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- EMDB-9501: Three-dimensional reconstruction of human LRP6 ectodomain complex... -

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Basic information

Entry
Database: EMDB / ID: EMD-9501
TitleThree-dimensional reconstruction of human LRP6 ectodomain complexed with Dkk1
Map data
Sample
  • Complex: Human LRP6 ectodomain complexed with full length of Dkk1
    • Complex: LRP6 ectodomain
      • Protein or peptide: Low-density lipoprotein receptor-related protein 6
      • Protein or peptide: Low-density lipoprotein receptor-related protein 6
    • Complex: Human Dickkopf-related protein1
      • Protein or peptide: Dickkopf-related protein 1
  • Ligand: N-ACETYL-D-GLUCOSAMINEN-Acetylglucosamine
  • Ligand: PHOSPHATE IONPhosphate
  • Ligand: ALPHA-L-FUCOSE
  • Ligand: GLYCEROL
  • Ligand: water
Function / homology
Function and homology information


negative regulation of mesodermal cell fate specification / regulation of endodermal cell fate specification / positive regulation of Wnt signaling pathway, calcium modulating pathway / negative regulation of Wnt-Frizzled-LRP5/6 complex assembly / positive regulation of midbrain dopaminergic neuron differentiation / negative regulation of presynapse assembly / Signaling by LRP5 mutants / Wnt signaling pathway involved in somitogenesis / regulation of dopaminergic neuron differentiation / motor learning ...negative regulation of mesodermal cell fate specification / regulation of endodermal cell fate specification / positive regulation of Wnt signaling pathway, calcium modulating pathway / negative regulation of Wnt-Frizzled-LRP5/6 complex assembly / positive regulation of midbrain dopaminergic neuron differentiation / negative regulation of presynapse assembly / Signaling by LRP5 mutants / Wnt signaling pathway involved in somitogenesis / regulation of dopaminergic neuron differentiation / motor learning / Wnt-Frizzled-LRP5/6 complex / negative regulation of cardiac muscle cell differentiation / Negative regulation of TCF-dependent signaling by WNT ligand antagonists / endoderm formation / synapse pruning / Signaling by RNF43 mutants / neural crest formation / receptor-mediated endocytosis involved in cholesterol transport / endocardial cushion development / regulation of receptor internalization / heart induction / receptor antagonist activity / kinase inhibitor activity / Wnt receptor activity / low-density lipoprotein particle receptor activity / toxin transmembrane transporter activity / co-receptor binding / negative regulation of smooth muscle cell apoptotic process / Wnt-protein binding / positive regulation of Wnt signaling pathway, planar cell polarity pathway / cellular response to cholesterol / midbrain dopaminergic neuron differentiation / negative regulation of protein serine/threonine kinase activity / dopaminergic neuron differentiation / heart valve development / frizzled binding / negative regulation of ossification / neural crest cell differentiation / Wnt signalosome / embryonic limb morphogenesis / face morphogenesis / Disassembly of the destruction complex and recruitment of AXIN to the membrane / low-density lipoprotein particle receptor binding / limb development / negative regulation of Wnt signaling pathway / negative regulation of SMAD protein signal transduction / mesoderm formation / negative regulation of BMP signaling pathway / hair follicle development / canonical Wnt signaling pathway / coreceptor activity / positive regulation of cell cycle / negative regulation of peptidyl-serine phosphorylation / regulation of neuron apoptotic process / response to retinoic acid / forebrain development / regulation of synaptic transmission, glutamatergic / Regulation of FZD by ubiquitination / negative regulation of protein binding / TCF dependent signaling in response to WNT / protein localization to plasma membrane / positive regulation of JNK cascade / negative regulation of canonical Wnt signaling pathway / growth factor activity / cell morphogenesis / cell-cell adhesion / response to peptide hormone / Wnt signaling pathway / positive regulation of DNA-binding transcription factor activity / negative regulation of neuron projection development / positive regulation of cytosolic calcium ion concentration / chemical synaptic transmission / cytoplasmic vesicle / early endosome membrane / learning or memory / membrane raft / signaling receptor binding / neuronal cell body / synapse / positive regulation of gene expression / negative regulation of apoptotic process / positive regulation of DNA-templated transcription / negative regulation of transcription by RNA polymerase II / cell surface / endoplasmic reticulum / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / extracellular space / extracellular region / identical protein binding / plasma membrane
