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- EMDB-23244: Structure of human SHLD2-SHLD3-REV7-TRIP13(E253Q) complex -

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Basic information

Entry
Database: EMDB / ID: EMD-23244
TitleStructure of human SHLD2-SHLD3-REV7-TRIP13(E253Q) complex
Map datacomposite map
Sample
  • Complex: SHLD2.3-REV7(4)-TRIP13(E253Q) complex with ATP-gamma-S
    • Protein or peptide: Pachytene checkpoint protein 2 homolog
    • Protein or peptide: Mitotic spindle assembly checkpoint protein MAD2B
    • Protein or peptide: Shieldin complex subunit 2, Shieldin complex subunit 3 chimera
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
KeywordsREV7 / SHLD2 / SHLD3 / TRIP13 / NUCLEAR PROTEIN
Function / homology
Function and homology information


somatic diversification of immunoglobulins involved in immune response / DNA damage response, signal transduction resulting in transcription / negative regulation of transcription regulatory region DNA binding / meiotic recombination checkpoint signaling / zeta DNA polymerase complex / synaptonemal complex assembly / positive regulation of isotype switching / positive regulation of extracellular matrix assembly / 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 / negative regulation of transcription regulatory region DNA binding / meiotic recombination checkpoint signaling / zeta DNA polymerase complex / synaptonemal complex assembly / positive regulation of isotype switching / positive regulation of extracellular matrix assembly / negative regulation of transcription by competitive promoter binding / negative regulation of cell-cell adhesion mediated by cadherin / JUN kinase binding / reciprocal meiotic recombination / oocyte maturation / female meiosis I / negative regulation of epithelial to mesenchymal transition / positive regulation of double-strand break repair via nonhomologous end joining / negative regulation of ubiquitin protein ligase activity / regulation of double-strand break repair via homologous recombination / oogenesis / mitotic spindle assembly checkpoint signaling / male meiosis I / telomere maintenance in response to DNA damage / spermatid development / negative regulation of double-strand break repair via homologous recombination / error-prone translesion synthesis / positive regulation of epithelial to mesenchymal transition / 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 / positive regulation of peptidyl-serine phosphorylation / site of double-strand break / 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 / Shieldin complex subunit 2, second OB fold domain / Pachytene checkpoint protein 2-like / Shieldin complex subunit 3 / Mad2-like ...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 / 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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsXie W / Patel DJ
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, 2021-
Header (metadata) releaseMar 3, 2021-
Map releaseMar 3, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 4
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7l9p
  • Surface level: 4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23244.png

Downloads & links

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Map

FileDownload / File: emd_23244.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcomposite map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.06 Å/pix.
x 240 pix.
= 255.36 Å		1.06 Å/pix.
x 240 pix.
= 255.36 Å		1.06 Å/pix.
x 240 pix.
= 255.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.064 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.0 / Movie #1: 4
Minimum - Maximum-21.230463 - 36.671756999999999
Average (Standard dev.)0.0020590862 (±1.0343122)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 255.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0641.0641.064
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z255.360255.360255.360
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ240240240
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-21.23036.6720.002

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Supplemental data

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Sample components

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Entire : SHLD2.3-REV7(4)-TRIP13(E253Q) complex with ATP-gamma-S

EntireName: SHLD2.3-REV7(4)-TRIP13(E253Q) complex with ATP-gamma-S
Components
  • Complex: SHLD2.3-REV7(4)-TRIP13(E253Q) complex with ATP-gamma-S
    • Protein or peptide: Pachytene checkpoint protein 2 homolog
    • Protein or peptide: Mitotic spindle assembly checkpoint protein MAD2B
    • Protein or peptide: Shieldin complex subunit 2, Shieldin complex subunit 3 chimera
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

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Supramolecule #1: SHLD2.3-REV7(4)-TRIP13(E253Q) complex with ATP-gamma-S

SupramoleculeName: SHLD2.3-REV7(4)-TRIP13(E253Q) complex with ATP-gamma-S
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Pachytene checkpoint protein 2 homolog

