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- EMDB-17994: Cryo-EM structure of a full-length HACE1 dimer -

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Basic information

Entry
Database: EMDB / ID: EMD-17994
TitleCryo-EM structure of a full-length HACE1 dimer
Map data
Sample
  • Complex: HACE1
    • Protein or peptide: E3 ubiquitin-protein ligase HACE1
KeywordsE3 / HECT / ubiquitin / LIGASE
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 / protein ubiquitination / nuclear body / 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: 4.73 Å
AuthorsDuering J / Wolter M / Dienemann C / Lorenz S
Funding support Germany, European Union, 2 items
OrganizationGrant numberCountry
Max Planck Society Germany
European Molecular Biology Organization (EMBO)EMBO ALTF 439-2022European Union
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structural mechanisms of autoinhibition and substrate recognition by the ubiquitin ligase HACE1.
Authors: Jonas Düring / Madita Wolter / Julia J Toplak / Camilo Torres / Olexandr Dybkov / Thornton J Fokkens / Katherine E Bohnsack / Henning Urlaub / Wieland Steinchen / Christian Dienemann / Sonja Lorenz /
Abstract: Ubiquitin ligases (E3s) are pivotal specificity determinants in the ubiquitin system by selecting substrates and decorating them with distinct ubiquitin signals. However, structure determination of ...Ubiquitin ligases (E3s) are pivotal specificity determinants in the ubiquitin system by selecting substrates and decorating them with distinct ubiquitin signals. However, structure determination of the underlying, specific E3-substrate complexes has proven challenging owing to their transient nature. In particular, it is incompletely understood how members of the catalytic cysteine-driven class of HECT-type ligases (HECTs) position substrate proteins for modification. Here, we report a cryogenic electron microscopy (cryo-EM) structure of the full-length human HECT HACE1, along with solution-based conformational analyses by small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry. Structure-based functional analyses in vitro and in cells reveal that the activity of HACE1 is stringently regulated by dimerization-induced autoinhibition. The inhibition occurs at the first step of the catalytic cycle and is thus substrate-independent. We use mechanism-based chemical crosslinking to reconstitute a complex of activated, monomeric HACE1 with its major substrate, RAC1, determine its structure by cryo-EM and validate the binding mode by solution-based analyses. Our findings explain how HACE1 achieves selectivity in ubiquitinating the active, GTP-loaded state of RAC1 and establish a framework for interpreting mutational alterations of the HACE1-RAC1 interplay in disease. More broadly, this work illuminates central unexplored aspects in the architecture, conformational dynamics, regulation and specificity of full-length HECTs.
History
DepositionJul 20, 2023-
Header (metadata) releaseJan 10, 2024-
Map releaseJan 10, 2024-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17994.png

Downloads & links

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Map

FileDownload / File: emd_17994.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.834 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-1.0752301 - 2.451095
Average (Standard dev.)0.0076297545 (±0.079607114)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 250.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_17994_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_17994_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

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

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Supramolecule #1: HACE1

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

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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: 102.506719 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: GMERAMEQLN RLTRSLRRAR TVELPEDNET AVYTLMPMVM ADQHRSVSEL LSNSKFDVNY AFGRVKRSLL HIAANCGSVE CLVLLLKKG ANPNYQDISG CTPLHLAARN GQKKCMSKLL EYSADVNICN NEGLTAIHWL AVNGRTELLH DLVQHVSDVD V EDAMGQTA ...String:
GMERAMEQLN RLTRSLRRAR TVELPEDNET AVYTLMPMVM ADQHRSVSEL LSNSKFDVNY AFGRVKRSLL HIAANCGSVE CLVLLLKKG ANPNYQDISG CTPLHLAARN GQKKCMSKLL EYSADVNICN NEGLTAIHWL AVNGRTELLH DLVQHVSDVD V EDAMGQTA LHVACQNGHK TTVQCLLDSG ADINRPNVSG ATPLYFACSH GQRDTAQILL LRGAKYLPDK NGVTPLDLCV QG GYGETCE VLIQYHPRLF QTIIQMTQNE DLRENMLRQV LEHLSQQSES QYLKILTSLA EVATTNGHKL LSLSSNYDAQ MKS LLRIVR MFCHVFRIGP SSPSNGIDMG YNGNKTPRSQ VFKPLELLWH SLDEWLVLIA TELMKNKRDS TEITSILLKQ KGQD QDAAS IPPFEPPGPG SYENLSTGTR ESKPDALAGR QEASADCQDV ISMTANRLSA VIQAFYMCCS CQMPPGMTSP RFIEF VCKH DEVLKCFVNR NPKIIFDHFH FLLECPELMS RFMHIIKAQP FKDRCEWFYE HLHSGQPDSD MVHRPVNEND ILLVHR DSI FRSSCEVVSK ANCAKLKQGI AVRFHGEEGM GQGVVREWFD ILSNEIVNPD YALFTQSADG TTFQPNSNSY VNPDHLN YF RFAGQILGLA LNHRQLVNIY FTRSFYKHIL GIPVNYQDVA SIDPEYAKNL QWILDNDISD LGLELTFSVE TDVFGAME E VPLKPGGGSI LVTQNNKAEY VQLVTELRMT RAIQPQINAF LQGFHMFIPP SLIQLFDEYE LELLLSGMPE IDVSDWIKN TEYTSGYERE DPVIQWFWEV VEDITQEERV LLLQFVTGSS RVPHGGFANI MGGSGLQNFT IAAVPYTPNL LPTSSTCINM LKLPEYPSK EILKDRLLVA LHCGSYGYTM A

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

Concentration0.7 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
50.0 mMHEPES
30.0 mMNaClSodium chloride
1.0 mMDTT
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 105000
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 29748 / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3639240
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.73 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4) / Number images used: 118791
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8pwl:
Cryo-EM structure of a full-length HACE1 dimer

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