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- PDB-8hae: Cryo-EM structure of HACE1 dimer -

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Basic information

Entry
Database: PDB / ID: 8hae
TitleCryo-EM structure of HACE1 dimer
ComponentsE3 ubiquitin-protein ligase HACE1
KeywordsANTITUMOR PROTEIN / E3 ubiquitin ligase / tumor suppressor / Post-translational modifier / Protein degradation
Function / homology
Function and homology information


HECT-type E3 ubiquitin transferase / Golgi cisterna membrane / Rac protein signal transduction / Golgi organization / protein K48-linked ubiquitination / regulation of cell migration / small GTPase binding / protein polyubiquitination / ubiquitin-protein transferase activity / ubiquitin protein ligase activity ...HECT-type E3 ubiquitin transferase / Golgi cisterna membrane / Rac protein signal transduction / Golgi organization / protein K48-linked ubiquitination / regulation of cell migration / small GTPase binding / protein polyubiquitination / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / Antigen processing: Ubiquitination & Proteasome degradation / ubiquitin-dependent protein catabolic process / membrane fusion / nuclear body / protein ubiquitination / Golgi membrane / endoplasmic reticulum / nucleus / cytoplasm
Similarity search - Function
: / Ankyrin repeats (many copies) / HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) / HECT domain profile. / Domain Homologous to E6-AP Carboxyl Terminus with / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. ...: / Ankyrin repeats (many copies) / HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) / HECT domain profile. / Domain Homologous to E6-AP Carboxyl Terminus with / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily
Similarity search - Domain/homology
E3 ubiquitin-protein ligase HACE1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.55 Å
AuthorsSingh, S. / Machida, S. / Tulsian, N.K. / Choong, Y.K. / Ng, J. / Shanker, S. / Yaochen, L.D. / Shi, J. / Sivaraman, J. / Machida, S.
Funding support Singapore, 1items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore) Singapore
CitationJournal: Adv Sci (Weinh) / Year: 2023
Title: Structural Basis for the Enzymatic Activity of the HACE1 HECT-Type E3 Ligase Through N-Terminal Helix Dimerization.
Authors: Sunil Singh / Satoru Machida / Nikhil Kumar Tulsian / Yeu Khai Choong / Joel Ng / Srihari Shankar / Yaochen Liu / Krisha Vashdev Chandiramani / Jian Shi / J Sivaraman /
Abstract: HACE1 is an ankyrin repeat (AKR) containing HECT-type E3 ubiquitin ligase that interacts with and ubiquitinates multiple substrates. While HACE1 is a well-known tumor suppressor, its structure and ...HACE1 is an ankyrin repeat (AKR) containing HECT-type E3 ubiquitin ligase that interacts with and ubiquitinates multiple substrates. While HACE1 is a well-known tumor suppressor, its structure and mode of ubiquitination are not understood. The authors present the cryo-EM structures of human HACE1 along with in vitro functional studies that provide insights into how the enzymatic activity of HACE1 is regulated. HACE1 comprises of an N-terminal AKR domain, a middle (MID) domain, and a C-terminal HECT domain. Its unique G-shaped architecture interacts as a homodimer, with monomers arranged in an antiparallel manner. In this dimeric arrangement, HACE1 ubiquitination activity is hampered, as the N-terminal helix of one monomer restricts access to the C-terminal domain of the other. The in vitro ubiquitination assays, hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis, mutagenesis, and in silico modeling suggest that the HACE1 MID domain plays a crucial role along with the AKRs in RAC1 substrate recognition.
History
DepositionOct 26, 2022Deposition site: PDBJ / Processing site: RCSB
Revision 1.0Jun 28, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 16, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Aug 30, 2023Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Oct 4, 2023Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: E3 ubiquitin-protein ligase HACE1
B: E3 ubiquitin-protein ligase HACE1


Theoretical massNumber of molelcules
Total (without water)204,8992
Polymers204,8992
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration static light scattering
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein E3 ubiquitin-protein ligase HACE1 / HECT domain and ankyrin repeat-containing E3 ubiquitin-protein ligase 1 / HECT-type E3 ubiquitin ...HECT domain and ankyrin repeat-containing E3 ubiquitin-protein ligase 1 / HECT-type E3 ubiquitin transferase HACE1


Mass: 102449.672 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HACE1, KIAA1320 / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: Q8IYU2, HECT-type E3 ubiquitin transferase

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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: HACE1 dimer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.2 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.13_2998: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.55 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 71331 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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