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- PDB-6wwa: Crystal structure of human SHLD2-SHLD3-REV7 complex -

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Basic information

Entry
Database: PDB / ID: 6wwa
TitleCrystal structure of human SHLD2-SHLD3-REV7 complex
Components
  • Mitotic spindle assembly checkpoint protein MAD2B
  • Shieldin complex subunit 2,Shieldin complex subunit 3 chimera
KeywordsNUCLEAR PROTEIN / REV7 / SHLD2 / SHLD3
Function / homology
Function and homology information


somatic diversification of immunoglobulins involved in immune response / DNA damage response, signal transduction resulting in transcription / zeta DNA polymerase complex / positive regulation of isotype switching / negative regulation of transcription by competitive promoter binding / negative regulation of cell-cell adhesion mediated by cadherin / JUN kinase binding / negative regulation of epithelial to mesenchymal transition / negative regulation of ubiquitin protein ligase activity / regulation of double-strand break repair via homologous recombination ...somatic diversification of immunoglobulins involved in immune response / DNA damage response, signal transduction resulting in transcription / zeta DNA polymerase complex / positive regulation of isotype switching / negative regulation of transcription by competitive promoter binding / negative regulation of cell-cell adhesion mediated by cadherin / JUN kinase binding / negative regulation of epithelial to mesenchymal transition / negative regulation of ubiquitin protein ligase activity / regulation of double-strand break repair via homologous recombination / positive regulation of double-strand break repair via nonhomologous end joining / mitotic spindle assembly checkpoint signaling / telomere maintenance in response to DNA damage / positive regulation of peptidyl-serine phosphorylation / error-prone translesion synthesis / negative regulation of double-strand break repair via homologous recombination / actin filament organization / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / regulation of cell growth / negative regulation of canonical Wnt signaling pathway / negative regulation of protein catabolic process / spindle / transcription corepressor activity / double-strand break repair / actin cytoskeleton / chromosome / site of double-strand break / RNA polymerase II-specific DNA-binding transcription factor binding / cell division / DNA repair / chromatin / positive regulation of DNA-templated transcription / nucleolus / negative regulation of transcription by RNA polymerase II / nucleoplasm / 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 / Shieldin complex subunit 2, second OB fold domain / Shieldin complex subunit 3 / Cell Cycle, Spindle Assembly Checkpoint Protein; Chain A / 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 / Shieldin complex subunit 2, second OB fold domain / Shieldin complex subunit 3 / Cell Cycle, Spindle Assembly Checkpoint Protein; Chain A / HORMA domain / Mad2-like / HORMA domain / HORMA domain / HORMA domain profile. / HORMA domain superfamily / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Shieldin complex subunit 3 / Shieldin complex subunit 2 / Mitotic spindle assembly checkpoint protein MAD2B
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.8 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
D: Mitotic spindle assembly checkpoint protein MAD2B
C: Mitotic spindle assembly checkpoint protein MAD2B
B: 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


Theoretical massNumber of molelcules
Total (without water)118,6506
Polymers118,6506
Non-polymers00
Water00
1
A: Mitotic spindle assembly checkpoint protein MAD2B
B: Mitotic spindle assembly checkpoint protein MAD2B
X: Shieldin complex subunit 2,Shieldin complex subunit 3 chimera


Theoretical massNumber of molelcules
Total (without water)59,3253
Polymers59,3253
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7550 Å2
ΔGint-40 kcal/mol
Surface area21730 Å2
MethodPISA
2
D: Mitotic spindle assembly checkpoint protein MAD2B
C: Mitotic spindle assembly checkpoint protein MAD2B
Y: Shieldin complex subunit 2,Shieldin complex subunit 3 chimera


