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Entry
Database: EMDB / ID: EMD-32837
TitleTethered peptide activation mechanism of adhesion GPCRs ADGRG2 and ADGRG4
Map dataCryo-EM structure of the ADGRG4-beta-Gs complex
Sample
  • Complex: Cryo-EM structure of the ADGR4-beta-Gs complex
    • Complex: Gs
      • Protein or peptide: mini-Gs
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
      • Protein or peptide: Adhesion G-protein coupled receptor G4,Uncharacterized protein
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
    • Complex: scFv16
      • Protein or peptide: scFv16
    • Complex: Nanobody-35Single-domain antibody
      • Protein or peptide: Nanobody-35Single-domain antibody
Function / homology
Function and homology information


plasma membrane => GO:0005886 / bioluminescence / generation of precursor metabolites and energy / G protein-coupled receptor activity / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G-protein activation ...plasma membrane => GO:0005886 / bioluminescence / generation of precursor metabolites and energy / G protein-coupled receptor activity / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / adenylate cyclase-activating G protein-coupled receptor signaling pathway / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / Glucagon-type ligand receptors / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / sensory perception of taste / GPER1 signaling / G-protein beta-subunit binding / heterotrimeric G-protein complex / Inactivation, recovery and regulation of the phototransduction cascade / extracellular vesicle / G alpha (12/13) signalling events / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / GTPase binding / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (i) signalling events / fibroblast proliferation / G alpha (s) signalling events / G alpha (q) signalling events / Ras protein signal transduction / cell population proliferation / Extra-nuclear estrogen signaling / membrane => GO:0016020 / cell surface receptor signaling pathway / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / synapse / protein-containing complex binding / signal transduction / extracellular exosome / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Pentaxin family / Pentraxin / C-reactive protein / pentaxin family / Pentraxin-related / Pentraxin (PTX) domain profile. / GAIN domain superfamily / GPCR proteolysis site, GPS, motif / GPS motif / GPS domain profile. / G-protein-coupled receptor proteolytic site domain / GPCR family 2, extracellular hormone receptor domain superfamily ...Pentaxin family / Pentraxin / C-reactive protein / pentaxin family / Pentraxin-related / Pentraxin (PTX) domain profile. / GAIN domain superfamily / GPCR proteolysis site, GPS, motif / GPS motif / GPS domain profile. / G-protein-coupled receptor proteolytic site domain / GPCR family 2, extracellular hormone receptor domain superfamily / G-protein coupled receptors family 2 signature 2. / GPCR, family 2, secretin-like, conserved site / GPCR, family 2, secretin-like / 7 transmembrane receptor (Secretin family) / GPCR, family 2-like / G-protein coupled receptors family 2 profile 2. / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / Concanavalin A-like lectin/glucanase domain superfamily / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Uncharacterized protein / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Adhesion G-protein coupled receptor G4
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others) / Lama glama (llama) / Psychromonas sp. B3M02 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGuo SC / Huang SM / He QT / Xiao P / Sun JP / Yu X
Funding support China, 2 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31971195 China
National Natural Science Foundation of China (NSFC)11922410 China
CitationJournal: Nature / Year: 2022
Title: Tethered peptide activation mechanism of the adhesion GPCRs ADGRG2 and ADGRG4.
Authors: Peng Xiao / Shengchao Guo / Xin Wen / Qing-Tao He / Hui Lin / Shen-Ming Huang / Lu Gou / Chao Zhang / Zhao Yang / Ya-Ni Zhong / Chuan-Cheng Yang / Yu Li / Zheng Gong / Xiao-Na Tao / Zhi- ...Authors: Peng Xiao / Shengchao Guo / Xin Wen / Qing-Tao He / Hui Lin / Shen-Ming Huang / Lu Gou / Chao Zhang / Zhao Yang / Ya-Ni Zhong / Chuan-Cheng Yang / Yu Li / Zheng Gong / Xiao-Na Tao / Zhi-Shuai Yang / Yan Lu / Shao-Long Li / Jun-Yan He / Chuanxin Wang / Lei Zhang / Liangliang Kong / Jin-Peng Sun / Xiao Yu /
Abstract: Adhesion G protein-coupled receptors (aGPCRs) constitute an evolutionarily ancient family of receptors that often undergo autoproteolysis to produce α and β subunits. A tethered agonism mediated by ...Adhesion G protein-coupled receptors (aGPCRs) constitute an evolutionarily ancient family of receptors that often undergo autoproteolysis to produce α and β subunits. A tethered agonism mediated by the 'Stachel sequence' of the β subunit has been proposed to have central roles in aGPCR activation. Here we present three cryo-electron microscopy structures of aGPCRs coupled to the G heterotrimer. Two of these aGPCRs are activated by tethered Stachel sequences-the ADGRG2-β-G complex and the ADGRG4-β-G complex (in which β indicates the β subunit of the aGPCR)-and the other is the full-length ADGRG2 in complex with the exogenous ADGRG2 Stachel-sequence-derived peptide agonist IP15 (ADGRG2(FL)-IP15-G). The Stachel sequences of both ADGRG2-β and ADGRG4-β assume a U shape and insert deeply into the seven-transmembrane bundles. Constituting the FXφφφXφ motif (in which φ represents a hydrophobic residue), five residues of ADGRG2-β or ADGRG4-β extend like fingers to mediate binding to the seven-transmembrane domain and activation of the receptor. The structure of the ADGRG2(FL)-IP15-G complex reveals the structural basis for the improved binding affinity of IP15 compared with VPM-p15 and indicates that rational design of peptidic agonists could be achieved by exploiting aGPCR-β structures. By converting the 'finger residues' to acidic residues, we develop a method to generate peptidic antagonists towards several aGPCRs. Collectively, our study provides structural and biochemical insights into the tethered activation mechanism of aGPCRs.
History
DepositionFeb 8, 2022-
Header (metadata) releaseApr 27, 2022-
Map releaseApr 27, 2022-
UpdateMay 11, 2022-
Current statusMay 11, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32837.png

