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- EMDB-35212: Cryo-EM structure of KCTD7 in complex with Cullin3 -

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Basic information

Entry
Database: EMDB / ID: EMD-35212
TitleCryo-EM structure of KCTD7 in complex with Cullin3
Map datacryoEM map of KCTD7/CUL3
Sample
  • Complex: complex of KCTD7 and Cullin-3
    • Complex: KCTD7
      • Protein or peptide: BTB/POZ domain-containing protein KCTD7
    • Complex: Cullin-3
      • Protein or peptide: Cullin-3
KeywordsCUL3 / ubiquitination / E3 ligase / SIGNALING PROTEIN
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 / Notch binding / RHOBTB1 GTPase cycle / fibroblast apoptotic process / negative regulation of Rho protein signal transduction / mitotic metaphase chromosome alignment / negative regulation of type I interferon production / Cul3-RING ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / 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 / regulation of cellular response to insulin stimulus / cyclin binding / intrinsic apoptotic signaling pathway / positive regulation of protein ubiquitination / integrin-mediated signaling pathway / Degradation of DVL / cellular response to amino acid stimulus / Hedgehog 'on' state / protein destabilization / 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 / ubiquitin protein ligase binding / positive regulation of cell population proliferation / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsJiang W / Wang W / Zheng S
Funding support China, 1 items
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, 2023-
Header (metadata) releaseJul 26, 2023-
Map releaseJul 26, 2023-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35212.png

Downloads & links

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Map

FileDownload / File: emd_35212.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationcryoEM map of KCTD7/CUL3
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 300 pix.
= 326.1 Å		1.09 Å/pix.
x 300 pix.
= 326.1 Å		1.09 Å/pix.
x 300 pix.
= 326.1 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.087 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.275
Minimum - Maximum-0.4003876 - 1.247889
Average (Standard dev.)-0.0009664841 (±0.036459763)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 326.1 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: cryoEM map of KCTD7/CUL3 after map sharpening

Fileemd_35212_additional_1.map
AnnotationcryoEM map of KCTD7/CUL3 after map sharpening
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map of KCTD7/CUL3

Fileemd_35212_half_map_1.map
Annotationhalf map of KCTD7/CUL3
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map of KCTD7/CUL3

Fileemd_35212_half_map_2.map
Annotationhalf map of KCTD7/CUL3
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : complex of KCTD7 and Cullin-3

EntireName: complex of KCTD7 and Cullin-3
Components
  • Complex: complex of KCTD7 and Cullin-3
    • Complex: KCTD7
      • Protein or peptide: BTB/POZ domain-containing protein KCTD7
    • Complex: Cullin-3
      • Protein or peptide: Cullin-3

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Supramolecule #1: complex of KCTD7 and Cullin-3

SupramoleculeName: complex of KCTD7 and Cullin-3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #2: KCTD7

SupramoleculeName: KCTD7 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Cullin-3

SupramoleculeName: Cullin-3 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2

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

MacromoleculeName: BTB/POZ domain-containing protein KCTD7 / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 34.146633 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DYKDDDDKMV VVTGREPDSR HSDGAMSSSE AEDDFLEPAT PTATQAGHGL PLLPQEFPEV VPLNIGGAHF TTRLSTLRRY EDTMLAAMF SGRHYIPTDS EGRYFIDRDG THFGDVLNFL RSGDLPPREH VRAVYKEAQY YAIGPLLEQL ENMQPLKGEK V RQAFLGLM ...String:
DYKDDDDKMV VVTGREPDSR HSDGAMSSSE AEDDFLEPAT PTATQAGHGL PLLPQEFPEV VPLNIGGAHF TTRLSTLRRY EDTMLAAMF SGRHYIPTDS EGRYFIDRDG THFGDVLNFL RSGDLPPREH VRAVYKEAQY YAIGPLLEQL ENMQPLKGEK V RQAFLGLM PYYKDHLERI VEIARLRAVQ RKARFAKLKV CVFKEEMPIT PYECPLLNSL RFERSESDGQ LFEHHCEVDV SF GPWEAVA DVYDLLHCLV TDLSAQGLTV DHQCIGVCDK HLVNHYYCKR PIYEFKITWW

UniProtKB: BTB/POZ domain-containing protein KCTD7

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Macromolecule #2: Cullin-3

MacromoleculeName: Cullin-3 / type: protein_or_peptide / ID: 2 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.146426 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MHHHHHHGSP MTMDEKYVNS IWDLLKNAIQ EIQRKNNSGL SFEELYRNAY TMVLHKHGEK LYTGLREVVT EHLINKVRED VLNSLNNNF LQTLNQAWND HQTAMVMIRD ILMYMDRVYV QQNNVENVYN LGLIIFRDQV VRYGCIRDHL RQTLLDMIAR E RKGEVVDR ...String:
MHHHHHHGSP MTMDEKYVNS IWDLLKNAIQ EIQRKNNSGL SFEELYRNAY TMVLHKHGEK LYTGLREVVT EHLINKVRED VLNSLNNNF LQTLNQAWND HQTAMVMIRD ILMYMDRVYV QQNNVENVYN LGLIIFRDQV VRYGCIRDHL RQTLLDMIAR E RKGEVVDR GAIRNACQML MILGLEGRSV YEEDFEAPFL EMSAEFFQME SQKFLAENSA SVYIKKVEAR INEEIERVMH CL DKSTEEP IVKVVERELI SKHMKTIVEM ENSGLVHMLK NGKTEDLGCM YKLFSRVPNG LKTMCECMSS YLREQGKALV SEE GEGKNP VDYRQGLDDL KSRFDRFLLE SFNNDRLFKQ TIAGDFEYFL NLNSRSPEYL

UniProtKB: Cullin-3

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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

Concentration0.32 mg/mL
BufferpH: 7.4 / Component:
ConcentrationName
25.0 mMHEPEs
150.0 mMNaCl
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 2.56 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 398489
Startup modelType of model: INSILICO MODEL
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 115147
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC

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