Similarity search - Function
: / : / : / : / Dickkopf-related protein 1/2/4, C-terminal subdomain 2 / Dickkopf-related protein 1/2/4, C-terminal subdomain 1 / Dickkopf, N-terminal cysteine-rich / Dickkopf-like protein / Dickkopf N-terminal cysteine-rich region / Low density lipoprotein receptor-related protein 5/6 ...: / : / : / : / Dickkopf-related protein 1/2/4, C-terminal subdomain 2 / Dickkopf-related protein 1/2/4, C-terminal subdomain 1 / Dickkopf, N-terminal cysteine-rich / Dickkopf-like protein / Dickkopf N-terminal cysteine-rich region / Low density lipoprotein receptor-related protein 5/6 / Low-density lipoprotein receptor repeat class B / LDL-receptor class B (LDLRB) repeat profile. / LDLR class B repeat / Low-density lipoprotein-receptor YWTD domain / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A, conserved site / LDL-receptor class A (LDLRA) domain signature. / LDL-receptor class A (LDLRA) domain profile. / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A repeat / LDL receptor-like superfamily / Six-bladed beta-propeller, TolB-like / Coagulation Factor Xa inhibitory site / Epidermal growth factor-like domain. / EGF-like domain signature 2. / EGF-like domain
Similarity search - Domain/homology
Low-density lipoprotein receptor-related protein 6 / Dickkopf-related protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 21.0 Å
AuthorsMatoba K / Mihara E / Tamura-Kawakami K / Hirai H / Thompson S / Iwasaki K / Takagi J
CitationJournal: Cell Rep / Year: 2017
Title: Conformational Freedom of the LRP6 Ectodomain Is Regulated by N-glycosylation and the Binding of the Wnt Antagonist Dkk1.
Authors: Kyoko Matoba / Emiko Mihara / Keiko Tamura-Kawakami / Naoyuki Miyazaki / Shintaro Maeda / Hidenori Hirai / Samuel Thompson / Kenji Iwasaki / Junichi Takagi /
Abstract: LDL-receptor-related protein 6 (LRP6) is a single-pass membrane glycoprotein with a large modular ectodomain and forms a higher order signaling platform upon binding Wnt ligands on the cell surface. ...LDL-receptor-related protein 6 (LRP6) is a single-pass membrane glycoprotein with a large modular ectodomain and forms a higher order signaling platform upon binding Wnt ligands on the cell surface. Although multiple crystal structures are available for fragments of the LRP6 ectodomain, we lack a consensus view on the overall molecular architecture of the full-length LRP6 and its dynamic aspects. Here, we used negative-stain electron microscopy to probe conformational states of the entire ectodomain of LRP6 in solution and found that the four-module ectodomain undergoes a large bending motion hinged at the junction between the second and the third modules. Importantly, the extent of inter-domain motion is modulated by evolutionarily conserved N-glycan chains proximal to the joint. We also found that the LRP6 ectodomain becomes highly compact upon complexation with the Wnt antagonist Dkk1, suggesting a potential role for the ectodomain conformational change in the regulation of receptor oligomerization and signaling.
History
DepositionJun 29, 2016-
Header (metadata) releaseNov 2, 2016-
Map releaseJan 18, 2017-
UpdateJan 18, 2017-
Current statusJan 18, 2017Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.3
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  • Surface view with fitted model
  • Atomic models: PDB-5gje
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9501.png

Downloads & links

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Map

FileDownload / File: emd_9501.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.2 Å/pix.
x 200 pix.
= 440. Å		2.2 Å/pix.
x 200 pix.
= 440. Å		2.2 Å/pix.
x 200 pix.
= 440. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.198 / Movie #1: 0.3
Minimum - Maximum-0.079741 - 1.0378006
Average (Standard dev.)0.00088656624 (±0.036129534)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 440.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.22.22.2
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z440.000440.000440.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-190-190-190
NX/NY/NZ380380380
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0801.0380.001

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Supplemental data

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Sample components

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Entire : Human LRP6 ectodomain complexed with full length of Dkk1