Downloads & links

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Map

FileDownload / File: emd_32837.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the ADGRG4-beta-Gs complex
Voxel sizeX=Y=Z: 1.5 Å
Density
Contour LevelBy AUTHOR: 0.045
Minimum - Maximum-0.25658363 - 0.3859321
Average (Standard dev.)5.0322433e-05 (±0.013754968)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 192.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Cryo-EM structure of the ADGR4-beta-Gs complex

EntireName: Cryo-EM structure of the ADGR4-beta-Gs complex
Components
  • Complex: Cryo-EM structure of the ADGR4-beta-Gs complex
    • Complex: Gs
      • Protein or peptide: mini-Gs
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
      • Protein or peptide: Adhesion G-protein coupled receptor G4,Uncharacterized protein
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
    • Complex: scFv16
      • Protein or peptide: scFv16
    • Complex: Nanobody-35Single-domain antibody
      • Protein or peptide: Nanobody-35Single-domain antibody

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Supramolecule #1: Cryo-EM structure of the ADGR4-beta-Gs complex

SupramoleculeName: Cryo-EM structure of the ADGR4-beta-Gs complex / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Gs

SupramoleculeName: Gs / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1-#3, #5
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Baculovirus expression vector pFastBac1-HM

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Supramolecule #3: scFv16

SupramoleculeName: scFv16 / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Baculovirus expression vector pFastBac1-HM

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Supramolecule #4: Nanobody-35

SupramoleculeName: Nanobody-35 / type: complex / Chimera: Yes / ID: 4 / Parent: 1 / Macromolecule list: #6
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia phage EcSzw-2 (virus)

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Macromolecule #1: mini-Gs

MacromoleculeName: mini-Gs / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.879465 KDa
Recombinant expressionOrganism: Baculovirus expression vector pFastBac1-HM
SequenceString: MGCTLSAEDK AAVERSKMIE KQLQKDKQVY RATHRLLLLG ADNSGKSTIV KQMRIYHVNG YSEEECKQYK AVVYSNTIQS IIAIIRAMG RLKIDFGDSA RADDARQLFV LAGAAEEGFM TAELAGVIKR LWKDSGVQAC FNRSREYQLN DSAAYYLNDL D RIAQPNYI ...String:
MGCTLSAEDK AAVERSKMIE KQLQKDKQVY RATHRLLLLG ADNSGKSTIV KQMRIYHVNG YSEEECKQYK AVVYSNTIQS IIAIIRAMG RLKIDFGDSA RADDARQLFV LAGAAEEGFM TAELAGVIKR LWKDSGVQAC FNRSREYQLN DSAAYYLNDL D RIAQPNYI PTQQDVLRTR VKTSGIFETK FQVDKVNFHM FDVGAQRDER RKWIQCFNDV TAIIFVVDSS DYNRLQEALN DF KSIWNNR WLRTISVILF LNKQDLLAEK VLAGKSKIED YFPEFARYTT PEDATPEPGE DPRVTRAKYF IRDEFLRIST ASG DGRHYC YPHFTCSVDT ENARRIFNDC RDIIQRMHLR QYELL

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Macromolecule #2: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 39.48916 KDa
Recombinant expressionOrganism: Baculovirus expression vector pFastBac1-HM
SequenceString: MHHHHHHLEV LFQGPGSSQS ELDQLRQEAE QLKNQIRDAR KACADATLSQ ITNNIDPVGR IQMRTRRTLR GHLAKIYAMH WGTDSRLLV SASQDGKLII WDSYTTNKVH AIPLRSSWVM TCAYAPSGNY VACGGLDNIC SIYNLKTREG NVRVSRELAG H TGYLSCCR ...String:
MHHHHHHLEV LFQGPGSSQS ELDQLRQEAE QLKNQIRDAR KACADATLSQ ITNNIDPVGR IQMRTRRTLR GHLAKIYAMH WGTDSRLLV SASQDGKLII WDSYTTNKVH AIPLRSSWVM TCAYAPSGNY VACGGLDNIC SIYNLKTREG NVRVSRELAG H TGYLSCCR FLDDNQIVTS SGDTTCALWD IETGQQTTTF TGHTGDVMSL SLAPDTRLFV SGACDASAKL WDVREGMCRQ TF TGHESDI NAICFFPNGN AFATGSDDAT CRLFDLRADQ ELMTYSHDNI ICGITSVSFS KSGRLLLAGY DDFNCNVWDA LKA DRAGVL AGHDNRVSCL GVTDDGMAVA TGSWDSFLKI WN

