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- PDB-6ve5: X-ray structure of human REV7 in complex with Shieldin3 (residues... -

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Basic information

Entry
Database: PDB / ID: 6ve5
TitleX-ray structure of human REV7 in complex with Shieldin3 (residues 41-74)
Components
  • Mitotic spindle assembly checkpoint protein MAD2B
  • Shieldin complex subunit 3
KeywordsPROTEIN BINDING / DNA damage response / DNA double-strand break repair / DNA end resection / homologous recombination / non-homologous end joining / Shieldin3 / SHLD3 / RINN1 / REV7 / 53BP1 / RIF1
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 / positive regulation of double-strand break repair via nonhomologous end joining ...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 / 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 / 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 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 / Mitotic spindle assembly checkpoint protein MAD2B
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsCui, G. / Botuyan, M.V. / Mer, G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA132878 United States
CitationJournal: J Biol Chem / Year: 2021
Title: Cryo-EM reveals conformational flexibility in apo DNA polymerase ζ.
Authors: Chloe Du Truong / Theodore A Craig / Gaofeng Cui / Maria Victoria Botuyan / Rachel A Serkasevich / Ka-Yi Chan / Georges Mer / Po-Lin Chiu / Rajiv Kumar /
Abstract: The translesion synthesis (TLS) DNA polymerases Rev1 and Polζ function together in DNA lesion bypass during DNA replication, acting as nucleotide inserter and extender polymerases, respectively. ...The translesion synthesis (TLS) DNA polymerases Rev1 and Polζ function together in DNA lesion bypass during DNA replication, acting as nucleotide inserter and extender polymerases, respectively. While the structural characterization of the Saccharomyces cerevisiae Polζ in its DNA-bound state has illuminated how this enzyme synthesizes DNA, a mechanistic understanding of TLS also requires probing conformational changes associated with DNA- and Rev1 binding. Here, we used single-particle cryo-electron microscopy to determine the structure of the apo Polζ holoenzyme. We show that compared with its DNA-bound state, apo Polζ displays enhanced flexibility that correlates with concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. We also identified a lysine residue that obstructs the DNA-binding channel in apo Polζ, suggesting a gating mechanism. The Polζ subunit Rev7 is a hub protein that directly binds Rev1 and is a component of several other protein complexes such as the shieldin DNA double-strand break repair complex. We analyzed the molecular interactions of budding yeast Rev7 in the context of Polζ and those of human Rev7 in the context of shieldin using a crystal structure of Rev7 bound to a fragment of the shieldin-3 protein. Overall, our study provides new insights into Polζ mechanism of action and the manner in which Rev7 recognizes partner proteins.
History
DepositionDec 28, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 13, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 30, 2021Group: 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 1.2Jul 14, 2021Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Jul 28, 2021Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.4Oct 11, 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

Downloads & links

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Assembly

Deposited unit
A: Mitotic spindle assembly checkpoint protein MAD2B
B: Shieldin complex subunit 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,9875
Polymers28,6982
Non-polymers2883
Water3,315184
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: equilibrium centrifugation
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2900 Å2
ΔGint-50 kcal/mol
Surface area12040 Å2
MethodPISA
Unit cell
Length a, b, c (Å)59.990, 59.990, 132.470
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number154
Space group name H-MP3221
Space group name HallP322"
Symmetry operation#1: x,y,z
#2: -y,x-y,z+2/3
#3: -x+y,-x,z+1/3
#4: x-y,-y,-z+1/3
#5: -x,-x+y,-z+2/3
#6: y,x,-z

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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: 24630.719 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAD2L2, MAD2B, REV7 / Plasmid: pETDuet-1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9UI95
#2: Protein/peptide Shieldin complex subunit 3 / REV7-interacting novel NHEJ regulator 1 / Shield complex subunit 3


Mass: 4067.683 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SHLD3, FLJ26957, RINN1 / Plasmid: pETDuet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q6ZNX1
#3: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: SO4 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 184 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.4 Å3/Da / Density % sol: 48.7 %
Crystal growTemperature: 288 K / Method: vapor diffusion / pH: 6.5
Details: Protein complex was in 5 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM DDT at 25 mg/mL;Reservoir solution was 0.1 M MES monohydrate, pH 6.5, 1.4 M MgSO4.6H20;Cryoprotection was done in 50% PEG 400

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-BM / Wavelength: 0.9794 Å
DetectorType: ADSC QUANTUM 210r / Detector: CCD / Date: Feb 15, 2019 / Details: Sagittal focusing 2nd crystal
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9794 Å / Relative weight: 1
ReflectionResolution: 2→29.25 Å / Num. obs: 19399 / % possible obs: 98.4 % / Redundancy: 22.6 % / Biso Wilson estimate: 24.07 Å2 / CC1/2: 0.999 / CC star: 1 / Rmerge(I) obs: 0.08271 / Rpim(I) all: 0.01825 / Rrim(I) all: 0.08478 / Net I/σ(I): 40.89
Reflection shellResolution: 2→2.05 Å / Rmerge(I) obs: 1.193 / Mean I/σ(I) obs: 4.11 / Num. unique obs: 1337 / CC1/2: 0.847 / CC star: 0.958 / Rpim(I) all: 0.2621 / Rrim(I) all: 1.222

