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- EMDB-34586: Cryo-EM structure of HACE1 dimer -

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Basic information

Entry
Database: EMDB / ID: EMD-34586
TitleCryo-EM structure of HACE1 dimer
Map data
Sample
  • Complex: HACE1 dimer
    • Protein or peptide: E3 ubiquitin-protein ligase HACE1
KeywordsE3 ubiquitin ligase / tumor suppressor / Post-translational modifier / Protein degradation / ANTITUMOR PROTEIN
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 / cell cycle / Golgi membrane / endoplasmic reticulum / nucleus / cytoplasm
Similarity search - Function
HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) / HECT domain profile. / Domain Homologous to E6-AP Carboxyl Terminus with / Ankyrin repeats (many copies) / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats ...HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) / HECT domain profile. / Domain Homologous to E6-AP Carboxyl Terminus with / Ankyrin repeats (many copies) / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.55 Å
AuthorsSingh S / Machida S / Tulsian NK / Choong YK / Ng J / Shanker S / Yaochen LD / Shi J / Sivaraman J
Funding support Singapore, 1 items
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, 2022-
Header (metadata) releaseJun 28, 2023-
Map releaseJun 28, 2023-
UpdateOct 4, 2023-
Current statusOct 4, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_34586.png

Downloads & links

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Map

FileDownload / File: emd_34586.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.8584 Å
Density
Contour LevelBy AUTHOR: 4.26
Minimum - Maximum-12.868067999999999 - 25.492159000000001
Average (Standard dev.)0.000000000006008 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 247.2192 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_34586_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_34586_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34586_half_map_2.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 : HACE1 dimer

EntireName: HACE1 dimer
Components
  • Complex: HACE1 dimer
    • Protein or peptide: E3 ubiquitin-protein ligase HACE1

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Supramolecule #1: HACE1 dimer

SupramoleculeName: HACE1 dimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 200 KDa

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Macromolecule #1: E3 ubiquitin-protein ligase HACE1

MacromoleculeName: E3 ubiquitin-protein ligase HACE1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: HECT-type E3 ubiquitin transferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 102.449672 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MERAMEQLNR LTRSLRRART VELPEDNETA VYTLMPMVMA DQHRSVSELL SNSKFDVNYA FGRVKRSLLH IAANCGSVEC LVLLLKKGA NPNYQDISGC TPLHLAARNG QKKCMSKLLE YSADVNICNN EGLTAIHWLA VNGRTELLHD LVQHVSDVDV E DAMGQTAL ...String:
MERAMEQLNR LTRSLRRART VELPEDNETA VYTLMPMVMA DQHRSVSELL SNSKFDVNYA FGRVKRSLLH IAANCGSVEC LVLLLKKGA NPNYQDISGC TPLHLAARNG QKKCMSKLLE YSADVNICNN EGLTAIHWLA VNGRTELLHD LVQHVSDVDV E DAMGQTAL HVACQNGHKT TVQCLLDSGA DINRPNVSGA TPLYFACSHG QRDTAQILLL RGAKYLPDKN GVTPLDLCVQ GG YGETCEV LIQYHPRLFQ TIIQMTQNED LRENMLRQVL EHLSQQSESQ YLKILTSLAE VATTNGHKLL SLSSNYDAQM KSL LRIVRM FCHVFRIGPS SPSNGIDMGY NGNKTPRSQV FKPLELLWHS LDEWLVLIAT ELMKNKRDST EITSILLKQK GQDQ DAASI PPFEPPGPGS YENLSTGTRE SKPDALAGRQ EASADCQDVI SMTANRLSAV IQAFYMCCSC QMPPGMTSPR FIEFV CKHD EVLKCFVNRN PKIIFDHFHF LLECPELMSR FMHIIKAQPF KDRCEWFYEH LHSGQPDSDM VHRPVNENDI LLVHRD SIF RSSCEVVSKA NCAKLKQGIA VRFHGEEGMG QGVVREWFDI LSNEIVNPDY ALFTQSADGT TFQPNSNSYV NPDHLNY FR FAGQILGLAL NHRQLVNIYF TRSFYKHILG IPVNYQDVAS IDPEYAKNLQ WILDNDISDL GLELTFSVET DVFGAMEE V PLKPGGGSIL VTQNNKAEYV QLVTELRMTR AIQPQINAFL QGFHMFIPPS LIQLFDEYEL ELLLSGMPEI DVSDWIKNT EYTSGYERED PVIQWFWEVV EDITQEERVL LLQFVTGSSR VPHGGFANIM GGSGLQNFTI AAVPYTPNLL PTSSTCINML KLPEYPSKE ILKDRLLVAL HCGSYGYTMA

UniProtKB: E3 ubiquitin-protein ligase HACE1

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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

BufferpH: 8
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec.
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.55 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 71331
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 3
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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