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Macromolecule #3: Adhesion G-protein coupled receptor G4,Uncharacterized protein

MacromoleculeName: Adhesion G-protein coupled receptor G4,Uncharacterized protein
type: protein_or_peptide / ID: 3
Details: residues 3081-3088 = His tag, residues 3089-3091 = linker, residues 3092-3098 = TEV site
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Psychromonas sp. B3M02 (bacteria)
Molecular weightTheoretical: 71.627969 KDa
Recombinant expressionOrganism: Baculovirus expression vector pFastBac1-HM
SequenceString: MKTIIALSYI FCLVFAHLTH FGVLMDLSRS TVDSVNEQIL ALITYTGCGI SSIFLGVAVV TYIAFHKLRK DYPAKILINL CTALLMLNL VFLINSWLSS FQKVGVCITA AVALHYFLLV SFTWMGLEAV HMYLALVKVF NIYIPNYILK FCLVGWGIPA I MVAITVSV ...String:
MKTIIALSYI FCLVFAHLTH FGVLMDLSRS TVDSVNEQIL ALITYTGCGI SSIFLGVAVV TYIAFHKLRK DYPAKILINL CTALLMLNL VFLINSWLSS FQKVGVCITA AVALHYFLLV SFTWMGLEAV HMYLALVKVF NIYIPNYILK FCLVGWGIPA I MVAITVSV KKDLYGTLSP TTPFCWIKDD SIFYISVVAY FCLIFLMNLS MFCTVLVQLN SVKSQIQKTR RKMILHDLKG TM SLTFLLG LTWGFAFFAW GPMRNFFLYL FAIFNTLQGF FIFVFHCVMK ESVREQWQIH LCCGWLRLDN SSDGSSRCQI KVG YKQEGL KKIFEHKLLT PSLKSTATSS TFKSLGSAQG TPSEISFPND DFDKDPYCSS PHHHHHHHHG SAENLYFQGM VSKG EEDNM AIIKEFMRFK VHMEGSVNGH EFEIEGEGEG RPYEGTQTAK LKVTKGGPLP FAWDILSPQF MYGSKAYVKH PADIP DYLK LSFPEGFKWE RVMNFEDGGV VTVTQDSSLQ DGEFIYKVKL RGTNFPSDGP VMQKKTMGWE ASSERMYPED GALKGE IKQ RLKLKDGGHY DAEVKTTYKA KKPVQLPGAY NVNIKLDITS HNEDYTIVEQ YERAEGRHST GGMDELYK

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Macromolecule #4: scFv16

MacromoleculeName: scFv16 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 26.610615 KDa
Recombinant expressionOrganism: Baculovirus expression vector pFastBac1-HM
SequenceString: DVQLVESGGG LVQPGGSRKL SCSASGFAFS SFGMHWVRQA PEKGLEWVAY ISSGSGTIYY ADTVKGRFTI SRDDPKNTLF LQMTSLRSE DTAMYYCVRS IYYYGSSPFD FWGQGTTLTV SSGGGGSGGG GSGGGGSDIV MTQATSSVPV TPGESVSISC R SSKSLLHS ...String:
DVQLVESGGG LVQPGGSRKL SCSASGFAFS SFGMHWVRQA PEKGLEWVAY ISSGSGTIYY ADTVKGRFTI SRDDPKNTLF LQMTSLRSE DTAMYYCVRS IYYYGSSPFD FWGQGTTLTV SSGGGGSGGG GSGGGGSDIV MTQATSSVPV TPGESVSISC R SSKSLLHS NGNTYLYWFL QRPGQSPQLL IYRMSNLASG VPDRFSGSGS GTAFTLTISR LEAEDVGVYY CMQHLEYPLT FG AGTKLEL KGS

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Macromolecule #5: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 7.861143 KDa
Recombinant expressionOrganism: Baculovirus expression vector pFastBac1-HM
SequenceString:
MASNNTASIA QARKLVEQLK MEANIDRIKV SKAAADLMAY CEAHAKEDPL LTPVPASENP FREKKFFCAI L

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Macromolecule #6: Nanobody-35

MacromoleculeName: Nanobody-35 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 13.885439 KDa
Recombinant expressionOrganism: Escherichia phage EcSzw-2 (virus)
SequenceString:
QVQLQESGGG LVQPGGSLRL SCAASGFTFS NYKMNWVRQA PGKGLEWVSD ISQSGASISY TGSVKGRFTI SRDNAKNTLY LQMNSLKPE DTAVYYCARC PAPFTRDCFD VTSTTYAYRG QGTQVTVSS

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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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 64.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7d77

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 619803

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