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- EMDB-37739: cryo-EM structure of neddylated CUL2-RBX1-ELOB-ELOC-FEM1B bound w... -

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Entry
Database: EMDB / ID: EMD-37739
Titlecryo-EM structure of neddylated CUL2-RBX1-ELOB-ELOC-FEM1B bound with the C-degron of CDK5R1
Map data
Sample
  • Complex: cryo-EM structure of neddylated CUL2-RBX1-ELOB-ELOC-FEM1B bound with the C-degron of CDK5R1
    • Protein or peptide: Elongin-B
    • Protein or peptide: E3 ubiquitin-protein ligase RBX1, N-terminally processed
    • Protein or peptide: Elongin-C
    • Protein or peptide: Cullin-2
    • Protein or peptide: NEDD8
    • Protein or peptide: Protein fem-1 homolog B
  • Ligand: ZINC ION
KeywordsE3 ubiquitin ligase / Pro/C-degron / LIGASE
Function / homology
Function and homology information


regulation of ubiquitin-protein transferase activity / epithelial cell maturation involved in prostate gland development / branching involved in prostate gland morphogenesis / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / cullin-RING ubiquitin ligase complex / cellular response to chemical stress / Cul7-RING ubiquitin ligase complex / regulation of DNA damage checkpoint / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway ...regulation of ubiquitin-protein transferase activity / epithelial cell maturation involved in prostate gland development / branching involved in prostate gland morphogenesis / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / cullin-RING ubiquitin ligase complex / cellular response to chemical stress / Cul7-RING ubiquitin ligase complex / regulation of DNA damage checkpoint / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / target-directed miRNA degradation / elongin complex / positive regulation of protein autoubiquitination / RNA polymerase II transcription initiation surveillance / protein neddylation / regulation of extrinsic apoptotic signaling pathway via death domain receptors / death receptor binding / NEDD8 ligase activity / VCB complex / negative regulation of response to oxidative stress / Cul5-RING ubiquitin ligase complex / SCF ubiquitin ligase complex / Cul2-RING ubiquitin ligase complex / negative regulation of type I interferon production / ubiquitin-ubiquitin ligase activity / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Cul4A-RING E3 ubiquitin ligase complex / Cul4-RING E3 ubiquitin ligase complex / Cul3-RING ubiquitin ligase complex / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / negative regulation of mitophagy / Prolactin receptor signaling / TGF-beta receptor signaling activates SMADs / cullin family protein binding / regulation of proteolysis / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / regulation of postsynapse assembly / HIV elongation arrest and recovery / Pausing and recovery of HIV elongation / anatomical structure morphogenesis / protein monoubiquitination / ubiquitin ligase complex / Tat-mediated elongation of the HIV-1 transcript / Formation of HIV-1 elongation complex containing HIV-1 Tat / ubiquitin-like ligase-substrate adaptor activity / protein K48-linked ubiquitination / Formation of HIV elongation complex in the absence of HIV Tat / Nuclear events stimulated by ALK signaling in cancer / RNA Polymerase II Transcription Elongation / Formation of RNA Pol II elongation complex / transcription-coupled nucleotide-excision repair / RNA Polymerase II Pre-transcription Events / positive regulation of TORC1 signaling / regulation of cellular response to insulin stimulus / intrinsic apoptotic signaling pathway / negative regulation of insulin receptor signaling pathway / post-translational protein modification / Regulation of BACH1 activity / T cell activation / transcription corepressor binding / TP53 Regulates Transcription of DNA Repair Genes / transcription initiation at RNA polymerase II promoter / cellular response to amino acid stimulus / Degradation of DVL / transcription elongation by RNA polymerase II / Degradation of GLI1 by the proteasome / Iron uptake and transport / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / Recognition of DNA damage by PCNA-containing replication complex / Negative regulation of NOTCH4 signaling / Vif-mediated degradation of APOBEC3G / Hedgehog 'on' state / G1/S transition of mitotic cell cycle / negative regulation of canonical Wnt signaling pathway / 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 / Inactivation of CSF3 (G-CSF) signaling / DNA Damage Recognition in GG-NER / Degradation of beta-catenin by the destruction complex / RING-type E3 ubiquitin transferase / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / Evasion by RSV of host interferon responses / modification-dependent protein catabolic process / NOTCH1 Intracellular Domain Regulates Transcription / protein modification process / Dual Incision in GG-NER / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / Constitutive Signaling by NOTCH1 PEST Domain Mutants / Constitutive Signaling by NOTCH1 HD+PEST Domain Mutants / Regulation of expression of SLITs and ROBOs / protein tag activity / Formation of Incision Complex in GG-NER / Interleukin-1 signaling / Orc1 removal from chromatin / protein polyubiquitination
Similarity search - Function
Nedd8-like ubiquitin / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / Cullin protein neddylation domain / : / Cullin, conserved site / Cullin family signature. / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin ...Nedd8-like ubiquitin / Zinc finger, RING-H2-type / RING-H2 zinc finger 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 / Elongin-C / Elongin B / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin alpha solenoid domain / Cullin family profile. / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / Ankyrin repeat / SKP1/BTB/POZ domain superfamily / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Zinc finger RING-type profile. / Ankyrin repeat-containing domain superfamily / Zinc finger, RING-type / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Zinc finger, RING/FYVE/PHD-type / Ubiquitin-like domain superfamily / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
E3 ubiquitin-protein ligase RBX1 / Cullin-2 / Elongin-C / Elongin-B / Ubiquitin-like protein NEDD8 / Protein fem-1 homolog B
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsChen X / Zhang K / Xu C
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nat Commun / Year: 2024
Title: Mechanism of Ψ-Pro/C-degron recognition by the CRL2 ubiquitin ligase.
Authors: Xinyan Chen / Anat Raiff / Shanshan Li / Qiong Guo / Jiahai Zhang / Hualin Zhou / Richard T Timms / Xuebiao Yao / Stephen J Elledge / Itay Koren / Kaiming Zhang / Chao Xu /
Abstract: The E3 ligase-degron interaction determines the specificity of the ubiquitin‒proteasome system. We recently discovered that FEM1B, a substrate receptor of Cullin 2-RING ligase (CRL2), recognizes C- ...The E3 ligase-degron interaction determines the specificity of the ubiquitin‒proteasome system. We recently discovered that FEM1B, a substrate receptor of Cullin 2-RING ligase (CRL2), recognizes C-degrons containing a C-terminal proline. By solving several cryo-EM structures of CRL2 bound to different C-degrons, we elucidate the dimeric assembly of the complex. Furthermore, we reveal distinct dimerization states of unmodified and neddylated CRL2 to uncover the NEDD8-mediated activation mechanism of CRL2. Our research also indicates that, FEM1B utilizes a bipartite mechanism to recognize both the C-terminal proline and an upstream aromatic residue within the substrate. These structural findings, complemented by in vitro ubiquitination and in vivo cell-based assays, demonstrate that CRL2-mediated polyubiquitination and subsequent protein turnover depend on both FEM1B-degron interactions and the dimerization state of the E3 ligase complex. Overall, this study deepens our molecular understanding of how Cullin-RING E3 ligase substrate selection mediates protein turnover.
History
DepositionOct 11, 2023-
Header (metadata) releaseApr 3, 2024-
Map releaseApr 3, 2024-
UpdateMay 8, 2024-
Current statusMay 8, 2024Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_37739.png

