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- PDB-6v9h: Ankyrin repeat and SOCS-box protein 9 (ASB9), ElonginB (ELOB), an... -

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Basic information

Entry
Database: PDB / ID: 6v9h
TitleAnkyrin repeat and SOCS-box protein 9 (ASB9), ElonginB (ELOB), and ElonginC (ELOC) bound to its substrate Brain-type Creatine Kinase (CKB)
Components
  • Ankyrin repeat and SOCS box protein 9
  • Creatine kinase B-type
  • Elongin-B
  • Elongin-C
KeywordsTRANSCRIPTION / Ankyrin repeat / Elongation Factor / Creatine Kinase / Ubiquitin
Function / homology
Function and homology information


futile creatine cycle / creatine kinase / Creatine metabolism / phosphocreatine biosynthetic process / creatine kinase activity / target-directed miRNA degradation / elongin complex / VCB complex / Cul5-RING ubiquitin ligase complex / RND3 GTPase cycle ...futile creatine cycle / creatine kinase / Creatine metabolism / phosphocreatine biosynthetic process / creatine kinase activity / target-directed miRNA degradation / elongin complex / VCB complex / Cul5-RING ubiquitin ligase complex / RND3 GTPase cycle / Cul2-RING ubiquitin ligase complex / 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 / 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 / substantia nigra development / transcription corepressor binding / Evasion by RSV of host interferon responses / transcription elongation by RNA polymerase II / transcription initiation at RNA polymerase II promoter / TP53 Regulates Transcription of DNA Repair Genes / Vif-mediated degradation of APOBEC3G / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / Inactivation of CSF3 (G-CSF) signaling / Regulation of expression of SLITs and ROBOs / positive regulation of protein catabolic process / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / Antigen processing: Ubiquitination & Proteasome degradation / protein-macromolecule adaptor activity / Neddylation / ubiquitin-dependent protein catabolic process / protein-containing complex assembly / protein ubiquitination / intracellular signal transduction / phosphorylation / ubiquitin protein ligase binding / regulation of transcription by RNA polymerase II / mitochondrion / extracellular space / extracellular exosome / nucleoplasm / ATP binding / plasma membrane / cytosol
Similarity search - Function
Ankyrin repeat and SOCS box protein 9/11, SOCS box domain / SOCS box / SOCS box-like domain superfamily / SOCS box domain / SOCS box domain profile. / SOCS_box / ATP:guanido phosphotransferase, N-terminal / ATP:guanido phosphotransferase, N-terminal domain superfamily / ATP:guanido phosphotransferase, N-terminal domain / Phosphagen kinase N-terminal domain profile. ...Ankyrin repeat and SOCS box protein 9/11, SOCS box domain / SOCS box / SOCS box-like domain superfamily / SOCS box domain / SOCS box domain profile. / SOCS_box / ATP:guanido phosphotransferase, N-terminal / ATP:guanido phosphotransferase, N-terminal domain superfamily / ATP:guanido phosphotransferase, N-terminal domain / Phosphagen kinase N-terminal domain profile. / ATP:guanido phosphotransferase active site / Phosphagen kinase active site signature. / ATP:guanido phosphotransferase / ATP:guanido phosphotransferase, catalytic domain / ATP:guanido phosphotransferase, C-terminal catalytic domain / Phosphagen kinase C-terminal domain profile. / Glutamine synthetase/guanido kinase, catalytic domain / Elongin B / Elongin-C / 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 / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Creatine kinase B-type / Elongin-C / Elongin-B / Ankyrin repeat and SOCS box protein 9
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsKomives, E.A. / Lumpkin, R.J. / Baker, R.W. / Leschziner, A.E.
Funding support United States, 1items
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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 29, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 11, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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  • Deposited structure unit
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  • EMDB-21120
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Assembly

Deposited unit
A: Creatine kinase B-type
B: Creatine kinase B-type
C: Ankyrin repeat and SOCS box protein 9
D: Elongin-C
E: Elongin-B


Theoretical massNumber of molelcules
Total (without water)143,7075
Polymers143,7075
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Number of models10

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Components

#1: Protein Creatine kinase B-type / Brain creatine kinase / B-CK / Creatine kinase B chain / Creatine phosphokinase B-type / CPK-B


Mass: 42699.207 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CKB, CKBB / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P12277, creatine kinase
#2: Protein Ankyrin repeat and SOCS box protein 9 / ASB-9


Mass: 34186.141 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ASB9 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q96DX5
#3: 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: 10974.616 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 BL21 (bacteria) / References: UniProt: Q15369
#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 BL21 (bacteria) / References: UniProt: Q15370

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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: Ankyrin repeat and SOCS-box protein 9 (ASB9), ElonginB (ELOB), and ElonginC (ELOC) bound to its substrate Brain-type Creatine Kinase (CKB)
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.14 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21
Buffer solutionpH: 7.5
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 4 second blot, blot force 20

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 7 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2Leginon3.3image acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
12cryoSPARC23D reconstruction
13Rosettamodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1309964
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 285105 / Details: Non-uniform refinement in cryoSPARC v2 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
13ZNG

3zng

1
23DRB

3drb

1

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