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- PDB-6bi7: Crystal structure of Rev7-WT/Rev3 as a monomer under high-salt co... -

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Basic information

Entry
Database: PDB / ID: 6bi7
TitleCrystal structure of Rev7-WT/Rev3 as a monomer under high-salt conditions
Components
  • DNA polymerase zeta catalytic subunit
  • Mitotic spindle assembly checkpoint protein MAD2B
KeywordsREPLICATION / DNA damage tolerance / translesion DNA synthesis
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 transcription by competitive promoter binding / negative regulation of cell-cell adhesion mediated by cadherin / 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 / 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 / negative regulation of epithelial to mesenchymal transition / JUN kinase binding / negative regulation of ubiquitin protein ligase activity / mitotic spindle assembly checkpoint signaling / positive regulation of double-strand break repair via nonhomologous end joining / 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 / actin filament organization / regulation of cell growth / double-strand break repair via homologous recombination / negative regulation of canonical Wnt signaling pathway / negative regulation of protein catabolic process / negative regulation of DNA-binding transcription factor activity / DNA-templated DNA replication / spindle / double-strand break repair / positive regulation of peptidyl-serine phosphorylation / site of double-strand break / chromosome / 4 iron, 4 sulfur cluster binding / RNA polymerase II-specific DNA-binding transcription factor binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / cell division / nucleotide binding / chromatin / nucleolus / positive regulation of DNA-templated transcription / negative regulation of transcription by RNA polymerase II / DNA binding / nucleoplasm / metal ion binding / nucleus / cytoplasm
Similarity search - Function
Domain of unknown function DUF4683 / Domain of unknown function (DUF4683) / DNA polymerase zeta catalytic subunit / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta / Mad2-like / HORMA domain / HORMA domain / HORMA domain profile. / HORMA domain superfamily ...Domain of unknown function DUF4683 / Domain of unknown function (DUF4683) / DNA polymerase zeta catalytic subunit / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta / Mad2-like / HORMA domain / HORMA domain / HORMA domain profile. / HORMA domain superfamily / DNA polymerase family B, thumb domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA polymerase zeta catalytic subunit / Mitotic spindle assembly checkpoint protein MAD2B
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.8 Å
AuthorsRizzo, A.A. / Korzhnev, D.M. / Hao, B. / Li, Y.
Funding support United States, 8items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)1615866 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM123239 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)R01-ES015818 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)P30-ES002109 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM099948 United States
Connecticut Regenerative Medicine Research Fund15-RMA- UCHC-03 United States
National Science Foundation (NSF, United States)DMR-1332208 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2018
Title: Rev7 dimerization is important for assembly and function of the Rev1/Polζ translesion synthesis complex.
Authors: Alessandro A Rizzo / Faye-Marie Vassel / Nimrat Chatterjee / Sanjay D'Souza / Yunfeng Li / Bing Hao / Michael T Hemann / Graham C Walker / Dmitry M Korzhnev /
Abstract: The translesion synthesis (TLS) polymerases Polζ and Rev1 form a complex that enables replication of damaged DNA. The Rev7 subunit of Polζ, which is a multifaceted HORMA (Hop1, Rev7, Mad2) protein ...The translesion synthesis (TLS) polymerases Polζ and Rev1 form a complex that enables replication of damaged DNA. The Rev7 subunit of Polζ, which is a multifaceted HORMA (Hop1, Rev7, Mad2) protein with roles in TLS, DNA repair, and cell-cycle control, facilitates assembly of this complex by binding Rev1 and the catalytic subunit of Polζ, Rev3. Rev7 interacts with Rev3 by a mechanism conserved among HORMA proteins, whereby an open-to-closed transition locks the ligand underneath the "safety belt" loop. Dimerization of HORMA proteins promotes binding and release of this ligand, as exemplified by the Rev7 homolog, Mad2. Here, we investigate the dimerization of Rev7 when bound to the two Rev7-binding motifs (RBMs) in Rev3 by combining in vitro analyses of Rev7 structure and interactions with a functional assay in a Rev7 cell line. We demonstrate that Rev7 uses the conventional HORMA dimerization interface both to form a homodimer when tethered by the two RBMs in Rev3 and to heterodimerize with other HORMA domains, Mad2 and p31 Structurally, the Rev7 dimer can bind only one copy of Rev1, revealing an unexpected Rev1/Polζ architecture. In cells, mutation of the Rev7 dimer interface increases sensitivity to DNA damage. These results provide insights into the structure of the Rev1/Polζ TLS assembly and highlight the function of Rev7 homo- and heterodimerization.
History
DepositionNov 1, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 1, 2018Provider: repository / Type: Initial release
Revision 1.1Aug 22, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Aug 29, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.pdbx_database_id_PubMed ..._citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.3Sep 5, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.4Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Oct 4, 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
B: DNA polymerase zeta catalytic subunit
C: Mitotic spindle assembly checkpoint protein MAD2B
D: DNA polymerase zeta catalytic subunit
E: Mitotic spindle assembly checkpoint protein MAD2B
F: DNA polymerase zeta catalytic subunit
G: Mitotic spindle assembly checkpoint protein MAD2B
H: DNA polymerase zeta catalytic subunit


Theoretical massNumber of molelcules
Total (without water)117,8618
Polymers117,8618
Non-polymers00
Water23413
1
A: Mitotic spindle assembly checkpoint protein MAD2B
B: DNA polymerase zeta catalytic subunit