EntireName: Human LRP6 ectodomain complexed with full length of Dkk1
Components
  • Complex: Human LRP6 ectodomain complexed with full length of Dkk1
    • Complex: LRP6 ectodomain
      • Protein or peptide: Low-density lipoprotein receptor-related protein 6
      • Protein or peptide: Low-density lipoprotein receptor-related protein 6
    • Complex: Human Dickkopf-related protein1
      • Protein or peptide: Dickkopf-related protein 1
  • Ligand: N-ACETYL-D-GLUCOSAMINEN-Acetylglucosamine
  • Ligand: PHOSPHATE IONPhosphate
  • Ligand: ALPHA-L-FUCOSE
  • Ligand: GLYCEROL
  • Ligand: water

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Supramolecule #1: Human LRP6 ectodomain complexed with full length of Dkk1

SupramoleculeName: Human LRP6 ectodomain complexed with full length of Dkk1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Human Dkk1 fused with human growth hormone at the N-terminal (hGH-Dkk1) was co-expressed with LRP6ec in the HEK293S GnT1- cells.
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293S GnTI- / Recombinant plasmid: pCDNA3.1/pEBMulti-Hyg
Molecular weightTheoretical: 210 KDa

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Supramolecule #2: LRP6 ectodomain

SupramoleculeName: LRP6 ectodomain / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293S GnTI- / Recombinant plasmid: pCDNA3.1

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Supramolecule #3: Human Dickkopf-related protein1

SupramoleculeName: Human Dickkopf-related protein1 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Details: N-terminal human growth hormone fused full length of Dkk1
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293S GnTI- / Recombinant plasmid: pEBMulti-Hyg

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Macromolecule #1: Low-density lipoprotein receptor-related protein 6

MacromoleculeName: Low-density lipoprotein receptor-related protein 6 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 69.034883 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: APLLLYANRR DLRLVDATNG KENATIVVGG LEDAAAVDFV FSHGLIYWSD VSEEAIKRTE FNKTESVQNV VVSGLLSPDG LACDWLGEK LYWTDSETNR IEVSNLDGSL RKVLFWQELD QPRAIALDPS SGFMYWTDWG EVPKIERAGM DGSSRFIIIN S EIYWPNGL ...String:
APLLLYANRR DLRLVDATNG KENATIVVGG LEDAAAVDFV FSHGLIYWSD VSEEAIKRTE FNKTESVQNV VVSGLLSPDG LACDWLGEK LYWTDSETNR IEVSNLDGSL RKVLFWQELD QPRAIALDPS SGFMYWTDWG EVPKIERAGM DGSSRFIIIN S EIYWPNGL TLDYEEQKLY WADAKLNFIH KSNLDGTNRQ AVVKGSLPHP FALTLFEDIL YWTDWSTHSI LACNKYTGEG LR EIHSDIF SPMDIHAFSQ QRQPNATNPC GIDNGGCSHL CLMSPVKPFY QCACPTGVKL LENGKTCKDG ATELLLLARR TDL RRISLD TPDFTDIVLQ LEDIRHAIAI DYDPVEGYIY WTDDEVRAIR RSFIDGSGSQ FVVTAQIAHP DGIAVDWVAR NLYW TDTGT DRIEVTRLNG TMRKILISED LEEPRAIVLD PMVGYMYWTD WGEIPKIERA ALDGSDRVVL VNTSLGWPNG LALDY DEGK IYWGDAKTDK IEVMNTDGTG RRVLVEDKIP HIFGFTLLGD YVYWTDWQRR SIERVHKRSA EREVIIDQLP DLMGLK ATN VHRVIGSNPC AEENGGCSHL CLYRPQGLRC ACPIGFELIS DMKTCIVP

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Macromolecule #2: Low-density lipoprotein receptor-related protein 6

