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- EMDB-30957: Cryo-EM structure of hDisp1NNN-3C-Cleavage -

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Basic information

Entry
Database: EMDB / ID: EMD-30957
TitleCryo-EM structure of hDisp1NNN-3C-Cleavage
Map data
Sample
  • Complex: hDisp1NNN-3C
    • Protein or peptide: Protein dispatched homolog 1
  • Protein or peptide: Protein dispatched homolog 1
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Keywordsmembrane protein / LIPID TRANSPORT
Function / homology
Function and homology information


patched ligand maturation / diaphragm development / embryonic pattern specification / peptide transmembrane transporter activity / dorsal/ventral pattern formation / determination of left/right symmetry / peptide transport / smoothened signaling pathway / regulation of protein secretion / protein homotrimerization ...patched ligand maturation / diaphragm development / embryonic pattern specification / peptide transmembrane transporter activity / dorsal/ventral pattern formation / determination of left/right symmetry / peptide transport / smoothened signaling pathway / regulation of protein secretion / protein homotrimerization / basolateral plasma membrane / membrane
Similarity search - Function
: / Protein patched/dispatched / Patched family / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain
Similarity search - Domain/homology
Protein dispatched homolog 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.68 Å
AuthorsLi W / Wang L
Funding support China, 2 items
OrganizationGrant numberCountry
Other government China
Other government China
CitationJournal: Nat Commun / Year: 2021
Title: Structural insights into proteolytic activation of the human Dispatched1 transporter for Hedgehog morphogen release.
Authors: Wanqiu Li / Linlin Wang / Bradley M Wierbowski / Mo Lu / Feitong Dong / Wenchen Liu / Sisi Li / Peiyi Wang / Adrian Salic / Xin Gong /
Abstract: The membrane protein Dispatched (Disp), which belongs to the RND family of small molecule transporters, is essential for Hedgehog (Hh) signaling, by catalyzing the extracellular release of palmitate- ...The membrane protein Dispatched (Disp), which belongs to the RND family of small molecule transporters, is essential for Hedgehog (Hh) signaling, by catalyzing the extracellular release of palmitate- and cholesterol-modified Hh ligands from producing cells. Disp function requires Furin-mediated proteolytic cleavage of its extracellular domain, but how this activates Disp remains obscure. Here, we employ cryo-electron microscopy to determine atomic structures of human Disp1 (hDisp1), before and after cleavage, and in complex with lipid-modified Sonic hedgehog (Shh) ligand. These structures, together with biochemical data, reveal that proteolytic cleavage opens the extracellular domain of hDisp1, removing steric hindrance to Shh binding. Structure-guided functional experiments demonstrate the role of hDisp1-Shh interactions in ligand release. Our results clarify the mechanisms of hDisp1 activation and Shh morphogen release, and highlight how a unique proteolytic cleavage event enabled acquisition of a protein substrate by a member of a family of small molecule transporters.
History
DepositionFeb 5, 2021-
Header (metadata) releaseDec 8, 2021-
Map releaseDec 8, 2021-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7e2h
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30957.png

Downloads & links

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Map

FileDownload / File: emd_30957.map.gz / Format: CCP4 / Size: 23.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 184 pix.
= 198.72 Å		1.08 Å/pix.
x 184 pix.
= 198.72 Å		1.08 Å/pix.
x 184 pix.
= 198.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.03
Minimum - Maximum-0.1350333 - 0.20365518
Average (Standard dev.)0.00060276076 (±0.0069585065)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions184184184
Spacing184184184
CellA=B=C: 198.72 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z184184184
origin x/y/z0.0000.0000.000
length x/y/z198.720198.720198.720
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS184184184
D min/max/mean-0.1350.2040.001

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Supplemental data

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Sample components

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Entire : hDisp1NNN-3C

EntireName: hDisp1NNN-3C
Components
  • Complex: hDisp1NNN-3C
    • Protein or peptide: Protein dispatched homolog 1
  • Protein or peptide: Protein dispatched homolog 1
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: hDisp1NNN-3C

SupramoleculeName: hDisp1NNN-3C / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 150 KDa

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Macromolecule #1: Protein dispatched homolog 1

