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- EMDB-62221: Cryo-EM structure of LGR4-RSPO2-ZNRF3 complex (2:2:2) -

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Entry
Database: EMDB / ID: EMD-62221
TitleCryo-EM structure of LGR4-RSPO2-ZNRF3 complex (2:2:2)
Map data
Sample
  • Complex: LGR4-RSPO2-ZNRF3 complex (2:2:2)
    • Protein or peptide: Leucine-rich repeat-containing G-protein coupled receptor 4
    • Protein or peptide: E3 ubiquitin-protein ligase ZNRF3
    • Protein or peptide: R-spondin-2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsWnt signal / MEMBRANE PROTEIN
Function / homology
Function and homology information


trachea cartilage morphogenesis / negative regulation of odontogenesis of dentin-containing tooth / lung growth / regulation of Wnt signaling pathway, planar cell polarity pathway / Wnt receptor catabolic process / metanephric glomerulus development / metanephric nephron tubule morphogenesis / negative regulation of non-canonical Wnt signaling pathway / epithelial cell proliferation involved in renal tubule morphogenesis / protein-hormone receptor activity ...trachea cartilage morphogenesis / negative regulation of odontogenesis of dentin-containing tooth / lung growth / regulation of Wnt signaling pathway, planar cell polarity pathway / Wnt receptor catabolic process / metanephric glomerulus development / metanephric nephron tubule morphogenesis / negative regulation of non-canonical Wnt signaling pathway / epithelial cell proliferation involved in renal tubule morphogenesis / protein-hormone receptor activity / intestinal stem cell homeostasis / BMP receptor binding / negative regulation of toll-like receptor signaling pathway / positive regulation of branching involved in ureteric bud morphogenesis / male genitalia development / bone remodeling / dopaminergic neuron differentiation / frizzled binding / embryonic forelimb morphogenesis / embryonic hindlimb morphogenesis / digestive tract development / limb development / negative regulation of cold-induced thermogenesis / negative regulation of cytokine production / epithelial tube branching involved in lung morphogenesis / bone mineralization / hair follicle development / positive regulation of Wnt signaling pathway / canonical Wnt signaling pathway / Regulation of FZD by ubiquitination / stem cell proliferation / circadian regulation of gene expression / negative regulation of canonical Wnt signaling pathway / G protein-coupled receptor activity / RING-type E3 ubiquitin transferase / Wnt signaling pathway / ubiquitin-protein transferase activity / osteoblast differentiation / ubiquitin protein ligase activity / transmembrane signaling receptor activity / positive regulation of canonical Wnt signaling pathway / heparin binding / ubiquitin-dependent protein catabolic process / spermatogenesis / protein ubiquitination / signaling receptor binding / innate immune response / cell surface / extracellular space / extracellular region / zinc ion binding / plasma membrane
Similarity search - Function