Downloads & links

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Map

FileDownload / File: emd_37739.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 512 pix.
= 419.84 Å		0.82 Å/pix.
x 512 pix.
= 419.84 Å		0.82 Å/pix.
x 512 pix.
= 419.84 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.7091322 - 1.1884904
Average (Standard dev.)0.00014417776 (±0.014358814)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 419.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_37739_half_map_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_37739_half_map_2.map
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Sample components

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Entire : cryo-EM structure of neddylated CUL2-RBX1-ELOB-ELOC-FEM1B bound w...

EntireName: cryo-EM structure of neddylated CUL2-RBX1-ELOB-ELOC-FEM1B bound with the C-degron of CDK5R1
Components
  • Complex: cryo-EM structure of neddylated CUL2-RBX1-ELOB-ELOC-FEM1B bound with the C-degron of CDK5R1
    • Protein or peptide: Elongin-B
    • Protein or peptide: E3 ubiquitin-protein ligase RBX1, N-terminally processed
    • Protein or peptide: Elongin-C
    • Protein or peptide: Cullin-2
    • Protein or peptide: NEDD8
    • Protein or peptide: Protein fem-1 homolog B
  • Ligand: ZINC ION

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Supramolecule #1: cryo-EM structure of neddylated CUL2-RBX1-ELOB-ELOC-FEM1B bound w...

SupramoleculeName: cryo-EM structure of neddylated CUL2-RBX1-ELOB-ELOC-FEM1B bound with the C-degron of CDK5R1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Elongin-B

MacromoleculeName: Elongin-B / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.147781 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MDVFLMIRRH KTTIFTDAKE SSTVFELKRI VEGILKRPPD EQRLYKDDQL LDDGKTLGEC GFTSQTARPQ APATVGLAFR ADDTFEALC IEPFSSPPEL PDVMKPQDSG SSANEQAVQ

UniProtKB: Elongin-B

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Macromolecule #2: E3 ubiquitin-protein ligase RBX1, N-terminally processed

MacromoleculeName: E3 ubiquitin-protein ligase RBX1, N-terminally processed
type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.19683 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SHMGAGKKRF EVKKWNAVAL WAWDIVVDNC AICRNHIMDL CIECQANQAS ATSEECTVAW GVCNHAFHFH CISRWLKTRQ VCPLDNREW EFQKYGH

UniProtKB: E3 ubiquitin-protein ligase RBX1

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Macromolecule #3: Elongin-C

MacromoleculeName: Elongin-C / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 10.84342 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MYVKLISSDG HEFIVKREHA LTSGTIKAML SGPGQFAENE TNEVNFREIP SHVLSKVCMY FTYKVRYTNS STEIPEFPIA PEIALELLM AANFLDC