MacromoleculeName: Protein dispatched homolog 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 29.497619 KDa
Recombinant expressionOrganism: Mammalian expression vector EGFP-MCS-pcDNA3.1 (others)
SequenceString: MAMSNGNNDF VVLSNSSIAT SAANPSPLTP CDGDHAAQQL TPKEATRTKV SPNGCLQLNG TVKSSFLPLD NQRMPQMLPQ CCHPCPYHH PLTSHSSHQE CHPEAGPAAP SALASCCMQP HSEYSASLCP NHSPVYQTTC CLQPSPSFCL HHPWPDHFQH Q PVQQHIAN ...String:
MAMSNGNNDF VVLSNSSIAT SAANPSPLTP CDGDHAAQQL TPKEATRTKV SPNGCLQLNG TVKSSFLPLD NQRMPQMLPQ CCHPCPYHH PLTSHSSHQE CHPEAGPAAP SALASCCMQP HSEYSASLCP NHSPVYQTTC CLQPSPSFCL HHPWPDHFQH Q PVQQHIAN IRPSRPFKLP KSYAALIADW PVVVLGMCTM FIVVCALVGV LVPELPDFSD PLLGFEPRGT AIGQRLVTWN NM VKNTGYK ATLANYPFKY ADEQASSLEV LFQ

UniProtKB: Protein dispatched homolog 1

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Macromolecule #2: Protein dispatched homolog 1

MacromoleculeName: Protein dispatched homolog 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 140.427594 KDa
Recombinant expressionOrganism: Mammalian expression vector EGFP-MCS-pcDNA3.1 (others)
SequenceString: GPGSEVDWNF HKDSFFCDVP SDRYSRVVFT SSGGETLWNL PAIKSMCNVD NSRIRSHPQF GDLCQRTTAA SCCPSWTLGN YIAILNNRS SCQKIVERDV SHTLKLLRTC AKHYQNGTLG PDCWDMAARR KDQLKCTNVP RKCTKYNAVY QILHYLVDKD F MTPKTADY ...String:
GPGSEVDWNF HKDSFFCDVP SDRYSRVVFT SSGGETLWNL PAIKSMCNVD NSRIRSHPQF GDLCQRTTAA SCCPSWTLGN YIAILNNRS SCQKIVERDV SHTLKLLRTC AKHYQNGTLG PDCWDMAARR KDQLKCTNVP RKCTKYNAVY QILHYLVDKD F MTPKTADY ATPALKYSML FSPTEKGESM MNIYLDNFEN WNSSDGVTTI TGIEFGIKHS LFQDYLLMDT VYPAIAIVIV LL VMCVYTK SMFITLMTMF AIISSLIVSY FLYRVVFHFE FFPFMNLTAL IILVGIGANN AFVLCDVWNY TKFDKPHAET SET VSITLQ HAALSMFVTS FTTAAAFYAN YVSNITAIRC FGVYAGTAIL VNYVLMVTWL PAVVVLHERY LLNIFTCFKK PQQQ IYDNK SCWTVACQKC HKVLFAISEA SRIFFEKVLP CIVIKFRYLW LFWFLALTVG GAYIVCINPK MKLPSLELSE FQVFR SSHP FERYDAEYKK LFMFERVHHG EELHMPITVI WGVSPEDNGN PLNPKSKGKL TLDSSFNIAS PASQAWILHF CQKLRN QTF FYQTDEQDFT SCFIETFKQW MENQDCDEPA LYPCCSHWSF PYKQEIFELC IKRAIMELER STGYHLDSKT PGPRFDI ND TIRAVVLEFQ STYLFTLAYE KMHQFYKEVD SWISSELSSA PEGLSNGWFV SNLEFYDLQD SLSDGTLIAM GLSVAVAF S VMLLTTWNII ISLYAIISIA GTIFVTVGSL VLLGWELNVL ESVTISVAVG LSVNFAVHYG VAYRLAPDPD REGKVIFSL SRVGSAMAMA ALTTFVAGAM MMPSTVLAYT QLGTFMMLIM CISWAFATFF FQCMCRCLGP QGTCGQIPLP KKLQCSAFSH ALSTSPSDK GQSKTHTINA YHLDPRGPKS ELEHEFYELE PLASHSCTAP EKTTYEETHI CSEFFNSQAK NLGMPVHAAY N SELSKSTE SDAGSALLQP PLEQHTVCHF FSLNQRCSCP DAYKHLNYGP HSCQQMGDCL CHQCSPTTSS FVQIQNGVAP LK ATHQAVE GFVHPITHIH HCPCLQGRVK PAGMQNSLPR NFFLHPVQHI QAQEKIGKTN VHSLQRSIEE HLPKMAEPSS FVC RSTGSL LKTCCDPENK QRELCKNRDV SNLESSGGTE NKAGGKVELS LSQTDASVNS EHFNQNEPKV LFNHLMGEAG CRSC PNNSQ SCGRIVRVKC NSVDCQMPNM EANVPAVLTH SELSGESLLI KTL

UniProtKB: Protein dispatched homolog 1

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Macromolecule #3: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 3 / Number of copies: 8 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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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

Concentration7.5 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.68 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 63043
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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