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- PDB-8i79: Cryo-EM structure of KCTD7 in complex with Cullin3 -

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Basic information

Entry
Database: PDB / ID: 8i79
TitleCryo-EM structure of KCTD7 in complex with Cullin3
Components
  • BTB/POZ domain-containing protein KCTD7
  • Cullin-3
KeywordsSIGNALING PROTEIN / CUL3 / ubiquitination / E3 ligase
Function / homology
Function and homology information


positive regulation of mitotic cell cycle phase transition / trophectodermal cellular morphogenesis / liver morphogenesis / POZ domain binding / regulation protein catabolic process at postsynapse / positive regulation of transporter activity / polar microtubule / nuclear protein quality control by the ubiquitin-proteasome system / anaphase-promoting complex-dependent catabolic process / COPII vesicle coating ...positive regulation of mitotic cell cycle phase transition / trophectodermal cellular morphogenesis / liver morphogenesis / POZ domain binding / regulation protein catabolic process at postsynapse / positive regulation of transporter activity / polar microtubule / nuclear protein quality control by the ubiquitin-proteasome system / anaphase-promoting complex-dependent catabolic process / COPII vesicle coating / intracellular glutamate homeostasis / stem cell division / Neddylation / RHOBTB3 ATPase cycle / Antigen processing: Ubiquitination & Proteasome degradation / embryonic cleavage / intracellular potassium ion homeostasis / cell projection organization / positive regulation of mitotic metaphase/anaphase transition / membrane hyperpolarization / fibroblast apoptotic process / Notch binding / RHOBTB1 GTPase cycle / negative regulation of Rho protein signal transduction / mitotic metaphase chromosome alignment / negative regulation of type I interferon production / ubiquitin ligase complex scaffold activity / Cul3-RING ubiquitin ligase complex / stress fiber assembly / positive regulation of cytokinesis / protein monoubiquitination / sperm flagellum / endoplasmic reticulum to Golgi vesicle-mediated transport / RHOBTB2 GTPase cycle / protein autoubiquitination / protein K48-linked ubiquitination / gastrulation / positive regulation of TORC1 signaling / intrinsic apoptotic signaling pathway / regulation of cellular response to insulin stimulus / cyclin binding / positive regulation of protein ubiquitination / integrin-mediated signaling pathway / cellular response to amino acid stimulus / Degradation of DVL / protein destabilization / Hedgehog 'on' state / protein homooligomerization / mitotic spindle / Wnt signaling pathway / spindle pole / protein polyubiquitination / Regulation of RAS by GAPs / G1/S transition of mitotic cell cycle / ubiquitin protein ligase activity / KEAP1-NFE2L2 pathway / cell migration / Antigen processing: Ubiquitination & Proteasome degradation / Neddylation / gene expression / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / postsynapse / Potential therapeutics for SARS / protein ubiquitination / inflammatory response / centrosome / glutamatergic synapse / positive regulation of cell population proliferation / ubiquitin protein ligase binding / Golgi apparatus / negative regulation of transcription by RNA polymerase II / extracellular exosome / nucleoplasm / identical protein binding / membrane / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Potassium channel tetramerisation-type BTB domain / BTB/POZ domain / Cullin protein neddylation domain / Cullin, conserved site / Cullin family signature. / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / Cullin ...Potassium channel tetramerisation-type BTB domain / BTB/POZ domain / Cullin protein neddylation domain / Cullin, conserved site / Cullin family signature. / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin family / Cullin family profile. / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / SKP1/BTB/POZ domain superfamily / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
Cullin-3 / BTB/POZ domain-containing protein KCTD7
Similarity search - Component
Biological speciesMus musculus (house mouse)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsJiang, W. / Wang, W. / Zheng, S.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China) China
CitationJournal: Sci Adv / Year: 2023
Title: Structural basis for the ubiquitination of G protein βγ subunits by KCTD5/Cullin3 E3 ligase.
Authors: Wentong Jiang / Wei Wang / Yinfei Kong / Sanduo Zheng /
Abstract: G protein-coupled receptor (GPCR) signaling is precisely controlled to avoid overstimulation that results in detrimental consequences. Gβγ signaling is negatively regulated by a Cullin3 (Cul3)- ...G protein-coupled receptor (GPCR) signaling is precisely controlled to avoid overstimulation that results in detrimental consequences. Gβγ signaling is negatively regulated by a Cullin3 (Cul3)-dependent E3 ligase, KCTD5, which triggers ubiquitination and degradation of free Gβγ. Here, we report the cryo-electron microscopy structures of the KCTD5-Gβγ fusion complex and the KCTD7-Cul3 complex. KCTD5 in pentameric form engages symmetrically with five copies of Gβγ through its C-terminal domain. The unique pentameric assembly of the KCTD5/Cul3 E3 ligase places the ubiquitin-conjugating enzyme (E2) and the modification sites of Gβγ in close proximity and allows simultaneous transfer of ubiquitin from E2 to five Gβγ subunits. Moreover, we show that ubiquitination of Gβγ by KCTD5 is important for fine-tuning cyclic adenosine 3´,5´-monophosphate signaling of GPCRs. Our studies provide unprecedented insights into mechanisms of substrate recognition by unusual pentameric E3 ligases and highlight the KCTD family as emerging regulators of GPCR signaling.
History
DepositionJan 31, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 26, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 3, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
F: BTB/POZ domain-containing protein KCTD7
B: Cullin-3
A: BTB/POZ domain-containing protein KCTD7
C: Cullin-3
D: BTB/POZ domain-containing protein KCTD7
E: Cullin-3
G: BTB/POZ domain-containing protein KCTD7
H: Cullin-3
I: BTB/POZ domain-containing protein KCTD7
J: Cullin-3


Theoretical massNumber of molelcules
Total (without water)391,46510
Polymers391,46510
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
BTB/POZ domain-containing protein KCTD7


Mass: 34146.633 Da / Num. of mol.: 5 / Mutation: H126Y
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Kctd7 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8BJK1
#2: Protein
Cullin-3 / CUL-3


Mass: 44146.426 Da / Num. of mol.: 5 / Mutation: I342R, L346D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CUL3 / Production host: Escherichia coli (E. coli) / References: UniProt: Q13618

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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-IDSource
1complex of KCTD7 and Cullin-3COMPLEXall0RECOMBINANT
2KCTD7COMPLEX#11RECOMBINANT
3Cullin-3COMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Mus musculus (house mouse)10090
32Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
32Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
Buffer component
IDConc.NameBuffer-ID
125 mMHEPEs1
2150 mMNaCl1
SpecimenConc.: 0.32 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 500 nm
Image recordingAverage exposure time: 2.56 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
EM software
IDNameCategory
1cryoSPARCparticle selection
4cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 398489
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 115147 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00316592
ELECTRON MICROSCOPYf_angle_d0.51222569
ELECTRON MICROSCOPYf_dihedral_angle_d4.1752379
ELECTRON MICROSCOPYf_chiral_restr0.0392578
ELECTRON MICROSCOPYf_plane_restr0.0042960

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