MacromoleculeName: Low-density lipoprotein receptor-related protein 6 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 69.810609 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: EAFLLFSRRA DIRRISLETN NNNVAIPLTG VKEASALDFD VTDNRIYWTD ISLKTISRAF MNGSALEHVV EFGLDYPEGM AVDWLGKNL YWADTGTNRI EVSKLDGQHR QVLVWKDLDS PRALALDPAE GFMYWTEWGG KPKIDRAAMD GSERTTLVPN V GRANGLTI ...String:
EAFLLFSRRA DIRRISLETN NNNVAIPLTG VKEASALDFD VTDNRIYWTD ISLKTISRAF MNGSALEHVV EFGLDYPEGM AVDWLGKNL YWADTGTNRI EVSKLDGQHR QVLVWKDLDS PRALALDPAE GFMYWTEWGG KPKIDRAAMD GSERTTLVPN V GRANGLTI DYAKRRLYWT DLDTNLIESS NMLGLNREVI ADDLPHPFGL TQYQDYIYWT DWSRRSIERA NKTSGQNRTI IQ GHLDYVM DILVFHSSRQ SGWNECASSN GHCSHLCLAV PVGGFVCGCP AHYSLNADNR TCSAPTTFLL FSQKSAINRM VID EQQSPD IILPIHSLRN VRAIDYDPLD KQLYWIDSRQ NMIRKAQEDG SQGFTVVVSS VPSQNLEIQP YDLSIDIYSR YIYW TCEAT NVINVTRLDG RSVGVVLKGE QDRPRAIVVN PEKGYMYFTN LQERSPKIER AALDGTEREV LFFSGLSKPI ALALD SRLG KLFWADSDLR RIESSDLSGA NRIVLEDSNI LQPVGLTVFE NWLYWIDKQQ QMIEKIDMTG REGRTKVQAR IAQLSD IHA VKELNLQEYR QHPCAQDNGG CSHICLVKGD GTTRCSCPMH LVLLQDELSC GEP

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Macromolecule #3: Dickkopf-related protein 1

MacromoleculeName: Dickkopf-related protein 1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 9.607076 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
KGQEGSVCLR SSDCASGLCC ARHFWSKICK PVLKEGQVCT KHRRKGSHGL EIFQRCYCGE GLSCRIQKDH HQASNSSRLH TCQRH

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Macromolecule #4: N-ACETYL-D-GLUCOSAMINE

MacromoleculeName: N-ACETYL-D-GLUCOSAMINE / type: ligand / ID: 4 / Number of copies: 13 / Formula: NAG
Molecular weightTheoretical: 221.208 Da

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Macromolecule #5: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION / Phosphate

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Macromolecule #6: ALPHA-L-FUCOSE

MacromoleculeName: ALPHA-L-FUCOSE / type: ligand / ID: 6 / Number of copies: 1 / Formula: FUC
Molecular weightTheoretical: 164.156 Da
Chemical component information

ChemComp-AFL:
ALPHA-L-FUCOSE / Fucose

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Macromolecule #7: GLYCEROL

MacromoleculeName: GLYCEROL / type: ligand / ID: 7 / Number of copies: 1 / Formula: GOL
Molecular weightTheoretical: 92.094 Da
Chemical component information

ChemComp-GOL:
GLYCEROL / Glycerol

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 13 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.2
StainingType: NONE / Material: Uranyl acetate
GridMaterial: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeHITACHI H-9500SD
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.8 mm / Nominal magnification: 80000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Image recordingFilm or detector model: TVIPS TEMCAM-F224 (2k x 2k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Digitization - Sampling interval: 24.0 µm / Average exposure time: 2.0 sec. / Average electron dose: 20.0 e/Å2

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Image processing

Particle selectionDetails: 10974 tilt pairs for RCT
CTF correctionSoftware - Name: spider
Details: This was not done for RCT but for following single-particle reconstruction
Startup modelType of model: RANDOM CONICAL TILT / Random conical tilt - Number images: 10974
Initial angle assignmentType: PROJECTION MATCHING
Projection matching processing - Number reference projections: 83
Software - Name: spider
Details: The projections generated from RCT were used for initial angle assignment
Final angle assignmentType: PROJECTION MATCHING
Projection matching processing - Number reference projections: 83
Software - Name: spider
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: spider / Number images used: 5390
DetailsThe selected images were band-pass filtered.

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Atomic model buiding 1

Initial model
PDB IDChain

4dg6

chain_id: A, residue_range: 1-611

3s2k

chain_id: B, residue_range: 631-1243

3s2k

chain_id: C, residue_range: 182-266
RefinementProtocol: RIGID BODY FIT
Output model

PDB-5gje:
Three-dimensional reconstruction of human LRP6 ectodomain complexed with Dkk1

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