E3 ubiquitin-protein ligase ZNRF3, Zinc finger, RING-type / ZNRF-3, ectodomain / ZNRF-3 Ectodomain / : / R-spondin, Fu-CRD domain / : / Furin-like repeat, cysteine-rich / Glycoprotein hormone receptor family / Ring finger domain / Leucine rich repeat N-terminal domain ...E3 ubiquitin-protein ligase ZNRF3, Zinc finger, RING-type / ZNRF-3, ectodomain / ZNRF-3 Ectodomain / : / R-spondin, Fu-CRD domain / : / Furin-like repeat, cysteine-rich / Glycoprotein hormone receptor family / Ring finger domain / Leucine rich repeat N-terminal domain / Leucine-rich repeat N-terminal domain / Leucine rich repeat N-terminal domain / Thrombospondin type-1 (TSP1) repeat superfamily / Thrombospondin type-1 (TSP1) repeat profile. / Thrombospondin type 1 repeats / Thrombospondin type-1 (TSP1) repeat / Leucine Rich Repeat / Leucine-rich repeats, bacterial type / Leucine-rich repeat, SDS22-like subfamily / Furin-like repeat / Furin-like repeats / Leucine rich repeat / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / Ring finger / Growth factor receptor cysteine-rich domain superfamily / Leucine-rich repeat / Leucine-rich repeat domain superfamily / Zinc finger RING-type profile. / Zinc finger, RING-type / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
R-spondin-2 / Leucine-rich repeat-containing G-protein coupled receptor 4 / E3 ubiquitin-protein ligase ZNRF3
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.59 Å
AuthorsPeng Y / Fujimura A / Asami J / Zhang Z / Shimizu T / Ohto U
Funding support Japan, 1 items
OrganizationGrant numberCountry
Ministry of Education, Culture, Sports, Science and Technology (Japan)22K15046 and 24K09349 (Z.Z.); 22H05184 and 23H00366 (T.S.); and 22H02556 (U.O.) Japan
CitationJournal: Nat Commun / Year: 2025
Title: Structural insights into Wnt/β-catenin signaling regulation by LGR4, R-spondin, and ZNRF3.
Authors: Yuxuan Peng / Akiko Fujimura / Jinta Asami / Zhikuan Zhang / Toshiyuki Shimizu / Umeharu Ohto /
Abstract: Leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) plays a critical role in regulating the wingless-related integration site (Wnt) signaling pathway and is essential for organ ...Leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) plays a critical role in regulating the wingless-related integration site (Wnt) signaling pathway and is essential for organ development and carcinogenesis. LGR4, along with its ligand R-spondin (RSPO), potentiates Wnt/β-catenin signaling by recruiting its signaling suppressor, E3 ligase Zinc and Ring Finger 3 (ZNRF3), and inducing its membrane clearance. However, detailed mechanisms underlying this process remain unknown. In this study, we present the cryo-electron microscopy structures of human LGR4, the LGR4-RSPO2 and LGR4-RSPO2-ZNRF3 complexes. Upon RSPO2 binding, LGR4 undergoes no significant conformational changes in its transmembrane and extracellular domain structures or their relative orientations. LGR4, RSPO2, and ZNRF3 assemble into a 2:2:2 complex with the ZNRF3 dimer enclosed at the center. This ternary arrangement and forced dimerization of ZNRF3 likely underpin how LGR4 and RSPO2 potentiate Wnt/β-catenin signaling by sequestering ZNRF3 from Wnt receptors and facilitating its auto-inactivation. This study provides a structural basis for understanding the regulatory mechanism of Wnt/β-catenin signaling through the LGR4-RSPO2-ZNRF3 pathway and may offer opportunities for future drug development targeting this axis.
History
DepositionOct 30, 2024-
Header (metadata) releaseOct 15, 2025-
Map releaseOct 15, 2025-
UpdateOct 15, 2025-
Current statusOct 15, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_62221.png