Theoretical massNumber of molelcules
Total (without water)59,3253
Polymers59,3253
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7700 Å2
ΔGint-44 kcal/mol
Surface area21510 Å2
MethodPISA
Unit cell
Length a, b, c (Å)331.842, 331.842, 331.842
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number211
Space group name H-MI432
Space group name HallI423
Symmetry operation#1: x,y,z
#2: x,-z,y
#3: x,z,-y
#4: z,y,-x
#5: -z,y,x
#6: -y,x,z
#7: y,-x,z
#8: z,x,y
#9: y,z,x
#10: -y,-z,x
#11: z,-x,-y
#12: -y,z,-x
#13: -z,-x,y
#14: -z,x,-y
#15: y,-z,-x
#16: x,-y,-z
#17: -x,y,-z
#18: -x,-y,z
#19: y,x,-z
#20: -y,-x,-z
#21: z,-y,x
#22: -z,-y,-x
#23: -x,z,y
#24: -x,-z,-y
#25: x+1/2,y+1/2,z+1/2
#26: x+1/2,-z+1/2,y+1/2
#27: x+1/2,z+1/2,-y+1/2
#28: z+1/2,y+1/2,-x+1/2
#29: -z+1/2,y+1/2,x+1/2
#30: -y+1/2,x+1/2,z+1/2
#31: y+1/2,-x+1/2,z+1/2
#32: z+1/2,x+1/2,y+1/2
#33: y+1/2,z+1/2,x+1/2
#34: -y+1/2,-z+1/2,x+1/2
#35: z+1/2,-x+1/2,-y+1/2
#36: -y+1/2,z+1/2,-x+1/2
#37: -z+1/2,-x+1/2,y+1/2
#38: -z+1/2,x+1/2,-y+1/2
#39: y+1/2,-z+1/2,-x+1/2
#40: x+1/2,-y+1/2,-z+1/2
#41: -x+1/2,y+1/2,-z+1/2
#42: -x+1/2,-y+1/2,z+1/2
#43: y+1/2,x+1/2,-z+1/2
#44: -y+1/2,-x+1/2,-z+1/2
#45: z+1/2,-y+1/2,x+1/2
#46: -z+1/2,-y+1/2,-x+1/2
#47: -x+1/2,z+1/2,y+1/2
#48: -x+1/2,-z+1/2,-y+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: 24323.348 Da / Num. of mol.: 4
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 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
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) / Strain (production host): BL21(DE3) / References: UniProt: Q86V20, 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: 6.69 Å3/Da / Density % sol: 81.6 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 5.6 / Details: 0.1 M Na acetate pH 5.6, 1.5 M Na formate

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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: PSI PILATUS 6M / Detector: PIXEL / Date: Dec 16, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 3.8→40 Å / Num. obs: 30856 / % possible obs: 99.74 % / Redundancy: 35.8 % / Biso Wilson estimate: 153.27 Å2 / CC1/2: 0.999 / CC star: 1 / Rmerge(I) obs: 0.4031 / Rpim(I) all: 0.06797 / Rrim(I) all: 0.4089 / Net I/σ(I): 10.3
Reflection shellResolution: 3.8→3.936 Å / Rmerge(I) obs: 4.971 / Mean I/σ(I) obs: 0.86 / Num. unique obs: 3027 / CC1/2: 0.472 / CC star: 0.801 / Rpim(I) all: 0.8316 / Rrim(I) all: 5.04

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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: 3.8→39.66 Å / SU ML: 0.5072 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 29.5099
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2663 1548 5.02 %
Rwork0.239 29276 -
obs0.2403 30824 99.89 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 189.45 Å2
Refinement stepCycle: LAST / Resolution: 3.8→39.66 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6776 0 0 0 6776
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0036940
X-RAY DIFFRACTIONf_angle_d0.69719438
X-RAY DIFFRACTIONf_chiral_restr0.0421102
X-RAY DIFFRACTIONf_plane_restr0.00511196
X-RAY DIFFRACTIONf_dihedral_angle_d21.65122626
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.8-3.920.371420.33552589X-RAY DIFFRACTION99.38
3.92-4.060.32231320.30662621X-RAY DIFFRACTION99.85
4.06-4.230.32541250.27732644X-RAY DIFFRACTION99.93
4.23-4.420.21671560.21722598X-RAY DIFFRACTION99.96
4.42-4.650.19551210.19192659X-RAY DIFFRACTION99.93
4.65-4.940.22151440.20092629X-RAY DIFFRACTION100
4.94-5.320.26751400.22822650X-RAY DIFFRACTION99.96
5.32-5.860.31161580.26272643X-RAY DIFFRACTION100
5.86-6.70.34211300.30392691X-RAY DIFFRACTION100
6.7-8.420.28081410.26332706X-RAY DIFFRACTION100
8.43-39.660.24211590.20812846X-RAY DIFFRACTION99.8
Refinement TLS params.Method: refined / Origin x: 109.905339802 Å / Origin y: 14.6069753103 Å / Origin z: 133.372128478 Å
111213212223313233
T0.783800293968 Å2-0.144711256971 Å20.0277986503535 Å2-1.964971193 Å2-0.0623853649043 Å2--1.39264665562 Å2
L6.21651019976 °21.62521692342 °20.689130812605 °2-0.671332053057 °20.0436430537042 °2--0.0430921510909 °2
S-0.149704739706 Å °0.781135836434 Å °-0.362771177164 Å °0.0708683020881 Å °0.263042463886 Å °0.379124772079 Å °0.034652451947 Å °0.279437900623 Å °-0.0752398197091 Å °
Refinement TLS groupSelection details: all

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