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- PDB-7zn7: Cryo-EM structure of RCMV-E E27 bound to human DDB1 (deltaBPB) an... -

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Basic information

Entry
Database: PDB / ID: 7zn7
TitleCryo-EM structure of RCMV-E E27 bound to human DDB1 (deltaBPB) and rat STAT2 CCD
Components
  • B27a
  • DNA damage-binding protein 1
  • Signal transducer and activator of transcription
KeywordsVIRUS / Interferon / ubiquitin-proteasome system / Cullin-RING ubiquitin ligases (CRL) / DDB1 / DCAFs / viral DCAF (vDCAF) / cytomegalovirus / STAT2 / IRF9
Function / homology
Function and homology information


Interleukin-20 family signaling / Interferon alpha/beta signaling / Regulation of IFNA/IFNB signaling / ISGF3 complex / positive regulation by virus of viral protein levels in host cell / epigenetic programming in the zygotic pronuclei / spindle assembly involved in female meiosis / Cul4-RING E3 ubiquitin ligase complex / UV-damage excision repair / biological process involved in interaction with symbiont ...Interleukin-20 family signaling / Interferon alpha/beta signaling / Regulation of IFNA/IFNB signaling / ISGF3 complex / positive regulation by virus of viral protein levels in host cell / epigenetic programming in the zygotic pronuclei / spindle assembly involved in female meiosis / Cul4-RING E3 ubiquitin ligase complex / UV-damage excision repair / biological process involved in interaction with symbiont / WD40-repeat domain binding / regulation of mitotic cell cycle phase transition / Cul4A-RING E3 ubiquitin ligase complex / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / negative regulation of type I interferon-mediated signaling pathway / regulation of mitochondrial fission / type I interferon-mediated signaling pathway / negative regulation of reproductive process / negative regulation of developmental process / ubiquitin-like protein ligase binding / cullin family protein binding / cell surface receptor signaling pathway via JAK-STAT / viral release from host cell / ectopic germ cell programmed cell death / positive regulation of viral genome replication / positive regulation of gluconeogenesis / proteasomal protein catabolic process / nucleotide-excision repair / Recognition of DNA damage by PCNA-containing replication complex / regulation of protein phosphorylation / DNA Damage Recognition in GG-NER / defense response / regulation of circadian rhythm / Dual Incision in GG-NER / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / response to peptide hormone / Wnt signaling pathway / Formation of Incision Complex in GG-NER / cytokine-mediated signaling pathway / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / positive regulation of protein catabolic process / rhythmic process / cellular response to UV / protein-macromolecule adaptor activity / site of double-strand break / Neddylation / regulation of cell population proliferation / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / defense response to virus / chromosome, telomeric region / damaged DNA binding / protein ubiquitination / DNA-binding transcription factor activity, RNA polymerase II-specific / RNA polymerase II cis-regulatory region sequence-specific DNA binding / DNA repair / apoptotic process / DNA damage response / protein-containing complex binding / nucleolus / regulation of transcription by RNA polymerase II / negative regulation of apoptotic process / positive regulation of transcription by RNA polymerase II / protein-containing complex / DNA binding / extracellular space / extracellular exosome / nucleoplasm / identical protein binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
U5-like protein, herpesvirus / Herpesvirus U5-like family / Signal transducer and activation of transcription 2, C-terminal / STAT2, SH2 domain / Signal transducer and activator of transcription 2 C terminal / STAT transcription factor, DNA-binding, N-terminal / STAT transcription factor, protein interaction / STAT transcription factor, all-alpha domain / STAT transcription factor, DNA-binding / STAT protein, all-alpha domain ...U5-like protein, herpesvirus / Herpesvirus U5-like family / Signal transducer and activation of transcription 2, C-terminal / STAT2, SH2 domain / Signal transducer and activator of transcription 2 C terminal / STAT transcription factor, DNA-binding, N-terminal / STAT transcription factor, protein interaction / STAT transcription factor, all-alpha domain / STAT transcription factor, DNA-binding / STAT protein, all-alpha domain / STAT protein, DNA binding domain / STAT protein, protein interaction domain / STAT protein, protein interaction domain / STAT transcription factor, N-terminal domain superfamily / Transcription factor STAT / STAT transcription factor, coiled coil / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / Mono-functional DNA-alkylating methyl methanesulfonate N-term / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / CPSF A subunit region / p53-like transcription factor, DNA-binding / SH2 domain / Src homology 2 (SH2) domain profile. / Src homology 2 domains / SH2 domain / SH2 domain superfamily / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
B27a / DNA damage-binding protein 1 / Signal transducer and activator of transcription
Similarity search - Component
Biological speciesHomo sapiens (human)
Murid betaherpesvirus 8
Rattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.78 Å
AuthorsLauer, S. / Spahn, C.M.T. / Schwefel, D.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)SCHW 1851/1-1 Germany
CitationJournal: EMBO J / Year: 2023
Title: Structural mechanism of CRL4-instructed STAT2 degradation via a novel cytomegaloviral DCAF receptor.
Authors: Vu Thuy Khanh Le-Trilling / Sofia Banchenko / Darius Paydar / Pia Madeleine Leipe / Lukas Binting / Simon Lauer / Andrea Graziadei / Robin Klingen / Christine Gotthold / Jörg Bürger / ...Authors: Vu Thuy Khanh Le-Trilling / Sofia Banchenko / Darius Paydar / Pia Madeleine Leipe / Lukas Binting / Simon Lauer / Andrea Graziadei / Robin Klingen / Christine Gotthold / Jörg Bürger / Thilo Bracht / Barbara Sitek / Robert Jan Lebbink / Anna Malyshkina / Thorsten Mielke / Juri Rappsilber / Christian Mt Spahn / Sebastian Voigt / Mirko Trilling / David Schwefel /
Abstract: Human cytomegalovirus (CMV) is a ubiquitously distributed pathogen whose rodent counterparts such as mouse and rat CMV serve as common infection models. Here, we conducted global proteome profiling ...Human cytomegalovirus (CMV) is a ubiquitously distributed pathogen whose rodent counterparts such as mouse and rat CMV serve as common infection models. Here, we conducted global proteome profiling of rat CMV-infected cells and uncovered a pronounced loss of the transcription factor STAT2, which is crucial for antiviral interferon signalling. Via deletion mutagenesis, we found that the viral protein E27 is required for CMV-induced STAT2 depletion. Cellular and in vitro analyses showed that E27 exploits host-cell Cullin4-RING ubiquitin ligase (CRL4) complexes to induce poly-ubiquitylation and proteasomal degradation of STAT2. Cryo-electron microscopy revealed how E27 mimics molecular surface properties of cellular CRL4 substrate receptors called DCAFs (DDB1- and Cullin4-associated factors), thereby displacing them from the catalytic core of CRL4. Moreover, structural analyses showed that E27 recruits STAT2 through a bipartite binding interface, which partially overlaps with the IRF9 binding site. Structure-based mutations in M27, the murine CMV homologue of E27, impair the interferon-suppressing capacity and virus replication in mouse models, supporting the conserved importance of DCAF mimicry for CMV immune evasion.
History
DepositionApr 20, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 9, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 15, 2023Group: Database references / Refinement description
Category: citation / citation_author / pdbx_initial_refinement_model
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DNA damage-binding protein 1
B: B27a
D: Signal transducer and activator of transcription
hetero molecules


