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- PDB-8k9i: Structure of CUL3-RBX1-KLHL22 complex without CUL3 NA motif -

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Basic information

Entry
Database: PDB / ID: 8k9i
TitleStructure of CUL3-RBX1-KLHL22 complex without CUL3 NA motif
Components
  • Cullin-3
  • E3 ubiquitin-protein ligase RBX1, N-terminally processed
  • Kelch-like protein 22
KeywordsLIGASE / Cullin Ring E3 ubiquitin ligase
Function / homology
Function and homology information


liver morphogenesis / positive regulation of mitotic cell cycle phase transition / trophectodermal cellular morphogenesis / POZ domain binding / nuclear protein quality control by the ubiquitin-proteasome system / regulation protein catabolic process at postsynapse / polar microtubule / anaphase-promoting complex-dependent catabolic process / COPII vesicle coating / cullin-RING-type E3 NEDD8 transferase ...liver morphogenesis / positive regulation of mitotic cell cycle phase transition / trophectodermal cellular morphogenesis / POZ domain binding / nuclear protein quality control by the ubiquitin-proteasome system / regulation protein catabolic process at postsynapse / polar microtubule / anaphase-promoting complex-dependent catabolic process / COPII vesicle coating / cullin-RING-type E3 NEDD8 transferase / stem cell division / cellular response to chemical stress / NEDD8 transferase activity / cellular response to L-leucine / RHOBTB3 ATPase cycle / cullin-RING ubiquitin ligase complex / embryonic cleavage / cell projection organization / positive regulation of mitotic metaphase/anaphase transition / Cul7-RING ubiquitin ligase complex / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / positive regulation of protein autoubiquitination / protein neddylation / Notch binding / NEDD8 ligase activity / RHOBTB1 GTPase cycle / Cul5-RING ubiquitin ligase complex / negative regulation of response to oxidative stress / ubiquitin-ubiquitin ligase activity / fibroblast apoptotic process / Cul4A-RING E3 ubiquitin ligase complex / SCF ubiquitin ligase complex / negative regulation of Rho protein signal transduction / Cul2-RING ubiquitin ligase complex / negative regulation of type I interferon production / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / mitotic metaphase chromosome alignment / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Cul3-RING ubiquitin ligase complex / intercellular bridge / mitotic spindle assembly checkpoint signaling / stress fiber assembly / Prolactin receptor signaling / positive regulation of cytokinesis / protein monoubiquitination / mitotic sister chromatid segregation / cullin family protein binding / ubiquitin-like ligase-substrate adaptor activity / sperm flagellum / protein autoubiquitination / RHOBTB2 GTPase cycle / protein K48-linked ubiquitination / Nuclear events stimulated by ALK signaling in cancer / endoplasmic reticulum to Golgi vesicle-mediated transport / gastrulation / 14-3-3 protein binding / positive regulation of TORC1 signaling / T cell activation / Regulation of BACH1 activity / negative regulation of autophagy / intrinsic apoptotic signaling pathway / cyclin binding / post-translational protein modification / positive regulation of protein ubiquitination / integrin-mediated signaling pathway / Degradation of DVL / Recognition of DNA damage by PCNA-containing replication complex / Degradation of GLI1 by the proteasome / Negative regulation of NOTCH4 signaling / cellular response to amino acid stimulus / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / Vif-mediated degradation of APOBEC3G / Hedgehog 'on' state / DNA Damage Recognition in GG-NER / Degradation of GLI2 by the proteasome / GLI3 is processed to GLI3R by the proteasome / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / protein destabilization / RING-type E3 ubiquitin transferase / Degradation of beta-catenin by the destruction complex / negative regulation of canonical Wnt signaling pathway / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / Dual Incision in GG-NER / Evasion by RSV of host interferon responses / NOTCH1 Intracellular Domain Regulates Transcription / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / mitotic spindle / Wnt signaling pathway / Regulation of expression of SLITs and ROBOs / Constitutive Signaling by NOTCH1 PEST Domain Mutants / Constitutive Signaling by NOTCH1 HD+PEST Domain Mutants / Formation of Incision Complex in GG-NER / spindle pole / Interleukin-1 signaling / Orc1 removal from chromatin / Dual incision in TC-NER / G1/S transition of mitotic cell cycle
Similarity search - Function