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Processing

Software
NameVersionClassification
PHENIX1.16_3549refinement
HKL-2000data reduction
HKL-2000data scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3ABD

3abd


Resolution: 2→29.25 Å / SU ML: 0.2082 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 24.0879
RfactorNum. reflection% reflection
Rfree0.2386 1919 10.01 %
Rwork0.2154 --
obs0.2178 19167 98.45 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 37.57 Å2
Refinement stepCycle: LAST / Resolution: 2→29.25 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1817 0 15 184 2016
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00421867
X-RAY DIFFRACTIONf_angle_d0.6522545
X-RAY DIFFRACTIONf_chiral_restr0.0466305
X-RAY DIFFRACTIONf_plane_restr0.0031321
X-RAY DIFFRACTIONf_dihedral_angle_d11.9345697
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2-2.050.31671320.29781207X-RAY DIFFRACTION98.89
2.05-2.10.28531340.27641198X-RAY DIFFRACTION99.55
2.1-2.160.24241400.24721247X-RAY DIFFRACTION99.64
2.16-2.230.28991320.24371213X-RAY DIFFRACTION99.04
2.23-2.310.2651330.24881232X-RAY DIFFRACTION98.56
2.31-2.40.24681340.23211195X-RAY DIFFRACTION97.29
2.4-2.510.30881340.24181218X-RAY DIFFRACTION97.83
2.51-2.650.30741320.25071187X-RAY DIFFRACTION97.13
2.65-2.810.291340.22621210X-RAY DIFFRACTION97.67
2.81-3.030.22621340.21281253X-RAY DIFFRACTION98.65
3.03-3.330.21321410.19871246X-RAY DIFFRACTION99.07
3.33-3.810.21081430.18611242X-RAY DIFFRACTION99.21
3.81-4.80.17351450.15881274X-RAY DIFFRACTION99.23
4.8-29.250.24521510.22931326X-RAY DIFFRACTION96.85
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
14.321261718230.08787217887970.1446584540831.74998266017-0.1766336116933.280688242140.1742649275960.2444852670820.163725133037-0.0142109144553-0.0356638252383-0.127787769146-0.1700044257120.23147980996-0.1248061604410.1151815890230.0322483508954-0.01849048675870.132940987092-0.01280732876740.168790752591-36.133377258925.45678690983.26106086789
26.98571574989-2.1978436732-2.917771602375.057123873161.419814720986.082518236-0.06898033391160.402042960878-0.8427018023060.3362239602290.0204550273052-0.006428314243840.8234806406750.06348321829070.0536445583580.2028806191250.0498610407329-0.1181135252650.259566886378-0.09139636567010.242812368067-30.385619807213.492639663610.1017651638
33.278906986060.391772756933-2.469912790564.16575372174-0.06662710618775.41144776180.482026895894-0.2897006921990.85189858460.200479539449-0.001127418134950.239911284853-0.60570510161-0.108530753172-0.3743431979080.195141327728-0.05016236981780.01395342183430.295324745071-0.07751257021260.326990866803-34.052432695831.896876838416.3892040659
44.52273071040.7787332713751.76905940842.814669222830.308113318633.45313796140.366807582635-0.215168122173-0.6408262074840.18101738722-0.00523480508593-0.1746339679720.5783688092550.285550738821-0.1394784160910.2215138920940.0649574184167-0.07824113644810.247431971298-0.03401176106340.259701033713-31.543640182713.846824039310.7222917724
54.888881730452.34293271009-4.010192603835.77011410341-4.559160470918.5182086095-0.009473383069230.680664573535-0.769202627155-0.079717497226-0.0132099704886-0.2603839998930.700779389274-0.1848277246130.1414981253650.3500783049870.0889585622047-0.1245072535530.232016769502-0.1494536692980.390026377345-35.34411844179.739745787810.307842488411
60.769522734819-0.132302000654-0.366061235536.983264718020.4127277184357.27140459340.04614051096260.3149050699860.403456204748-1.100433628790.381387960039-0.928127743242-0.5302297739371.566597323340.4790477353580.42994061202-0.2482309917180.07150328720960.9727604816260.1630959703220.359298359695-28.406683181126.9008893962-15.7496949042
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 7 through 76 )
2X-RAY DIFFRACTION2chain 'A' and (resid 77 through 103 )
3X-RAY DIFFRACTION3chain 'A' and (resid 104 through 135 )
4X-RAY DIFFRACTION4chain 'A' and (resid 136 through 208 )
5X-RAY DIFFRACTION5chain 'B' and (resid 48 through 61 )
6X-RAY DIFFRACTION6chain 'B' and (resid 62 through 74 )

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