UniProtKB: Elongin-C

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Macromolecule #4: Cullin-2

MacromoleculeName: Cullin-2 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 87.11475 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SASWSHPQFE KGGGSGGGSG TSLKPRVVDF DETWNKLLTT IKAVVMLEYV ERATWNDRFS DIYALCVAYP EPLGERLYTE TKIFLENHV RHLHKRVLES EEQVLVMYHR YWEEYSKGAD YMDCLYRYLN TQFIKKNGGG PLMEIGELAL DMWRKLMVEP L QAILIRML ...String:
SASWSHPQFE KGGGSGGGSG TSLKPRVVDF DETWNKLLTT IKAVVMLEYV ERATWNDRFS DIYALCVAYP EPLGERLYTE TKIFLENHV RHLHKRVLES EEQVLVMYHR YWEEYSKGAD YMDCLYRYLN TQFIKKNGGG PLMEIGELAL DMWRKLMVEP L QAILIRML LREIKNDRGG EDPNQKVIHG VINSFVHVEQ YKKKFPLKFY QEIFESPFLT ETGEYYKQEA SNLLQESNCS QY MEKVLGR LKDEEIRCRK YLHPSSYTKV IHECQQRMVA DHLQFLHAEC HNIIRQEKKN DMANMYVLLR AVSTGLPHMI QEL QNHIHD EGLRATSNLT QENMPTLFVE SVLEVHGKFV QLINTVLNGD QHFMSALDKA LTSVVNYREP KSVCKAPELL AKYC DNLLK KSAKGMTENE VEDRLTSFIT VFKYIDDKDV FQKFYARMLA KRLIHGLSMS MDSEEAMINK LKQACGYEFT SKLHR MYTD MSVSADLNNK FNNFIKNQDT VIDLGISFQI YVLQAGAWPL TQAPSSTFAI PQELEKSVQM FELFYSQHFS GRKLTW LHY LCTGEVKMNY LGKPYVAMVT TYQMAVLLAF NNSETVSYKE LQDSTQMNEK ELTKTIKSLL DVKMINHDSE KEDIDAE SS FSLNMNFSSK RTKFKITTSM QKDTPQEMEQ TRSAVDEDRK MYLQAAIVRI MKARKVLRHN ALIQEVISQS RARFNPSI S MIKKCIEVLI DKQYIERSQA SADEYSYVA

UniProtKB: Cullin-2

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Macromolecule #5: NEDD8

MacromoleculeName: NEDD8 / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 8.573978 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MLIKVKTLTG KEIEIDIEPT DKVERIKERV EEKEGIPPQQ QRLIYSGKQM NDEKTAADYK ILGGSVLHLV LALRGG

UniProtKB: Ubiquitin-like protein NEDD8

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Macromolecule #6: Protein fem-1 homolog B

MacromoleculeName: Protein fem-1 homolog B / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 70.355062 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEGLAGYVYK AASEGKVLTL AALLLNRSES DIRYLLGYVS QQGGQRSTPL IIAARNGHAK VVRLLLEHYR VQTQQTGTVR FDGYVIDGA TALWCAAGAG HFEVVKLLVS HGANVNHTTV TNSTPLRAAC FDGRLDIVKY LVENNANISI ANKYDNTCLM I AAYKGHTD ...String:
MEGLAGYVYK AASEGKVLTL AALLLNRSES DIRYLLGYVS QQGGQRSTPL IIAARNGHAK VVRLLLEHYR VQTQQTGTVR FDGYVIDGA TALWCAAGAG HFEVVKLLVS HGANVNHTTV TNSTPLRAAC FDGRLDIVKY LVENNANISI ANKYDNTCLM I AAYKGHTD VVRYLLEQRA DPNAKAHCGA TALHFAAEAG HIDIVKELIK WRAAIVVNGH GMTPLKVAAE SCKADVVELL LS HADCDRR SRIEALELLG ASFANDRENY DIIKTYHYLY LAMLERFQDG DNILEKEVLP PIHAYGNRTE CRNPQELESI RQD RDALHM EGLIVRERIL GADNIDVSHP IIYRGAVYAD NMEFEQCIKL WLHALHLRQK GNRNTHKDLL RFAQVFSQMI HLNE TVKAP DIECVLRCSV LEIEQSMNRV KNISDADVHN AMDNYECNLY TFLYLVCIST KTQCSEEDQC KINKQIYNLI HLDPR TREG FTLLHLAVNS NTPVDDFHTN DVCSFPNALV TKLLLDCGAE VNAVDNEGNS ALHIIVQYNR PISDFLTLHS IIISLV EAG AHTDMTNKQN KTPLDKSTTG VSEILLKTQM KMSLKCLAAR AVRANDINYQ DQIPRTLEEF VGFH

UniProtKB: Protein fem-1 homolog B

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 6 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration5 mg/mL
BufferpH: 7.5
GridMaterial: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 4232 / Average electron dose: 57.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.9 µm / Nominal defocus min: 1.5 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 447970
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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