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- PDB-7sn8: Cryo-EM structure of Drosophila Integrator cleavage module (IntS4... -

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Basic information

Entry
Database: PDB / ID: 7sn8
TitleCryo-EM structure of Drosophila Integrator cleavage module (IntS4-IntS9-IntS11) in complex with IP6
Components
  • Integrator complex subunit 11
  • Integrator complex subunit 4
  • Integrator complex subunit 9
KeywordsNUCLEASE / integrator / inositol hexakisphosphate
Function / homology
Function and homology information


RNA polymerase II transcribes snRNA genes / snRNA processing / integrator complex / snRNA 3'-end processing / Hydrolases; Acting on ester bonds; Endoribonucleases producing 3'-phosphomonoesters / RNA endonuclease activity / nucleus / cytoplasm
Similarity search - Function
Integrator complex subunit 9 / Integrator complex subunit 11, MBL-fold / Metallo-beta-lactamase superfamily domain / Beta-Casp domain / Beta-Casp domain / Beta-Casp domain / Zn-dependent metallo-hydrolase, RNA specificity domain / Zn-dependent metallo-hydrolase RNA specificity domain / Metallo-beta-lactamase superfamily / Metallo-beta-lactamase ...Integrator complex subunit 9 / Integrator complex subunit 11, MBL-fold / Metallo-beta-lactamase superfamily domain / Beta-Casp domain / Beta-Casp domain / Beta-Casp domain / Zn-dependent metallo-hydrolase, RNA specificity domain / Zn-dependent metallo-hydrolase RNA specificity domain / Metallo-beta-lactamase superfamily / Metallo-beta-lactamase / Ribonuclease Z/Hydroxyacylglutathione hydrolase-like / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
INOSITOL HEXAKISPHOSPHATE / Integrator complex subunit 9 / Integrator complex subunit 11 / Integrator complex subunit 4
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsLin, M. / Tong, L.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM134539 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118093 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RP170593 United States
CitationJournal: Nat Commun / Year: 2022
Title: Inositol hexakisphosphate is required for Integrator function.
Authors: Min-Han Lin / Madeline K Jensen / Nathan D Elrod / Kai-Lieh Huang / Kevin A Welle / Eric J Wagner / Liang Tong /
Abstract: Integrator is a multi-subunit protein complex associated with RNA polymerase II (Pol II), with critical roles in noncoding RNA 3'-end processing and transcription attenuation of a broad collection of ...Integrator is a multi-subunit protein complex associated with RNA polymerase II (Pol II), with critical roles in noncoding RNA 3'-end processing and transcription attenuation of a broad collection of mRNAs. IntS11 is the endonuclease for RNA cleavage, as a part of the IntS4-IntS9-IntS11 Integrator cleavage module (ICM). Here we report a cryo-EM structure of the Drosophila ICM, at 2.74 Å resolution, revealing stable association of an inositol hexakisphosphate (IP) molecule. The IP binding site is located in a highly electropositive pocket at an interface among all three subunits of ICM, 55 Å away from the IntS11 active site and generally conserved in other ICMs. We also confirmed IP association with the same site in human ICM. IP binding is not detected in ICM samples harboring mutations in this binding site. Such mutations or disruption of IP biosynthesis significantly reduced Integrator function in snRNA 3'-end processing and mRNA transcription attenuation. Our structural and functional studies reveal that IP is required for Integrator function in Drosophila, humans, and likely other organisms.
History
DepositionOct 27, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 12, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: Integrator complex subunit 4
K: Integrator complex subunit 11
I: Integrator complex subunit 9
hetero molecules


Theoretical massNumber of molelcules
Total (without water)256,5556
Polymers255,7643
Non-polymers7913
Water0
1


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

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Components

#1: Protein Integrator complex subunit 4 /


Mass: 114728.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: IntS4, l(1)G0095, CG12113 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9W3E1
#2: Protein Integrator complex subunit 11 /


Mass: 67683.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: IntS11, CG1972 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: Q9VAH9, Hydrolases; Acting on ester bonds; Endoribonucleases producing 3'-phosphomonoesters
#3: Protein Integrator complex subunit 9 /


Mass: 73351.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: IntS9, CG5222 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q95TS5
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#5: Chemical ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE / Phytic acid


Mass: 660.035 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H18O24P6 / Feature type: SUBJECT OF INVESTIGATION
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: Integrator cleavage module with inositol hexakisphosphate
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 51.35 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 4395

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 620438 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00613067
ELECTRON MICROSCOPYf_angle_d0.8717648
ELECTRON MICROSCOPYf_dihedral_angle_d19.9381734
ELECTRON MICROSCOPYf_chiral_restr0.0562014
ELECTRON MICROSCOPYf_plane_restr0.0062233

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