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- EMDB-36367: Cryo-EM structure of KCTD5 in complex with Gbeta gamma subunits -

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Basic information

Entry
Database: EMDB / ID: EMD-36367
TitleCryo-EM structure of KCTD5 in complex with Gbeta gamma subunits
Map dataEM density map of KCTD5_Gng2/Gnb1 complex
Sample
  • Complex: Cryo-EM structure of KCTD5 in complex with G protein betagamma subunits
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
    • Protein or peptide: BTB/POZ domain-containing protein KCTD5
Keywordsubiquitination / GPCR signaling / PROTEIN BINDING
Function / homology
Function and homology information


Cul3-RING ubiquitin ligase complex / cullin family protein binding / protein homooligomerization / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / G-protein activation ...Cul3-RING ubiquitin ligase complex / cullin family protein binding / protein homooligomerization / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / G-protein activation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / G beta:gamma signalling through BTK / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / sensory perception of taste / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / adenylate cyclase-activating dopamine receptor signaling pathway / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / GPER1 signaling / cellular response to prostaglandin E stimulus / Inactivation, recovery and regulation of the phototransduction cascade / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / GTPase binding / retina development in camera-type eye / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (i) signalling events / fibroblast proliferation / G alpha (s) signalling events / G alpha (q) signalling events / proteasome-mediated ubiquitin-dependent protein catabolic process / Ras protein signal transduction / cell population proliferation / Extra-nuclear estrogen signaling / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / synapse / protein-containing complex binding / signal transduction / extracellular exosome / membrane / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Potassium channel tetramerisation-type BTB domain / BTB/POZ domain / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / SKP1/BTB/POZ domain superfamily / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain ...Potassium channel tetramerisation-type BTB domain / BTB/POZ domain / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / SKP1/BTB/POZ domain superfamily / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / BTB/POZ domain-containing protein KCTD5
Similarity search - Component
Biological speciesMus musculus (house mouse) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.27 Å
AuthorsZheng S / Jiang W / Wang W / Kong Y
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
DepositionJun 1, 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_36367.png

Downloads & links

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Map

FileDownload / File: emd_36367.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM density map of KCTD5_Gng2/Gnb1 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 350 pix.
= 301. Å		0.86 Å/pix.
x 350 pix.
= 301. Å		0.86 Å/pix.
x 350 pix.
= 301. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.186
Minimum - Maximum-0.55195594 - 1.3624479
Average (Standard dev.)-0.0015593425 (±0.042879235)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 301.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: EM density map of KCTD5 Gng2/Gnb1 complex after sharpening

Fileemd_36367_additional_1.map
AnnotationEM density map of KCTD5_Gng2/Gnb1 complex after sharpening
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map of KCTD5 Gng2/Gnb1 complex

Fileemd_36367_half_map_1.map
Annotationhalf map of KCTD5_Gng2/Gnb1 complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map of KCTD5 Gng2/Gnb1 complex

Fileemd_36367_half_map_2.map
Annotationhalf map of KCTD5_Gng2/Gnb1 complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of KCTD5 in complex with G protein betagamma su...

EntireName: Cryo-EM structure of KCTD5 in complex with G protein betagamma subunits
Components
  • Complex: Cryo-EM structure of KCTD5 in complex with G protein betagamma subunits
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
    • Protein or peptide: BTB/POZ domain-containing protein KCTD5

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Supramolecule #1: Cryo-EM structure of KCTD5 in complex with G protein betagamma su...

SupramoleculeName: Cryo-EM structure of KCTD5 in complex with G protein betagamma subunits
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.088762 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MYPYDVPDYA YPYDVPDYAY PYDVPDYAGS GSMSELDQLR QEAEQLKNQI RDARKACADA TLSQITNNID PVGRIQMRTR RTLRGHLAK IYAMHWGTDS RLLVSASQDG KLIIWDSYTT NKVHAIPLRS SWVMTCAYAP SGNYVACGGL DNICSIYNLK T REGNVRVS ...String:
MYPYDVPDYA YPYDVPDYAY PYDVPDYAGS GSMSELDQLR QEAEQLKNQI RDARKACADA TLSQITNNID PVGRIQMRTR RTLRGHLAK IYAMHWGTDS RLLVSASQDG KLIIWDSYTT NKVHAIPLRS SWVMTCAYAP SGNYVACGGL DNICSIYNLK T REGNVRVS RELAGHTGYL SCCRFLDDNQ IVTSSGDTTC ALWDIETGQQ TTTFTGHTGD VMSLSLAPDT RLFVSGACDA SA KLWDVRE GMCRQTFTGH ESDINAICFF PNGNAFATGS DDATCRLFDL RADQELMTYS HDNIICGITS VSFSKSGRLL LAG YDDFNC NVWDALKADR AGVLAGHDNR VSCLGVTDDG MAVATGSWDS FLKIWN

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

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Macromolecule #2: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
type: protein_or_peptide / ID: 2 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 10.228731 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
GSALEVLFQG PGAGALEVLF QGPGSMASNN TASIAQARKL VEQLKMEANI DRIKVSKAAA DLMAYCEAHA KEDPLLTPVP ASENPFREK KFFSAIL

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

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Macromolecule #3: BTB/POZ domain-containing protein KCTD5

MacromoleculeName: BTB/POZ domain-containing protein KCTD5 / type: protein_or_peptide / ID: 3 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 30.780623 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGSSHHHHHH GSEDQVDPRL IDGKDYKDDD DKLEVLFQGP MAENHCELLP PAPSGLGAGL GGGLCRRCSA GMGALAQRPG GVSKWVRLN VGGTYFLTTR QTLCRDPKSF LYRLCQADPD LDSDKDETGA YLIDRDPTYF GPVLNYLRHG KLVINKDLAE E GVLEEAEF ...String:
MGSSHHHHHH GSEDQVDPRL IDGKDYKDDD DKLEVLFQGP MAENHCELLP PAPSGLGAGL GGGLCRRCSA GMGALAQRPG GVSKWVRLN VGGTYFLTTR QTLCRDPKSF LYRLCQADPD LDSDKDETGA YLIDRDPTYF GPVLNYLRHG KLVINKDLAE E GVLEEAEF YNITSLIKLV KDKIRERDSR ISQMPVKHVY RVLQCQEEEL TQMVSTMSDG WKFEQLVSIG SSYNYGNEDQ AE FLCVVSK ELHNTPYGTT SEPSEKAKIL QERGSRM

UniProtKB: BTB/POZ domain-containing protein KCTD5

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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:
ConcentrationFormulaName
25.0 mMHEPESHEPES
150.0 mMNaClSodium Chloride
GridMaterial: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec.
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 electron dose: 49.71 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2484234
Startup modelType of model: INSILICO MODEL
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 99265
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 10 / Software - Name: cryoSPARC

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Atomic model buiding 1

Initial model
PDB IDChain

3dry

source_name: PDB, initial_model_type: experimental model

6m8s

source_name: PDB, initial_model_type: experimental model
RefinementSpace: RECIPROCAL / Overall B value: 188.6
Output model

PDB-8jkb:
Cryo-EM structure of KCTD5 in complex with Gbeta gamma subunits

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