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- PDB-6ww9: Crystal structure of human REV7(R124A)-SHLD3(35-58) complex -

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Basic information

Entry
Database: PDB / ID: 6ww9
TitleCrystal structure of human REV7(R124A)-SHLD3(35-58) complex
Components
  • Mitotic spindle assembly checkpoint protein MAD2B
  • Shieldin complex subunit 3
KeywordsNUCLEAR PROTEIN / REV7 / SHLD3
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 / 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 ...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 / 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 / JUN kinase binding / negative regulation of ubiquitin protein ligase activity / 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 / positive regulation of epithelial to mesenchymal transition / error-prone translesion synthesis / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / actin filament organization / regulation of cell growth / 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 / site of double-strand break / positive regulation of peptidyl-serine phosphorylation / chromosome / RNA polymerase II-specific DNA-binding transcription factor binding / cell division / DNA repair / chromatin / nucleolus / positive regulation of DNA-templated transcription / negative regulation of transcription by RNA polymerase II / nucleoplasm / nucleus / cytoplasm
Similarity search - Function
Shieldin complex subunit 3 / Mad2-like / HORMA domain / HORMA domain / HORMA domain profile. / HORMA domain superfamily
Similarity search - Domain/homology
Shieldin complex subunit 3 / Mitotic spindle assembly checkpoint protein MAD2B
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.7 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
DepositionMay 8, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 3, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Mitotic spindle assembly checkpoint protein MAD2B
X: Shieldin complex subunit 3
B: Mitotic spindle assembly checkpoint protein MAD2B
Y: Shieldin complex subunit 3


Theoretical massNumber of molelcules
Total (without water)56,6644
Polymers56,6644
Non-polymers00
Water0
1
A: Mitotic spindle assembly checkpoint protein MAD2B
X: Shieldin complex subunit 3


Theoretical massNumber of molelcules
Total (without water)28,3322
Polymers28,3322
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3120 Å2
ΔGint-22 kcal/mol
Surface area10650 Å2
MethodPISA
2
B: Mitotic spindle assembly checkpoint protein MAD2B
Y: Shieldin complex subunit 3


Theoretical massNumber of molelcules
Total (without water)28,3322
Polymers28,3322
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2950 Å2
ΔGint-19 kcal/mol
Surface area11090 Å2
MethodPISA
Unit cell
Length a, b, c (Å)75.723, 75.723, 220.087
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Space group name HallP4nw2abw
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+3/4
#3: y+1/2,-x+1/2,z+1/4
#4: x+1/2,-y+1/2,-z+1/4
#5: -x+1/2,y+1/2,-z+3/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2

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Components

#1: Protein Mitotic spindle assembly checkpoint protein MAD2B / Mitotic arrest deficient 2-like protein 2 / MAD2-like protein 2 / REV7 homolog / hREV7


Mass: 24237.232 Da / Num. of mol.: 2 / Mutation: R124A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAD2L2, MAD2B, REV7 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q9UI95
#2: Protein/peptide Shieldin complex subunit 3 / REV7-interacting novel NHEJ regulator 1 / Shield complex subunit 3


Mass: 4094.845 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SHLD3, FLJ26957, RINN1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q6ZNX1

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.78 Å3/Da / Density % sol: 55.82 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 5.6
Details: 0.2 M ammonium acetate, 0.1 M Na citrate pH 5.6, 30% PEG4000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9792 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Jun 20, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 2.7→29.04 Å / Num. obs: 18113 / % possible obs: 97.75 % / Redundancy: 4.3 % / Biso Wilson estimate: 63.8 Å2 / CC1/2: 0.991 / CC star: 0.998 / Rmerge(I) obs: 0.1652 / Rpim(I) all: 0.08723 / Rrim(I) all: 0.1884 / Net I/σ(I): 8.87
Reflection shellResolution: 2.7→2.796 Å / Redundancy: 4.3 % / Rmerge(I) obs: 1.319 / Mean I/σ(I) obs: 1.42 / Num. unique obs: 1755 / CC1/2: 0.453 / CC star: 0.79 / Rpim(I) all: 0.6845 / Rrim(I) all: 1.497 / % possible all: 99.26

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Processing

Software
NameVersionClassification
PHENIX1.18_3855refinement
XDSdata reduction
XDSdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3ABD

3abd


Resolution: 2.7→29.04 Å / SU ML: 0.3107 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 27.6071
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2444 892 4.95 %
Rwork0.2147 17119 -
obs0.2163 18011 97.87 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 66.35 Å2
Refinement stepCycle: LAST / Resolution: 2.7→29.04 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3526 0 0 0 3526
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00243636
X-RAY DIFFRACTIONf_angle_d0.52864948
X-RAY DIFFRACTIONf_chiral_restr0.0398571
X-RAY DIFFRACTIONf_plane_restr0.0036628
X-RAY DIFFRACTIONf_dihedral_angle_d21.68611377
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.7-2.870.35031620.29382769X-RAY DIFFRACTION98.45
2.87-3.090.36551180.28562808X-RAY DIFFRACTION98.15
3.09-3.40.32251300.26192857X-RAY DIFFRACTION98.74
3.4-3.890.25141620.21372796X-RAY DIFFRACTION97.17
3.89-4.90.1881530.17272858X-RAY DIFFRACTION97.51
4.9-29.040.22851670.20483031X-RAY DIFFRACTION97.26

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