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- PDB-9omf: Cryo-EM structure of neddylated PCMTD1-ELOBC-CUL5-RBX2 (N8-CRL5-P... -

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Basic information

Entry
Database: PDB / ID: 9omf
TitleCryo-EM structure of neddylated PCMTD1-ELOBC-CUL5-RBX2 (N8-CRL5-PCMTD1)
Components
  • Cullin-5
  • Elongin-B
  • Elongin-C
  • Protein-L-isoaspartate O-methyltransferase domain-containing protein 1
  • RING-box protein 2
KeywordsPROTEIN BINDING / CUL5-RING ubiquitin ligase complex
Function / homology
Function and homology information


radial glia guided migration of Purkinje cell / cerebral cortex radially oriented cell migration / protein-L-isoaspartate (D-aspartate) O-methyltransferase activity / Inactivation of CSF3 (G-CSF) signaling / ERBB2 signaling pathway / regulation of neuron migration / Neddylation / reelin-mediated signaling pathway / cullin-RING-type E3 NEDD8 transferase / target-directed miRNA degradation ...radial glia guided migration of Purkinje cell / cerebral cortex radially oriented cell migration / protein-L-isoaspartate (D-aspartate) O-methyltransferase activity / Inactivation of CSF3 (G-CSF) signaling / ERBB2 signaling pathway / regulation of neuron migration / Neddylation / reelin-mediated signaling pathway / cullin-RING-type E3 NEDD8 transferase / target-directed miRNA degradation / VCB complex / elongin complex / Antigen processing: Ubiquitination & Proteasome degradation / protein K11-linked ubiquitination / protein neddylation / NEDD8 ligase activity / Cul5-RING ubiquitin ligase complex / SCF ubiquitin ligase complex / intrinsic apoptotic signaling pathway in response to oxidative stress / Cul2-RING ubiquitin ligase complex / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / apoptotic mitochondrial changes / ubiquitin ligase complex scaffold activity / cullin family protein binding / site of DNA damage / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / HIV elongation arrest and recovery / Pausing and recovery of HIV elongation / Tat-mediated elongation of the HIV-1 transcript / ubiquitin-like ligase-substrate adaptor activity / Formation of HIV-1 elongation complex containing HIV-1 Tat / Formation of HIV elongation complex in the absence of HIV Tat / RNA Polymerase II Transcription Elongation / Formation of RNA Pol II elongation complex / RNA Polymerase II Pre-transcription Events / intrinsic apoptotic signaling pathway / transcription corepressor binding / transcription initiation at RNA polymerase II promoter / TP53 Regulates Transcription of DNA Repair Genes / transcription elongation by RNA polymerase II / Vif-mediated degradation of APOBEC3G / Inactivation of CSF3 (G-CSF) signaling / RING-type E3 ubiquitin transferase / Evasion by RSV of host interferon responses / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / calcium channel activity / G1/S transition of mitotic cell cycle / Regulation of expression of SLITs and ROBOs / Downregulation of ERBB2 signaling / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / Antigen processing: Ubiquitination & Proteasome degradation / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / signaling receptor activity / Neddylation / protein-containing complex assembly / ubiquitin-dependent protein catabolic process / protein-macromolecule adaptor activity / proteasome-mediated ubiquitin-dependent protein catabolic process / protein ubiquitination / ubiquitin protein ligase binding / regulation of transcription by RNA polymerase II / negative regulation of apoptotic process / zinc ion binding / nucleoplasm / nucleus / membrane / cytosol / cytoplasm
Similarity search - Function
Protein-L-isoaspartate(D-aspartate) O-methyltransferase / Protein-L-isoaspartate(D-aspartate) O-methyltransferase (PCMT) / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / Cullin protein neddylation domain / : / Cullin, conserved site / Elongin-C / Cullin family signature. / Elongin B ...Protein-L-isoaspartate(D-aspartate) O-methyltransferase / Protein-L-isoaspartate(D-aspartate) O-methyltransferase (PCMT) / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / Cullin protein neddylation domain / : / Cullin, conserved site / Elongin-C / Cullin family signature. / Elongin B / 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. / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / SKP1/BTB/POZ domain superfamily / Zinc finger RING-type profile. / Zinc finger, RING-type / 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 / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
Elongin-C / Elongin-B / Cullin-5 / Protein-L-isoaspartate O-methyltransferase domain-containing protein 1 / RING-box protein 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9.72 Å
AuthorsPang, E.Z. / Zhao, B. / Flowers, C. / Oroudjeva, E. / Winters, J.B. / Pandey, V. / Sawaya, M.R. / Wohlschlegel, W. / Loo, J.A. / Rodriguez, J.A. / Clarke, S.G.
Funding support United States, 8items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM136614 United States
National Science Foundation (NSF, United States)MCB-1714569 United States
National Science Foundation (NSF, United States)DMR-1548924 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM128867 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)S10RR028893 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM145286 United States
Department of Energy (DOE, United States)DE-FC02-02ER63421 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM145388 United States
CitationJournal: Biorxiv / Year: 2025
Title: Structural basis for L-isoaspartyl-containing protein recognition by the PCMTD1 cullin-RING E3 ubiquitin ligase
Authors: Pang, E.Z. / Zhao, B. / Flowers, C. / Oroudjeva, E. / Winter, J.B. / Pandey, V. / Sawaya, M.R. / Wohlschlegel, J. / Loo, J.A. / Rodriguez, J.A. / Clarke, S.G.
History
DepositionMay 13, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 4, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protein-L-isoaspartate O-methyltransferase domain-containing protein 1
B: Cullin-5
C: RING-box protein 2
D: Elongin-B
E: Elongin-C


