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- PDB-9oma: Cryo-EM structure of PCMTD1-ELOBC-CUL5-RBX2 (CRL5-PCMTD1) -

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Basic information

Entry
Database: PDB / ID: 9oma
TitleCryo-EM structure of PCMTD1-ELOBC-CUL5-RBX2 (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


protein-L-isoaspartate (D-aspartate) O-methyltransferase activity / ERBB2 signaling pathway / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / reelin-mediated signaling pathway / regulation of neuron migration / target-directed miRNA degradation / elongin complex / protein neddylation / protein K11-linked ubiquitination ...protein-L-isoaspartate (D-aspartate) O-methyltransferase activity / ERBB2 signaling pathway / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / reelin-mediated signaling pathway / regulation of neuron migration / target-directed miRNA degradation / elongin complex / protein neddylation / protein K11-linked ubiquitination / NEDD8 ligase activity / VCB complex / response to redox state / Cul5-RING ubiquitin ligase complex / SCF ubiquitin ligase complex / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Cul2-RING ubiquitin ligase complex / 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 / Formation of HIV-1 elongation complex containing HIV-1 Tat / ubiquitin-like ligase-substrate adaptor activity / Formation of HIV elongation complex in the absence of HIV Tat / endoplasmic reticulum unfolded protein response / RNA Polymerase II Transcription Elongation / Formation of RNA Pol II elongation complex / RNA Polymerase II Pre-transcription Events / intrinsic apoptotic signaling pathway / post-translational protein modification / transcription corepressor binding / TP53 Regulates Transcription of DNA Repair Genes / transcription initiation at RNA polymerase II promoter / transcription elongation by RNA polymerase II / G1/S transition of mitotic cell cycle / Vif-mediated degradation of APOBEC3G / RING-type E3 ubiquitin transferase / Inactivation of CSF3 (G-CSF) signaling / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / Evasion by RSV of host interferon responses / calcium channel activity / 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 / copper ion binding / ubiquitin protein ligase binding / regulation of transcription by RNA polymerase II / 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 / Cullin family signature. / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain ...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 / 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 / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.14 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: Eric Z Pang / Boyu Zhao / Cameron Flowers / Elizabeth Oroudjeva / Jasmine B Winter / Vijaya Pandey / Michael R Sawaya / James Wohlschlegel / Joseph A Loo / Jose A Rodriguez / Steven G Clarke /
Abstract: A major type of spontaneous protein damage that accumulates with age is the formation of kinked polypeptide chains with L-isoaspartyl residues. Mitigating this damage is necessary for maintaining ...A major type of spontaneous protein damage that accumulates with age is the formation of kinked polypeptide chains with L-isoaspartyl residues. Mitigating this damage is necessary for maintaining proteome stability and prolonging organismal survival. While repair through methylation by PCMT1 has been previously shown to suppress L-isoaspartyl accumulation, we provide an additional mechanism for L-isoaspartyl maintenance through PCMTD1, a cullin-RING ligase (CRL). We combined cryo-EM, native mass spectrometry, and biochemical assays to provide insight on how the assembly and architecture of PCMTD1 in the context of a CRL complex fulfils this alternative mechanism. We show that the PCMTD1 CRL complex specifically binds L-isoaspartyl residues when bound to AdoMet. This work provides evidence for a growing class of E3 ubiquitin ligases that recognize spontaneous covalent modifications as potential substrates for ubiquitylation and subsequent proteasomal degradation.
History
DepositionMay 13, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 4, 2025Provider: repository / Type: Initial release
Revision 1.1Jun 18, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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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, mass spectrometry, Native mass spectrometry validates pentameric assembly, gel filtration, All protein components in complex elute in single peak, immunoprecipitation, ...Evidence: electron microscopy, mass spectrometry, Native mass spectrometry validates pentameric assembly, gel filtration, All protein components in complex elute in single peak, immunoprecipitation, Immunoprecipitation of PCMTD1 co-immunoprecipitates Elongins B and C, CUL5, 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 / CKII beta-binding protein 1 / CKBBP1 / RING finger protein 7 / Regulator of cullins 2 / ...Rbx2 / CKII beta-binding protein 1 / CKBBP1 / RING finger protein 7 / Regulator of cullins 2 / Sensitive to apoptosis gene protein


Mass: 12721.500 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RNF7, RBX2, ROC2, SAG / Production host: Escherichia coli (E. coli)
References: UniProt: Q9UBF6, 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

Component
IDNameTypeEntity IDParent-IDSource
1Pentameric complex of PCMTD1, Elongins B and C, Cullin-5, and RING-box protein 2 (CRL5-PCMTD1)COMPLEXall0RECOMBINANT
2PCMTD1-ELOBC substrate receptor complexCOMPLEX#1, #4-#51RECOMBINANT
3PCMTD1COMPLEX#12RECOMBINANT
4CUL5-RBX2 catalytic coreCOMPLEX1RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
11169 kDa/nmYES
21NO
33
41NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
54Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Escherichia coli (E. coli)562BL21(DE3)
32Escherichia coli (E. coli)562BL21(DE3)
43Escherichia coli (E. coli)562BL21(DE3)
54Escherichia coli (E. coli)562BL21(DE3)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameBuffer-ID
150 mMHEPES1
2150 mMsodium chloride1
31 mMDTT1
SpecimenConc.: 2.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was monodisperse and freshly 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: 2.1 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 5447

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4particle selection
7UCSF ChimeraXmodel fitting
8Rosettamodel fitting
9Cootmodel fitting
11cryoSPARC4initial Euler assignment
12cryoSPARC4final Euler assignment
14cryoSPARC43D reconstruction
15PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1302046
3D reconstructionResolution: 4.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 352937 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: After initial fitting in ChimeraX, model was energy minimized against the 3D map with Rosetta FastRelax, adjusted in Coot, and refined in Phenix.
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDChain-IDSource nameTypeAccession codeDetailsInitial refinement model-ID
1AAlphaFoldin silico model
26V9I

6v9i

BBPDBexperimental model6V9IInitial model consisted of CUL5 from PDB entry 6V9I. Side-chain packing was performed with FASPR.2
36V9I

6v9i

CCPDBexperimental model6V9IInitial model consisted of RBX2 from PDB entry 6V9I. Side-chain packing was performed with FASPR.2
48FVI

8fvi

DDPDBexperimental model8FVIInitial model consisted of ELOB from PDB entry 8FVI3
58FVI

8fvi

EEPDBexperimental model8FVIInitial model consisted of ELOC from PDB entry 8FVI3
RefinementHighest resolution: 4.14 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00510516
ELECTRON MICROSCOPYf_angle_d0.64614178
ELECTRON MICROSCOPYf_dihedral_angle_d5.3331369
ELECTRON MICROSCOPYf_chiral_restr0.041573
ELECTRON MICROSCOPYf_plane_restr0.0051819

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