
TitleCryo-EM structure of human CPSF-160-WDR33-CPSF-30-PAS RNA complex at 3.4 A 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*C)-3')
  • pre-mRNA 3' end processing protein WDR33
KeywordsRNA BINDING PROTEIN/RNA / Recognition of the AAUAAA polyadenylation signal (PAS) / Hoogsteen base pair / zinc finger / RNA BINDING PROTEIN-RNA complex
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 / fibrillar center / endoribonuclease activity / mRNA polyadenylation / 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)
Simian virus 40
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.4 Å resolution
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
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jun 6, 2018 / Release: Jun 27, 2018

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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
E: RNA (5'-R(P*AP*AP*UP*AP*AP*AP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)261,8257
Polyers261,6284
Non-polymers1963
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)15770
ΔGint (kcal/M)-142
Surface area (Å2)64020

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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: 161074.234 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Gene: CPSF1, CPSF160 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q10570
#2: Protein/peptide 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: (gene. exp.) Homo sapiens (human) / Gene: WDR33, WDC146 / Production host: Trichoplusia ni (cabbage looper) / 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: 27646.055 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Gene: CPSF4, CPSF30, NAR, NEB1 / Production host: Escherichia coli (E. coli) / References: UniProt: O95639
#4: RNA chain RNA (5'-R(P*AP*AP*UP*AP*AP*AP*C)-3')


Mass: 5361.317 Da / Num. of mol.: 1 / Source: (synth.) Simian virus 40
#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
1Quaternary complex of human CPSF-160-WDR33-CPSF-30 with AAUAAA polyadenylation signalCOMPLEX1, 2, 3, 40MULTIPLE SOURCES
2CPSF-160-WDR33COMPLEX1, 21RECOMBINANT
3CPSF-30COMPLEX31RECOMBINANT
4AAUAAA polyadenylation signalCOMPLEX41NATURAL
Molecular weight
IDValueEntity assembly IDExperimental value
10.255 MDa1YES
20.225 MDa1
30.027 MDa1
40.005 MDa1
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
129606Homo sapiens (human)
239606Homo sapiens (human)
3410633Simian virus 40
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganism
127111Trichoplusia ni (cabbage looper)
23562Escherichia coli (E. coli)
34562Escherichia coli (E. coli)
Buffer solutionDetails: 25 mM Tris-HCl, pH 7.9, 380 mM NaCl, 5 mM DTT / pH: 7.9
Buffer componentConc.: 0.38 mM / Name: sodium chloride / Formula: NaCl
SpecimenConc.: 0.18 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins

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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 / Nominal magnification: 225000 / Calibrated magnification: 46729 / 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 mm
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) / Number of grids imaged: 1 / Number of real images: 2468
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameCategory
1Gautomatchparticle selection
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 1144122
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 173632 / Symmetry type: POINT
Atomic model buildingPDB-ID: 2RHK
Pdb chain ID: C / Pdb chain residue range: 56-116
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01013775
ELECTRON MICROSCOPYf_angle_d1.02018701
ELECTRON MICROSCOPYf_dihedral_angle_d8.1518188
ELECTRON MICROSCOPYf_chiral_restr0.0602078
ELECTRON MICROSCOPYf_plane_restr0.0082361

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