Theoretical massNumber of molelcules
Total (without water)168,9645
Polymers168,9645
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, gel filtration, All protein components in complex elute in single peak, neddylation validated by immunoblotting, and neddylated CRL5-PCMTD1 elutes ...Evidence: electron microscopy, not applicable, gel filtration, All protein components in complex elute in single peak, neddylation validated by immunoblotting, and neddylated CRL5-PCMTD1 elutes earlier compared to CRL5-PCMTD1, immunoprecipitation, Immunoprecipitation of PCMTD1 co-immunoprecipitates Elongins B and C, CUL5, NEDD8, and RBX2
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Protein-L-isoaspartate O-methyltransferase domain-containing protein 1


Mass: 40814.348 Da / Num. of mol.: 1 / Mutation: N312I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PCMTD1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q96MG8
#2: Protein Cullin-5 / CUL-5 / Vasopressin-activated calcium-mobilizing receptor 1 / VACM-1


Mass: 91436.680 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CUL5, VACM1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q93034
#3: Protein RING-box protein 2 / Rbx2 / RING finger protein 7 / Sensitive to apoptosis gene protein


Mass: 12721.500 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Rnf7, Rbx2, Sag / Production host: Escherichia coli (E. coli)
References: UniProt: Q9WTZ1, RING-type E3 ubiquitin transferase, cullin-RING-type E3 NEDD8 transferase
#4: Protein Elongin-B / EloB / Elongin 18 kDa subunit / RNA polymerase II transcription factor SIII subunit B / SIII p18 / ...EloB / Elongin 18 kDa subunit / RNA polymerase II transcription factor SIII subunit B / SIII p18 / Transcription elongation factor B polypeptide 2


Mass: 13147.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ELOB, TCEB2 / Production host: Escherichia coli (E. coli) / References: UniProt: Q15370
#5: Protein Elongin-C / EloC / Elongin 15 kDa subunit / RNA polymerase II transcription factor SIII subunit C / SIII p15 / ...EloC / Elongin 15 kDa subunit / RNA polymerase II transcription factor SIII subunit C / SIII p15 / Transcription elongation factor B polypeptide 1


Mass: 10843.420 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ELOC, TCEB1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q15369
Has protein modificationY

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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: Neddylated complex of PCMTD1, Elongins B and C, Cullin-5, and RING-box protein 2 (N8-CRL5-PCMTD1)
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameBuffer-ID
150 mMHEPES1
2150 mMsodium chloride1
31 mMDTT1
SpecimenConc.: 2.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was monodisperse and freshly neddylated then gel-filtrated prior to sample vitrification
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: SPT Labtech self-wicking R1.2/0.8
VitrificationInstrument: SPT LABTECH CHAMELEON / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingAverage exposure time: 2.64 sec. / Electron dose: 50 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of real images: 15192

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4particle selection
7Rosettamodel fitting
9cryoSPARC4initial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARC4classification
12cryoSPARC43D reconstruction
13PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1731846
3D reconstructionResolution: 9.72 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 7521 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Energy minimized against 3D map of N8-CRL5-PCMTD1 with model of CRL5-PCMTD1 (PDB: 9OMA) then refined in Phenix
Atomic model buildingPDB-ID: 9OMA

9oma


Accession code: 9OMA
Details: Initial model consisted of the complete complex for PDB entry 9OMA
Source name: PDB / Type: experimental model
RefinementHighest resolution: 9.72 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)

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