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- PDB-7l9p: Structure of human SHLD2-SHLD3-REV7-TRIP13(E253Q) complex -

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Basic information

Entry
Database: PDB / ID: 7l9p
TitleStructure of human SHLD2-SHLD3-REV7-TRIP13(E253Q) complex
Components
  • Mitotic spindle assembly checkpoint protein MAD2B
  • Pachytene checkpoint protein 2 homolog
  • Shieldin complex subunit 2, Shieldin complex subunit 3 chimera
KeywordsNUCLEAR PROTEIN / REV7 / SHLD2 / SHLD3 / TRIP13
Function / homology
Function and homology information


somatic diversification of immunoglobulins involved in immune response / DNA damage response, signal transduction resulting in transcription / telomere maintenance in response to DNA damage / negative regulation of transcription regulatory region DNA binding / zeta DNA polymerase complex / meiotic recombination checkpoint signaling / synaptonemal complex assembly / positive regulation of isotype switching / positive regulation of extracellular matrix assembly / negative regulation of epithelial to mesenchymal transition ...somatic diversification of immunoglobulins involved in immune response / DNA damage response, signal transduction resulting in transcription / telomere maintenance in response to DNA damage / negative regulation of transcription regulatory region DNA binding / zeta DNA polymerase complex / meiotic recombination checkpoint signaling / synaptonemal complex assembly / positive regulation of isotype switching / positive regulation of extracellular matrix assembly / negative regulation of epithelial to mesenchymal transition / negative regulation of transcription by competitive promoter binding / negative regulation of cell-cell adhesion mediated by cadherin / oocyte maturation / JUN kinase binding / reciprocal meiotic recombination / female meiosis I / negative regulation of ubiquitin protein ligase activity / regulation of double-strand break repair via homologous recombination / oogenesis / negative regulation of double-strand break repair via homologous recombination / mitotic spindle assembly checkpoint signaling / positive regulation of double-strand break repair via nonhomologous end joining / male meiosis I / spermatid development / positive regulation of epithelial to mesenchymal transition / error-prone translesion synthesis / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / male germ cell nucleus / actin filament organization / regulation of cell growth / transcription coregulator activity / negative regulation of canonical Wnt signaling pathway / negative regulation of DNA-binding transcription factor activity / negative regulation of protein catabolic process / spindle / double-strand break repair / actin cytoskeleton / site of double-strand break / positive regulation of peptidyl-serine phosphorylation / chromosome / spermatogenesis / RNA polymerase II-specific DNA-binding transcription factor binding / transcription by RNA polymerase II / cell division / DNA repair / chromatin / nucleolus / positive regulation of DNA-templated transcription / negative regulation of transcription by RNA polymerase II / ATP hydrolysis activity / nucleoplasm / ATP binding / identical protein binding / nucleus / cytoplasm
Similarity search - Function
Shieldin complex subunit 2 / Protein FAM35A, C-terminal domain / : / Shieldin complex subunit 2, C-terminal / Shieldin complex subunit 2, first OB fold domain / Pachytene checkpoint protein 2-like / Shieldin complex subunit 3 / Mad2-like / HORMA domain / HORMA domain ...Shieldin complex subunit 2 / Protein FAM35A, C-terminal domain / : / Shieldin complex subunit 2, C-terminal / Shieldin complex subunit 2, first OB fold domain / Pachytene checkpoint protein 2-like / Shieldin complex subunit 3 / Mad2-like / HORMA domain / HORMA domain / HORMA domain profile. / HORMA domain superfamily / ClpA/B family / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Pachytene checkpoint protein 2 homolog / Shieldin complex subunit 3 / Shieldin complex subunit 2 / Mitotic spindle assembly checkpoint protein MAD2B
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsXie, W. / Patel, D.J.
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Molecular mechanisms of assembly and TRIP13-mediated remodeling of the human Shieldin complex.
Authors: Wei Xie / Shengliu Wang / Juncheng Wang / M Jason de la Cruz / Guotai Xu / Maurizio Scaltriti / Dinshaw J Patel /
Abstract: The Shieldin complex, composed of REV7, SHLD1, SHLD2, and SHLD3, protects DNA double-strand breaks (DSBs) to promote nonhomologous end joining. The AAA ATPase TRIP13 remodels Shieldin to regulate DNA ...The Shieldin complex, composed of REV7, SHLD1, SHLD2, and SHLD3, protects DNA double-strand breaks (DSBs) to promote nonhomologous end joining. The AAA ATPase TRIP13 remodels Shieldin to regulate DNA repair pathway choice. Here we report crystal structures of human SHLD3-REV7 binary and fused SHLD2-SHLD3-REV7 ternary complexes, revealing that assembly of Shieldin requires fused SHLD2-SHLD3 induced conformational heterodimerization of open (O-REV7) and closed (C-REV7) forms of REV7. We also report the cryogenic electron microscopy (cryo-EM) structures of the ATPγS-bound fused SHLD2-SHLD3-REV7-TRIP13 complexes, uncovering the principles underlying the TRIP13-mediated disassembly mechanism of the Shieldin complex. We demonstrate that the N terminus of REV7 inserts into the central channel of TRIP13, setting the stage for pulling the unfolded N-terminal peptide of C-REV7 through the central TRIP13 hexameric channel. The primary interface involves contacts between the safety-belt segment of C-REV7 and a conserved and negatively charged loop of TRIP13. This process is mediated by ATP hydrolysis-triggered rotatory motions of the TRIP13 ATPase, thereby resulting in the disassembly of the Shieldin complex.
History
DepositionJan 4, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 3, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Pachytene checkpoint protein 2 homolog
B: Pachytene checkpoint protein 2 homolog
C: Pachytene checkpoint protein 2 homolog
D: Pachytene checkpoint protein 2 homolog
E: Pachytene checkpoint protein 2 homolog
F: Pachytene checkpoint protein 2 homolog
G: Mitotic spindle assembly checkpoint protein MAD2B
I: Mitotic spindle assembly checkpoint protein MAD2B
J: Mitotic spindle assembly checkpoint protein MAD2B
K: Mitotic spindle assembly checkpoint protein MAD2B
Y: Shieldin complex subunit 2, Shieldin complex subunit 3 chimera
X: Shieldin complex subunit 2, Shieldin complex subunit 3 chimera
hetero molecules