Kelch-like protein 22 / Kelch motif / Galactose oxidase, central domain / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / BTB-kelch protein / BTB/Kelch-associated / BTB And C-terminal Kelch / Cullin protein neddylation domain / BTB And C-terminal Kelch ...Kelch-like protein 22 / Kelch motif / Galactose oxidase, central domain / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / BTB-kelch protein / BTB/Kelch-associated / BTB And C-terminal Kelch / Cullin protein neddylation domain / BTB And C-terminal Kelch / Cullin, conserved site / Cullin family signature. / Cullin / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin protein neddylation domain / Kelch / Kelch repeat type 1 / Kelch motif / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin family / Cullin family profile. / Kelch-type beta propeller / BTB/POZ domain / BTB domain profile. / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / SKP1/BTB/POZ domain superfamily / Zinc finger RING-type profile. / Zinc finger, RING-type / Zinc finger, RING/FYVE/PHD-type / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
E3 ubiquitin-protein ligase RBX1 / Cullin-3 / Kelch-like protein 22
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsWang, W. / Ling, L. / Dai, Z. / Zuo, P. / Yin, Y.
Funding support China, 4items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)2021YFA1300601 China
National Natural Science Foundation of China (NSFC)82030081 China
National Natural Science Foundation of China (NSFC)81874235 China
National Natural Science Foundation of China (NSFC)31800626 China
CitationJournal: Nat Commun / Year: 2024
Title: A conserved N-terminal motif of CUL3 contributes to assembly and E3 ligase activity of CRL3.
Authors: Weize Wang / Ling Liang / Zonglin Dai / Peng Zuo / Shang Yu / Yishuo Lu / Dian Ding / Hongyi Chen / Hui Shan / Yan Jin / Youdong Mao / Yuxin Yin /
Abstract: The CUL3-RING E3 ubiquitin ligases (CRL3s) play an essential role in response to extracellular nutrition and stress stimuli. The ubiquitin ligase function of CRL3s is activated through dimerization. ...The CUL3-RING E3 ubiquitin ligases (CRL3s) play an essential role in response to extracellular nutrition and stress stimuli. The ubiquitin ligase function of CRL3s is activated through dimerization. However, how and why such a dimeric assembly is required for its ligase activity remains elusive. Here, we report the cryo-EM structure of the dimeric CRL3 complex and reveal a conserved N-terminal motif in CUL3 that contributes to the dimerization assembly and the E3 ligase activity of CRL3. We show that deletion of the CUL3 N-terminal motif impairs dimeric assembly and the E3 ligase activity of both CRL3 and several other CRL3s. In addition, we found that the dynamics of dimeric assembly of CRL3 generates a variable ubiquitination zone, potentially facilitating substrate recognition and ubiquitination. These findings demonstrate that a CUL3 N-terminal motif participates in the assembly process and provide insights into the assembly and activation of CRL3s.
History
DepositionAug 1, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 29, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: Cullin-3
K: Kelch-like protein 22
L: Kelch-like protein 22
R: E3 ubiquitin-protein ligase RBX1, N-terminally processed


Theoretical massNumber of molelcules
Total (without water)140,4534
Polymers140,4534
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Cullin-3


Mass: 87432.312 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CUL3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q13618
#2: Protein Kelch-like protein 22


Mass: 20365.340 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KLHL22 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q53GT1
#3: Protein E3 ubiquitin-protein ligase RBX1, N-terminally processed / E3 ubiquitin-protein transferase RBX1 / N-terminally processed


Mass: 12289.977 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RBX1, RNF75, ROC1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62877

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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

ComponentName: CUL3-RBX1-KLHL22 complex without CUL3 NA motif / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMtris(hydroxymethyl)aminomethaneTris1
31 mM2-Amino-2-(hydroxymethyl)propane-1,3-diolTCEP1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: DIFFRACTION / Nominal defocus max: 2000 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
4cryoSPARC4.2CTF correction
10cryoSPARC4.2initial Euler assignment
11cryoSPARC4.2final Euler assignment
12cryoSPARC4.2classification
13cryoSPARC4.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 74279
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 74279 / Symmetry type: POINT

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