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- EMDB-70631: Focused map of CUL5-RBX2: Cryo-EM structure of PCMTD1-ELOBC-CUL5-... -

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Basic information

Entry
Database: EMDB / ID: EMD-70631
TitleFocused map of CUL5-RBX2: Cryo-EM structure of PCMTD1-ELOBC-CUL5-RBX2 (CRL5-PCMTD1)
Map dataFocused map of CUL5-RBX2 within CRL5-PCMTD1 sharpened via EMReady
Sample
  • Complex: Pentameric complex of PCMTD1, Elongins B and C, Cullin-5, and RING-box protein 2 (CRL5-PCMTD1)
    • Complex: PCMTD1-ELOBC substrate receptor complex
      • Complex: PCMTD1
    • Complex: CUL5-RBX2 catalytic core
KeywordsCUL5-RING ubiquitin ligase complex / PROTEIN BINDING
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.51 Å
AuthorsPang EZ / Zhao B / Flowers C / Oroudjeva E / Winters JB / Pandey V / Sawaya MR / Wohlschlegel W / Loo JA / Rodriguez JA / Clarke SG
Funding support United States, 8 items
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.
ETOC BLURB: Limiting the accrual of L-isoaspartyl damaged proteins is essential during aging. While this is thought to be mediated solely by the repair activity of the protein, PCMT1, Pang al. now ...ETOC BLURB: Limiting the accrual of L-isoaspartyl damaged proteins is essential during aging. While this is thought to be mediated solely by the repair activity of the protein, PCMT1, Pang al. now demonstrate that a related protein, PCMTD1, functions as a cullin-RING ligase to selectively target L-isoaspartyl-damaged substrates for potential regulation by the ubiquitylation-proteosomal system.
HIGHLIGHTS: Atomic cryo-EM structure of CRL5-PCMTD1 determinedArchitecture of PCMTD1 when complexed as a CRL supports ubiquitylation activityPCMTD1 recognizes L-isoaspartyl residues as a recruitment ...HIGHLIGHTS: Atomic cryo-EM structure of CRL5-PCMTD1 determinedArchitecture of PCMTD1 when complexed as a CRL supports ubiquitylation activityPCMTD1 recognizes L-isoaspartyl residues as a recruitment motif for potential CRL activityRecognition of L-isoaspartyl residues is dependent on cofactor engagement.
History
DepositionMay 14, 2025-
Header (metadata) releaseJun 4, 2025-
Map releaseJun 4, 2025-
UpdateJun 18, 2025-
Current statusJun 18, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_70631.png

Downloads & links

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Map

FileDownload / File: emd_70631.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFocused map of CUL5-RBX2 within CRL5-PCMTD1 sharpened via EMReady
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 480 pix.
= 412.8 Å		0.86 Å/pix.
x 480 pix.
= 412.8 Å		0.86 Å/pix.
x 480 pix.
= 412.8 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: 3.0
Minimum - Maximum-0.14053117 - 19.586130000000001
Average (Standard dev.)-0.028289048 (±0.17111482)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 412.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_70631_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened focused map of CUL5-RBX2 generated in cryoSPARC

Fileemd_70631_additional_1.map
AnnotationUnsharpened focused map of CUL5-RBX2 generated in cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_70631_half_map_1.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_70631_half_map_2.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Pentameric complex of PCMTD1, Elongins B and C, Cullin-5, and RIN...

EntireName: Pentameric complex of PCMTD1, Elongins B and C, Cullin-5, and RING-box protein 2 (CRL5-PCMTD1)
Components
  • Complex: Pentameric complex of PCMTD1, Elongins B and C, Cullin-5, and RING-box protein 2 (CRL5-PCMTD1)
    • Complex: PCMTD1-ELOBC substrate receptor complex
      • Complex: PCMTD1
    • Complex: CUL5-RBX2 catalytic core

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Supramolecule #1: Pentameric complex of PCMTD1, Elongins B and C, Cullin-5, and RIN...

SupramoleculeName: Pentameric complex of PCMTD1, Elongins B and C, Cullin-5, and RING-box protein 2 (CRL5-PCMTD1)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: PCMTD1-ELOBC substrate receptor complex

SupramoleculeName: PCMTD1-ELOBC substrate receptor complex / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #4-#5
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: PCMTD1

SupramoleculeName: PCMTD1 / type: complex / ID: 3 / Parent: 2 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: CUL5-RBX2 catalytic core

SupramoleculeName: CUL5-RBX2 catalytic core / type: complex / ID: 4 / Parent: 1
Source (natural)Organism: Homo sapiens (human)

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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

Concentration2.8 mg/mL
BufferpH: 7.4
Component:
ConcentrationName
50.0 mMHEPES
150.0 mMsodium chloride
1.0 mMDTT
GridModel: SPT Labtech self-wicking R1.2/0.8 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: SPT LABTECH CHAMELEON
DetailsSample was monodisperse and freshly gel-filtrated prior to sample vitrification

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 5447 / Average exposure time: 2.1 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1302046
CTF correctionType: NONE
Startup modelType of model: INSILICO MODEL / Details: Ab initio model generated in cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.51 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4) / Number images used: 352937
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
ChainPDB IDDetails
chain_id: A, source_name: AlphaFold, initial_model_type: in silico model
chain_id: B, source_name: PDB, initial_model_type: experimental model

6v9i

Initial model consisted of CUL5 from PDB entry 6V9I. Side-chain packing was performed with FASPR.
chain_id: C, source_name: PDB, initial_model_type: experimental model

6v9i

Initial model consisted of RBX2 from PDB entry 6V9I. Side-chain packing was performed with FASPR.
chain_id: D, source_name: PDB, initial_model_type: experimental model

8fvi

Initial model consisted of ELOB from PDB entry 8FVI
chain_id: E, source_name: PDB, initial_model_type: experimental model

8fvi

Initial model consisted of ELOC from PDB entry 8FVI
DetailsAfter initial fitting in ChimeraX, model was energy minimized against the 3D map with Rosetta FastRelax, adjusted in Coot, and refined in Phenix.
RefinementSpace: REAL / Protocol: OTHER

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