Downloads & links

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Map

FileDownload / File: emd_62221.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.25 Å/pix.
x 280 pix.
= 348.6 Å		1.25 Å/pix.
x 280 pix.
= 348.6 Å		1.25 Å/pix.
x 280 pix.
= 348.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.245 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.137
Minimum - Maximum-0.32820752 - 0.6349937
Average (Standard dev.)0.00080949906 (±0.017049134)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 348.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_62221_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_62221_half_map_2.map
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Sample components

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Entire : LGR4-RSPO2-ZNRF3 complex (2:2:2)

EntireName: LGR4-RSPO2-ZNRF3 complex (2:2:2)
Components
  • Complex: LGR4-RSPO2-ZNRF3 complex (2:2:2)
    • Protein or peptide: Leucine-rich repeat-containing G-protein coupled receptor 4
    • Protein or peptide: E3 ubiquitin-protein ligase ZNRF3
    • Protein or peptide: R-spondin-2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: LGR4-RSPO2-ZNRF3 complex (2:2:2)

SupramoleculeName: LGR4-RSPO2-ZNRF3 complex (2:2:2) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Leucine-rich repeat-containing G-protein coupled receptor 4

MacromoleculeName: Leucine-rich repeat-containing G-protein coupled receptor 4
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 93.372047 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MPGPLGLLCF LALGLLGSAG PSGAAPPLCA APCSCDGDRR VDCSGKGLTA VPEGLSAFTQ ALDISMNNIT QLPEDAFKNF PFLEELQLA GNDLSFIHPK ALSGLKELKV LTLQNNQLKT VPSEAIRGLS ALQSLRLDAN HITSVPEDSF EGLVQLRHLW L DDNSLTEV ...String:
MPGPLGLLCF LALGLLGSAG PSGAAPPLCA APCSCDGDRR VDCSGKGLTA VPEGLSAFTQ ALDISMNNIT QLPEDAFKNF PFLEELQLA GNDLSFIHPK ALSGLKELKV LTLQNNQLKT VPSEAIRGLS ALQSLRLDAN HITSVPEDSF EGLVQLRHLW L DDNSLTEV PVHPLSNLPT LQALTLALNK ISSIPDFAFT NLSSLVVLHL HNNKIRSLSQ HCFDGLDNLE TLDLNYNNLG EF PQAIKAL PSLKELGFHS NSISVIPDGA FDGNPLLRTI HLYDNPLSFV GNSAFHNLSD LHSLVIRGAS MVQQFPNLTG TVH LESLTL TGTKISSIPN NLCQEQKMLR TLDLSYNNIR DLPSFNGCHA LEEISLQRNQ IYQIKEGTFQ GLISLRILDL SRNL IHEIH SRAFATLGPI TNLDVSFNEL TSFPTEGLNG LNQLKLVGNF KLKEALAAKD FVNLRSLSVP YAYQCCAFWG CDSYA NLNT EDNSLQDHSV AQEKGTADAA NVTSTLENEE HSQIIIHCTP STGAFKPCEY LLGSWMIRLT VWFIFLVALF FNLLVI LTT FASCTSLPSS KLFIGLISVS NLFMGIYTGI LTFLDAVSWG RFAEFGIWWE TGSGCKVAGF LAVFSSESAI FLLMLAT VE RSLSAKDIMK NGKSNHLKQF RVAALLAFLG ATVAGCFPLF HRGEYSASPL CLPFPTGETP SLGFTVTLVL LNSLAFLL M AVIYTKLYCN LEKEDLSENS QSSMIKHVAW LIFTNCIFFC PVAFFSFAPL ITAISISPEI MKSVTLIFFP LPACLNPVL YVFFNPKFKE DWKLLKRRVT KKENLYFQGD YKDDDDKHHH HHHHH

UniProtKB: Leucine-rich repeat-containing G-protein coupled receptor 4

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Macromolecule #2: E3 ubiquitin-protein ligase ZNRF3

MacromoleculeName: E3 ubiquitin-protein ligase ZNRF3 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: RING-type E3 ubiquitin transferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.888084 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: METGLRWLLL VAVLKGVQCK ETAFVEVVLF ESSPSGDYTT YTTGLTGRFS RAGATLSAEG EIVQMHPLGL CNNNDEEDLY EYGWVGVVK LEQPELDPKP CLTVLGKAKR AVQRGATAVI FDVSENPEAI DQLNQGSEDP LKRPVVYVKG ADAIKLMNIV N KQKVARAR ...String:
METGLRWLLL VAVLKGVQCK ETAFVEVVLF ESSPSGDYTT YTTGLTGRFS RAGATLSAEG EIVQMHPLGL CNNNDEEDLY EYGWVGVVK LEQPELDPKP CLTVLGKAKR AVQRGATAVI FDVSENPEAI DQLNQGSEDP LKRPVVYVKG ADAIKLMNIV N KQKVARAR IQHRPPRQPT EYFDMGIFLA FFVVVSLVCL ILLVKIKLKQ RRSQNSMNRL AVQALEKMET RKF

UniProtKB: E3 ubiquitin-protein ligase ZNRF3

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Macromolecule #3: R-spondin-2

MacromoleculeName: R-spondin-2 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 18.953715 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
MQFRLFSFAL IILNCMDYSH CQGNRWRRSK RASYVSNPIC KGCLSCSKDN GCSRCQQKLF FFLRREGMRQ YGECLHSCPS GYYGHRAPD MNRCARCRIE NCDSCFSKDF CTKCKVGFYL HRGRCFDECP DGFAPLEETM ECVEENLYFQ GHHHHHHHHH H

UniProtKB: R-spondin-2

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 4 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 48.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.59 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 139067
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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