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- PDB-7bfq: Structure of the Integrator cleavage module with extended INTS4 a... -

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Basic information

Entry
Database: PDB / ID: 7bfq
TitleStructure of the Integrator cleavage module with extended INTS4 and rigid body docked INTS9/11 CTD
Components
  • Integrator complex subunit 11
  • Integrator complex subunit 4
  • Integrator complex subunit 9
  • Unknown
KeywordsNUCLEAR PROTEIN / Nuclease / Integrator / 3'-end processing
Function / homology
Function and homology information


snRNA processing / integrator complex / snRNA 3'-end processing / regulation of transcription elongation by RNA polymerase II / Hydrolases; Acting on ester bonds; Endoribonucleases producing 3'-phosphomonoesters / RNA polymerase II transcribes snRNA genes / RNA endonuclease activity / negative regulation of transforming growth factor beta receptor signaling pathway / blood microparticle / nucleolus ...snRNA processing / integrator complex / snRNA 3'-end processing / regulation of transcription elongation by RNA polymerase II / Hydrolases; Acting on ester bonds; Endoribonucleases producing 3'-phosphomonoesters / RNA polymerase II transcribes snRNA genes / RNA endonuclease activity / negative regulation of transforming growth factor beta receptor signaling pathway / blood microparticle / nucleolus / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
HEAT repeat associated with sister chromatid cohesion / : / : / Integrator IntS9, C-terminal domain / Integrator IntS11, C-terminal domain / Integrator complex subunit 9 / Integrator complex subunit 11, MBL-fold / Sister chromatid cohesion protein PDS5 protein / Metallo-beta-lactamase superfamily domain / Beta-Casp domain ...HEAT repeat associated with sister chromatid cohesion / : / : / Integrator IntS9, C-terminal domain / Integrator IntS11, C-terminal domain / Integrator complex subunit 9 / Integrator complex subunit 11, MBL-fold / Sister chromatid cohesion protein PDS5 protein / 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 / HEAT repeats / Metallo-beta-lactamase superfamily / Metallo-beta-lactamase / Ribonuclease Z/Hydroxyacylglutathione hydrolase-like / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
Integrator complex subunit 11 / Integrator complex subunit 4 / Integrator complex subunit 9
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.15 Å
AuthorsPfleiderer, M.M. / Galej, W.P.
CitationJournal: Mol Cell / Year: 2021
Title: Structure of the catalytic core of the Integrator complex.
Authors: Moritz M Pfleiderer / Wojciech P Galej /
Abstract: The Integrator is a specialized 3' end-processing complex involved in cleavage and transcription termination of a subset of nascent RNA polymerase II transcripts, including small nuclear RNAs (snRNAs) ...The Integrator is a specialized 3' end-processing complex involved in cleavage and transcription termination of a subset of nascent RNA polymerase II transcripts, including small nuclear RNAs (snRNAs). We provide evidence of the modular nature of the Integrator complex by biochemically characterizing its two subcomplexes, INTS5/8 and INTS10/13/14. Using cryoelectron microscopy (cryo-EM), we determined a 3.5-Å-resolution structure of the INTS4/9/11 ternary complex, which constitutes Integrator's catalytic core. Our structure reveals the spatial organization of the catalytic nuclease INTS11, bound to its catalytically impaired homolog INTS9 via several interdependent interfaces. INTS4, a helical repeat protein, plays a key role in stabilizing nuclease domains and other components. In this assembly, all three proteins form a composite electropositive groove, suggesting a putative RNA binding path within the complex. Comparison with other 3' end-processing machineries points to distinct features and a unique architecture of the Integrator's catalytic module.
History
DepositionJan 4, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 24, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 31, 2021Group: Database references / Refinement description / Category: citation / refine_ls_shell
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _refine_ls_shell.R_factor_R_free / _refine_ls_shell.number_reflns_R_free

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Structure visualization

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Assembly

Deposited unit
A: Integrator complex subunit 9
C: Integrator complex subunit 4
B: Integrator complex subunit 11
U: Unknown


Theoretical massNumber of molelcules
Total (without water)271,3174
Polymers271,3174
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Integrator complex subunit 9 / / Int9 / Protein related to CPSF subunits of 74 kDa / RC-74


Mass: 73891.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: INTS9, RC74 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9NV88
#2: Protein Integrator complex subunit 4 / / Int4


Mass: 110164.773 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: INTS4, MSTP093 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q96HW7
#3: Protein Integrator complex subunit 11 / / Int11 / Cleavage and polyadenylation-specific factor 3-like protein / CPSF3-like protein / Protein ...Int11 / Cleavage and polyadenylation-specific factor 3-like protein / CPSF3-like protein / Protein related to CPSF subunits of 68 kDa / RC-68


Mass: 72690.008 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: INTS11, CPSF3L, RC68 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: Q5TA45, Hydrolases; Acting on ester bonds; Endoribonucleases producing 3'-phosphomonoesters
#4: Protein Unknown


Mass: 14570.941 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Trichoplusia ni (cabbage looper)

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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: Structure of the Integrator cleavage module with extended INTS4 and rigid body docked INTS9/11 CTD
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMPotassium chlorideKCl1
220 mMHEPES-KOH1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
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: 165000 X / Nominal defocus max: 500 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 5 sec. / Electron dose: 44 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 19268
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 40

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Processing

SoftwareName: REFMAC / Version: 5.8.0253 / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
3SerialEMimage acquisition
5RELION3CTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 9124445
3D reconstructionResolution: 4.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 20001 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL
RefinementResolution: 3.5→110.39 Å / Cor.coef. Fo:Fc: 0.762 / SU B: 62.878 / SU ML: 0.823 / ESU R: 1.094
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.41498 --
obs0.41498 58007 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 148.632 Å2
Baniso -1Baniso -2Baniso -3
1--2.62 Å2-2.59 Å23.8 Å2
2--0.91 Å2-3.56 Å2
3---1.72 Å2
Refinement stepCycle: 1 / Total: 10068
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.01210263
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg1.6671.62813957
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.75851349
ELECTRON MICROSCOPYr_dihedral_angle_2_deg31.49823.03429
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.491151634
ELECTRON MICROSCOPYr_dihedral_angle_4_deg18.1041543
ELECTRON MICROSCOPYr_chiral_restr0.1160.21407
ELECTRON MICROSCOPYr_gen_planes_refined0.0090.027701
ELECTRON MICROSCOPYr_gen_planes_other
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it20.74114.2015423
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it35.18521.2336763
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it30.48516.4174840
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined71.97940401
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.5→3.591 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork2.06 4345 -
obs--100 %

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