Theoretical massNumber of molelcules
Total (without water)267,2524
Polymers267,1863
Non-polymers651
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5760 Å2
ΔGint-20 kcal/mol
Surface area58200 Å2
MethodPISA

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Components

#1: Protein DNA damage-binding protein 1 / DDB p127 subunit / DNA damage-binding protein a / DDBa / Damage-specific DNA-binding protein 1 / ...DDB p127 subunit / DNA damage-binding protein a / DDBa / Damage-specific DNA-binding protein 1 / HBV X-associated protein 1 / XAP-1 / UV-damaged DNA-binding factor / UV-damaged DNA-binding protein 1 / UV-DDB 1 / XPE-binding factor / XPE-BF / Xeroderma pigmentosum group E-complementing protein / XPCe


Mass: 93671.461 Da / Num. of mol.: 1 / Mutation: delta396-705 GNGNSG
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DDB1, XAP1 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q16531
#2: Protein B27a / E27a


Mass: 76341.898 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Murid betaherpesvirus 8 / Strain: isolate England / Gene: E27a, B27a / Plasmid: pHisSUMO / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta2 / References: UniProt: K7Y9Z1
#3: Protein Signal transducer and activator of transcription


Mass: 97172.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Stat2 / Plasmid: pHisSUMO / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta2 / References: UniProt: Q5XI26
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSourceDetails
1Ternary complex of RCMV-E E27 with human DDB1 (deltaBPB) and rat STAT2COMPLEX#1-#30RECOMBINANT
2DNA damage-binding protein 1COMPLEX#11RECOMBINANTdelta396-705 GNGNSG
3E27COMPLEX#21RECOMBINANT
4Signal transducer and activator of transcriptionCOMPLEX#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.267 MDaNO
210.1 MDaNO
310.1 MDaNO
410.1 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Murid betaherpesvirus 81261657
32Homo sapiens (human)9606
43Murid betaherpesvirus 81261657
54Rattus norvegicus (Norway rat)10116
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainCell
21Escherichia coli BL21(DE3) (bacteria)469008Rosetta2(DE3)
32Spodoptera frugiperda (fall armyworm)7108Sf9
43Escherichia coli BL21(DE3) (bacteria)469008Rosetta2(DE3)
54Escherichia coli BL21(DE3) (bacteria)469008Rosetta2(DE3)
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTris HydrochlorideTris-HClTris1
2150 mMSodium ChlorideNaClSodium chloride1
34 mMMagnesium ChlorideMgCl21
40.5 mMTCEPTCEP1
SpecimenConc.: 0.13 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 99 % / Chamber temperature: 277.15 K / Details: 45 seconds adsorption 2 seconds blot

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 31000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2 mm
Image recordingAverage exposure time: 10 sec. / Electron dose: 62 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 4 / Num. of real images: 8069
Image scansMovie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2Leginonimage acquisition
4CTFFIND4CTF correction
7Coot0.9.5model fitting
9RELION3.0.7initial Euler assignment
10RELION3.0.7final Euler assignment
11RELION3.0.7classification
12cryoSPARC3.13D reconstruction
13PHENIX1.19.2-4158model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 974291
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 389784 / Algorithm: FOURIER SPACE / Details: non-uniform refinement / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 112 / Protocol: FLEXIBLE FIT / Space: REAL
Target criteria: Fast gradient-driven minimization of combined map and restraints target
Atomic model buildingPDB-ID: 6ZUE

6zue

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00610941
ELECTRON MICROSCOPYf_angle_d1.57214768
ELECTRON MICROSCOPYf_dihedral_angle_d7.091467
ELECTRON MICROSCOPYf_chiral_restr0.0631675
ELECTRON MICROSCOPYf_plane_restr0.0121903

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