  • defined by software
  • Evidence: SAXS, Supports formation the Rev7 dimer, mass spectrometry, Coelution and subsequent LC-MS/MS confirms the interaction between Rev7 and Rev3, equilibrium centrifugation, Supports the formation of the Rev7 dimer
  • 29.5 kDa, 2 polymers
  • Search similar-shape structures of this assembly by Omokage search (details)
Theoretical massNumber of molelcules
Total (without water)29,4652
Polymers29,4652
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2680 Å2
ΔGint-22 kcal/mol
Surface area11000 Å2
MethodPISA
2
C: Mitotic spindle assembly checkpoint protein MAD2B
D: DNA polymerase zeta catalytic subunit


Theoretical massNumber of molelcules
Total (without water)29,4652
Polymers29,4652
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2720 Å2
ΔGint-21 kcal/mol
Surface area11210 Å2
MethodPISA
3
E: Mitotic spindle assembly checkpoint protein MAD2B
F: DNA polymerase zeta catalytic subunit


Theoretical massNumber of molelcules
Total (without water)29,4652
Polymers29,4652
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2280 Å2
ΔGint-21 kcal/mol
Surface area10680 Å2
MethodPISA
4
G: Mitotic spindle assembly checkpoint protein MAD2B
H: DNA polymerase zeta catalytic subunit


Theoretical massNumber of molelcules
Total (without water)29,4652
Polymers29,4652
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2210 Å2
ΔGint-20 kcal/mol
Surface area9500 Å2
MethodPISA
Unit cell
Length a, b, c (Å)89.696, 89.696, 284.841
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number152
Space group name H-MP3121

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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: 26187.352 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAD2L2, MAD2B, REV7 / Plasmid: pETDuet / Details (production host): MCS1 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 / Variant (production host): DE3 / References: UniProt: Q9UI95
#2: Protein/peptide
DNA polymerase zeta catalytic subunit / Protein reversionless 3-like / hREV3


Mass: 3278.004 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: codon-optimized gBlock from IDT / Source: (gene. exp.) Homo sapiens (human) / Gene: REV3L, POLZ, REV3 / Plasmid: pETDuet / Details (production host): MCS2 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 / Variant (production host): DE3 / References: UniProt: O60673, DNA-directed DNA polymerase
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 13 / Source method: isolated from a natural source / Formula: H2O

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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: 2.81 Å3/Da / Density % sol: 56.17 %
Crystal growTemperature: 289.15 K / Method: vapor diffusion, hanging drop
Details: hRev7/3 at 60 mg/mL in 5 mM HEPES, 100 mM NaCl, 10 mM DTT, pH=7.4 was mixed in a 1:1 ratio with a well solution consisting of 100 mM sodium citrate, 1M LiCl, 7.5% (w/v) PEG6000 at pH=4.75. ...Details: hRev7/3 at 60 mg/mL in 5 mM HEPES, 100 mM NaCl, 10 mM DTT, pH=7.4 was mixed in a 1:1 ratio with a well solution consisting of 100 mM sodium citrate, 1M LiCl, 7.5% (w/v) PEG6000 at pH=4.75. Crystals were frozen in the reservoir solution with the addition of 20% (w/v) sucrose

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Data collection

DiffractionMean temperature: 77 K
Diffraction sourceSource: SYNCHROTRON / Site: CHESS / Beamline: F1 / Wavelength: 0.976 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Mar 3, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976 Å / Relative weight: 1
ReflectionResolution: 2.8→284.84 Å / Num. obs: 33727 / % possible obs: 99.8 % / Redundancy: 9.6 % / Rpim(I) all: 0.044 / Net I/σ(I): 21.1
Reflection shellResolution: 2.8→2.85 Å / Redundancy: 9.5 % / Mean I/σ(I) obs: 2.2 / Num. unique obs: 1631 / CC1/2: 0.946 / Rpim(I) all: 0.168 / % possible all: 99.9

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Processing

Software
NameClassification
REFMACrefinement
HKL-2000data processing
DENZOdata reduction
SCALEPACKdata scaling
PHASERphasing
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6BC8

6bc8


Resolution: 2.8→284.84 Å / Cor.coef. Fo:Fc: 0.909 / Cor.coef. Fo:Fc free: 0.858 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.8 / ESU R Free: 0.411
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.3213 1715 5.1 %RANDOM
Rwork0.2754 ---
obs0.2778 33727 99.75 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 239.78 Å2 / Biso mean: 94.6819 Å2 / Biso min: 34.11 Å2
Baniso -1Baniso -2Baniso -3
1--3.5 Å2-1.75 Å20 Å2
2---3.5 Å20 Å2
3---11.36 Å2
Refinement stepCycle: LAST / Resolution: 2.8→284.84 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6426 0 0 13 6439
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0110.0196558
X-RAY DIFFRACTIONr_bond_other_d00.026614
X-RAY DIFFRACTIONr_angle_refined_deg1.5271.9748883
X-RAY DIFFRACTIONr_angle_other_deg3.518315235
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.6175765
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.70124.913289
X-RAY DIFFRACTIONr_dihedral_angle_3_deg17.212151226
X-RAY DIFFRACTIONr_dihedral_angle_4_deg22.3021528
X-RAY DIFFRACTIONr_chiral_restr0.0880.21060
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.0217005
X-RAY DIFFRACTIONr_gen_planes_other0.0060.021375
X-RAY DIFFRACTIONr_mcbond_it7.6579.1583129
X-RAY DIFFRACTIONr_mcbond_other7.6569.1563128
X-RAY DIFFRACTIONr_mcangle_it11.89513.6873868
LS refinement shellResolution: 2.798→2.871 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.355 111 -
Rwork0.391 2319 -
all-2430 -
obs--99.67 %

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