Theoretical massNumber of molelcules
Total (without water)412,64117
Polymers410,02512
Non-polymers2,6165
Water0
1


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

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Components

#1: Protein
Pachytene checkpoint protein 2 homolog / Human papillomavirus type 16 E1 protein-binding protein / HPV16 E1 protein-binding protein / ...Human papillomavirus type 16 E1 protein-binding protein / HPV16 E1 protein-binding protein / Thyroid hormone receptor interactor 13 / Thyroid receptor-interacting protein 13 / TRIP-13


Mass: 48562.547 Da / Num. of mol.: 6 / Mutation: E253Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TRIP13, PCH2 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q15645
#2: Protein
Mitotic spindle assembly checkpoint protein MAD2B / Mitotic arrest deficient 2-like protein 2 / MAD2-like protein 2 / REV7 homolog / hREV7


Mass: 24323.348 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: closed form / Source: (gene. exp.) Homo sapiens (human) / Gene: MAD2L2, MAD2B, REV7 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9UI95
#3: Protein Shieldin complex subunit 2, Shieldin complex subunit 3 chimera / Protein FAM35A / RINN1-REV7-interacting novel NHEJ regulator 2 / Shield complex subunit 2 / REV7- ...Protein FAM35A / RINN1-REV7-interacting novel NHEJ regulator 2 / Shield complex subunit 2 / REV7-interacting novel NHEJ regulator 1 / Shield complex subunit 3


Mass: 10678.139 Da / Num. of mol.: 2
Fragment: SHLD2 (UNP residues 5-19) + linker + SHLD3 (UNP residues 2-58)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SHLD2, FAM35A, RINN2, SHLD3, FLJ26957, RINN1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q86V20, UniProt: Q6ZNX1
#4: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
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: SHLD2.3-REV7(4)-TRIP13(E253Q) complex with ATP-gamma-S
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.3
Details: 20 mM HEPES, pH 7.3, 300 mM NaCl, 5 mM MgCl2, 0.1 mM ATP-gamma-S, 1 mM DTT
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 1.5-second blot, blot force of 0

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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
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.075 sec. / Electron dose: 53 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 40

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Processing

SoftwareName: PHENIX / Version: 1.18_3855: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 104023 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00523102
ELECTRON MICROSCOPYf_angle_d0.96731467
ELECTRON MICROSCOPYf_dihedral_angle_d13.9313078
ELECTRON MICROSCOPYf_chiral_restr0.0553820
ELECTRON MICROSCOPYf_plane_restr0.0053936

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