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- EMDB-21121: cryo-EM structure of Cullin5 bound to RING-box protein 2 (Cul5-Rbx2) -

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Basic information

Entry
Database: EMDB / ID: EMD-21121
Titlecryo-EM structure of Cullin5 bound to RING-box protein 2 (Cul5-Rbx2)
Map datahalf map 2
Sample
  • Complex: cryo-EM structure of Cullin5 bound to RING-box protein 2 (Cul5-Rbx2)
    • Protein or peptide: Immunoglobulin G-binding protein G,Cullin-5
    • Protein or peptide: RING-box protein 2
  • Ligand: ZINC ION
Function / homology
Function and homology information


radial glia guided migration of Purkinje cell / cerebral cortex radially oriented cell migration / ERBB2 signaling pathway / Neddylation / Antigen processing: Ubiquitination & Proteasome degradation / cullin-RING ubiquitin ligase complex / protein neddylation / NEDD8 ligase activity / IgG binding / Cul5-RING ubiquitin ligase complex ...radial glia guided migration of Purkinje cell / cerebral cortex radially oriented cell migration / ERBB2 signaling pathway / Neddylation / Antigen processing: Ubiquitination & Proteasome degradation / cullin-RING ubiquitin ligase complex / protein neddylation / NEDD8 ligase activity / IgG binding / Cul5-RING ubiquitin ligase complex / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / SCF ubiquitin ligase complex / intrinsic apoptotic signaling pathway in response to oxidative stress / apoptotic mitochondrial changes / cullin family protein binding / site of DNA damage / intrinsic apoptotic signaling pathway / Vif-mediated degradation of APOBEC3G / G1/S transition of mitotic cell cycle / calcium channel activity / Inactivation of CSF3 (G-CSF) signaling / Downregulation of ERBB2 signaling / ubiquitin-protein transferase activity / activation of cysteine-type endopeptidase activity involved in apoptotic process / ubiquitin protein ligase activity / protein-macromolecule adaptor activity / Antigen processing: Ubiquitination & Proteasome degradation / signaling receptor activity / Neddylation / ubiquitin-dependent protein catabolic process / protein ubiquitination / ubiquitin protein ligase binding / negative regulation of apoptotic process / zinc ion binding / extracellular region / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
IgG-binding B / B domain / M protein-type anchor domain / GA-like domain / GA-like domain / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / Cullin protein neddylation domain / Cullin, conserved site / Cullin family signature. ...IgG-binding B / B domain / M protein-type anchor domain / GA-like domain / GA-like domain / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / Cullin protein neddylation domain / Cullin, conserved site / Cullin family signature. / Cullin / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin protein neddylation domain / Immunoglobulin/albumin-binding domain superfamily / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin family / Cullin family profile. / YSIRK Gram-positive signal peptide / LPXTG cell wall anchor motif / Gram-positive cocci surface proteins LPxTG motif profile. / LPXTG cell wall anchor domain / 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
Immunoglobulin G-binding protein G / Cullin-5 / RING-box protein 2
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.2 Å
AuthorsKomives EA / Lumpkin RJ / Baker RW / Leschziner AE
Funding support United States, 1 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)1817774 United States
CitationJournal: Nat Commun / Year: 2020
Title: Structure and dynamics of the ASB9 CUL-RING E3 Ligase.
Authors: Ryan J Lumpkin / Richard W Baker / Andres E Leschziner / Elizabeth A Komives /
Abstract: The Cullin 5 (CUL5) Ring E3 ligase uses adaptors Elongins B and C (ELOB/C) to bind different SOCS-box-containing substrate receptors, determining the substrate specificity of the ligase. The 18- ...The Cullin 5 (CUL5) Ring E3 ligase uses adaptors Elongins B and C (ELOB/C) to bind different SOCS-box-containing substrate receptors, determining the substrate specificity of the ligase. The 18-member ankyrin and SOCS box (ASB) family is the largest substrate receptor family. Here we report cryo-EM data for the substrate, creatine kinase (CKB) bound to ASB9-ELOB/C, and for full-length CUL5 bound to the RING protein, RBX2, which binds various E2s. To date, no full structures are available either for a substrate-bound ASB nor for CUL5. Hydrogen-deuterium exchange (HDX-MS) mapped onto a full structural model of the ligase revealed long-range allostery extending from the substrate through CUL5. We propose a revised allosteric mechanism for how CUL-E3 ligases function. ASB9 and CUL5 behave as rigid rods, connected through a hinge provided by ELOB/C transmitting long-range allosteric crosstalk from the substrate through CUL5 to the RBX2 flexible linker.
History
DepositionDec 13, 2019-
Header (metadata) releaseApr 29, 2020-
Map releaseApr 29, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
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  • Surface view colored by radius
  • Surface level: 0.5
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  • Surface view with fitted model
  • Atomic models: PDB-6v9i
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21121.png