MacromoleculeName: Pachytene checkpoint protein 2 homolog / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 48.562547 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SDEAVGDLKQ ALPCVAESPT VHVEVHQRGS STAKKEDINL SVRKLLNRHN IVFGDYTWTE FDEPFLTRNV QSVSIIDTEL KVKDSQPID LSACTVALHI FQLNEDGPSS ENLEEETENI IAANHWVLPA AEFHGLWDSL VYDVEVKSHL LDYVMTTLLF S DKNVNSNL ...String:
SDEAVGDLKQ ALPCVAESPT VHVEVHQRGS STAKKEDINL SVRKLLNRHN IVFGDYTWTE FDEPFLTRNV QSVSIIDTEL KVKDSQPID LSACTVALHI FQLNEDGPSS ENLEEETENI IAANHWVLPA AEFHGLWDSL VYDVEVKSHL LDYVMTTLLF S DKNVNSNL ITWNRVVLLH GPPGTGKTSL CKALAQKLTI RLSSRYRYGQ LIEINSHSLF SKWFSESGKL VTKMFQKIQD LI DDKDALV FVLIDQVESL TAARNACRAG TEPSDAIRVV NAVLTQIDQI KRHSNVVILT TSNITEKIDV AFVDRADIKQ YIG PPSAAA IFKIYLSCLE ELMKCQIIYP RQQLLTLREL EMIGFIENNV SKLSLLLNDI SRKSEGLSGR VLRKLPFLAH ALYV QAPTV TIEGFLQALS LAVDKQFEER KKLAAYI

UniProtKB: Pachytene checkpoint protein 2 homolog

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Macromolecule #2: Mitotic spindle assembly checkpoint protein MAD2B

MacromoleculeName: Mitotic spindle assembly checkpoint protein MAD2B / type: protein_or_peptide / ID: 2 / Details: closed form / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.323348 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: STTLTRQDLN FGQVVADVLC EFLEVAVHLI LYVREVYPVG IFQKRKKYNV PVQMSCHPEL NQYIQDTLHC VKPLLEKNDV EKVVVVILD KEHRPVEKFV FEITQPPLLS ISSDSLLSHV EQLLRAFILK ISVCDAVLDH NPPGCTFTVL VHTREAATRN M EKIQVIKD ...String:
STTLTRQDLN FGQVVADVLC EFLEVAVHLI LYVREVYPVG IFQKRKKYNV PVQMSCHPEL NQYIQDTLHC VKPLLEKNDV EKVVVVILD KEHRPVEKFV FEITQPPLLS ISSDSLLSHV EQLLRAFILK ISVCDAVLDH NPPGCTFTVL VHTREAATRN M EKIQVIKD FPWILADEQD VHMHDPRLIP LKTMTSDILK MQLYVEERAH KGS

UniProtKB: Mitotic spindle assembly checkpoint protein MAD2B

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Macromolecule #3: Shieldin complex subunit 2, Shieldin complex subunit 3 chimera

MacromoleculeName: Shieldin complex subunit 2, Shieldin complex subunit 3 chimera
type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 10.678139 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSQVHIFWGA PIAPLKGSGS GSGSGSGSGS GSTTEVILHY RPCESDPTQL PKIAEKAIQD FPTRPLSRFI PWFPYDGSKL PLRPKRSPP ASREEIMATL

UniProtKB: Shieldin complex subunit 2, Shieldin complex subunit 3

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Macromolecule #4: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 5 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 7.3
Details: 20 mM HEPES, pH 7.3, 300 mM NaCl, 5 mM MgCl2, 0.1 mM ATP-gamma-S, 1 mM DTT
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 1.5-second blot, blot force of 0.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 40 / Average exposure time: 0.075 sec. / Average electron dose: 53.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: -2.5 µm / Nominal defocus min: -1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6f0x


Details: TRIP13-p31-substrate model
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 104023
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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