Downloads & links

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Map

FileDownload / File: emd_21121.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhalf map 2
Voxel sizeX=Y=Z: 1.16 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-0.4154094 - 1.7585512
Average (Standard dev.)0.0060616485 (±0.07242459)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 232.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.161.161.16
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z232.000232.000232.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ350350350
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.4151.7590.006

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Supplemental data

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Additional map: unfiltered map

Fileemd_21121_additional.map
Annotationunfiltered map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_21121_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unfiltered map

Fileemd_21121_half_map_2.map
Annotationunfiltered map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : cryo-EM structure of Cullin5 bound to RING-box protein 2 (Cul5-Rbx2)

EntireName: cryo-EM structure of Cullin5 bound to RING-box protein 2 (Cul5-Rbx2)
Components
  • Complex: cryo-EM structure of Cullin5 bound to RING-box protein 2 (Cul5-Rbx2)
    • Protein or peptide: Immunoglobulin G-binding protein G,Cullin-5
    • Protein or peptide: RING-box protein 2
  • Ligand: ZINC ION

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Supramolecule #1: cryo-EM structure of Cullin5 bound to RING-box protein 2 (Cul5-Rbx2)

SupramoleculeName: cryo-EM structure of Cullin5 bound to RING-box protein 2 (Cul5-Rbx2)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21
Molecular weightTheoretical: 100 KDa

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Macromolecule #1: Immunoglobulin G-binding protein G,Cullin-5

MacromoleculeName: Immunoglobulin G-binding protein G,Cullin-5 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 100.4565 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MGSSHHHHHH SQDPMEYKLI LNGKTLKGET TTEAVDAATA EKVFKQYAND NGVDGEWTYD DATKTFTVTE IPTTENLYFQ GEFMATSNL LKNKGSLQFE DKWDFMRPIV LKLLRQESVT KQQWFDLFSD VHAVCLWDDK GPAKIHQALK EDILEFIKQA Q ARVLSHQD ...String:
MGSSHHHHHH SQDPMEYKLI LNGKTLKGET TTEAVDAATA EKVFKQYAND NGVDGEWTYD DATKTFTVTE IPTTENLYFQ GEFMATSNL LKNKGSLQFE DKWDFMRPIV LKLLRQESVT KQQWFDLFSD VHAVCLWDDK GPAKIHQALK EDILEFIKQA Q ARVLSHQD DTALLKAYIV EWRKFFTQCD ILPKPFCQLE ITLMGKQGSN KKSNVEDSIV RKLMLDTWNE SIFSNIKNRL QD SAMKLVH AERLGEAFDS QLVIGVRESY VNLCSNPEDK LQIYRDNFEK AYLDSTERFY RTQAPSYLQQ NGVQNYMKYA DAK LKEEEK RALRYLETRR ECNSVEALME CCVNALVTSF KETILAECQG MIKRNETEKL HLMFSLMDKV PNGIEPMLKD LEEH IISAG LADMVAAAET ITTDSEKYVE QLLTLFNRFS KLVKEAFQDD PRFLTARDKA YKAVVNDATI FKLELPLKQK GVGLK TQPE SKCPELLANY CDMLLRKTPL SKKLTSEEIE AKLKEVLLVL KYVQNKDVFM RYHKAHLTRR LILDISADSE IEENMV EWL REVGMPADYV NKLARMFQDI KVSEDLNQAF KEMHKNNKLA LPADSVNIKI LNAGAWSRSS EKVFVSLPTE LEDLIPE VE EFYKKNHSGR KLHWHHLMSN GIITFKNEVG QYDLEVTTFQ LAVLFAWNQR PREKISFENL KLATELPDAE LRRTLWSL V AFPKLKRQVL LYEPQVNSPK DFTEGTLFSV NQEFSLIKNA KVQKRGKINL IGRLQLTTER MREEENEGIV QLRILRTQE AIIQIMKMRK KISNAQLQTE LVEILKNMFL PQKKMIKEQI EWLIEHKYIR RDESDINTFI YMA

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Macromolecule #2: RING-box protein 2

MacromoleculeName: RING-box protein 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 12.7215 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MADVEDGEEP CVLSSHSGSA GSKSGGDKMF SLKKWNAVAM WSWDVECDTC AICRVQVMDA CLRCQAENKQ EDCVVVWGEC NHSFHNCCM SLWVKQNNRC PLCQQDWVVQ RIGK

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 3 / 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

Concentration0.5 mg/mL
BufferpH: 7.5
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 4 second blot time, blot force 20.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 36000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 8.0 sec. / Average electron dose: 59.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: OTHER / Details: Ab initio model generation in cryoSPARC v2
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER / Details: Non-uniform refinement in cryoSPARC v2.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Details: Non-uniform refinement in cryoSPARC v2 / Number images used: 46877

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Atomic model buiding 1

Initial model(PDB ID:

4jgh

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3dpl

)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6v9i:
cryo-EM structure of Cullin5 bound to RING-box protein 2 (Cul5-Rbx2)

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