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- PDB-7rpk: Cryo-EM structure of murine Dispatched in complex with Sonic hedgehog -

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Basic information

Entry
Database: PDB / ID: 7rpk
TitleCryo-EM structure of murine Dispatched in complex with Sonic hedgehog
Components
  • Protein dispatched homolog 1
  • Sonic hedgehog protein
KeywordsMEMBRANE PROTEIN / RND transporter / Hedgehog binding / Sterol sensing domain / sodium binding
Function / homology
Function and homology information


forebrain regionalization / cell proliferation in external granule layer / zona limitans intrathalamica formation / positive regulation of neurotrophin production / positive regulation of photoreceptor cell differentiation / epithelial-mesenchymal signaling involved in prostate gland development / fungiform papilla development / Release of Hh-Np from the secreting cell / digestive tract mesoderm development / fungiform papilla morphogenesis ...forebrain regionalization / cell proliferation in external granule layer / zona limitans intrathalamica formation / positive regulation of neurotrophin production / positive regulation of photoreceptor cell differentiation / epithelial-mesenchymal signaling involved in prostate gland development / fungiform papilla development / Release of Hh-Np from the secreting cell / digestive tract mesoderm development / fungiform papilla morphogenesis / ventral spinal cord interneuron specification / tongue morphogenesis / respiratory tube development / Ligand-receptor interactions / patched ligand maturation / Activation of SMO / positive regulation of oligodendrocyte progenitor proliferation / trachea development / positive regulation of skeletal muscle cell proliferation / right lung development / left lung development / primary prostatic bud elongation / regulation of mesenchymal cell proliferation involved in prostate gland development / mesenchymal smoothened signaling pathway involved in prostate gland development / positive regulation of sclerotome development / tracheoesophageal septum formation / negative regulation of ureter smooth muscle cell differentiation / positive regulation of ureter smooth muscle cell differentiation / negative regulation of kidney smooth muscle cell differentiation / positive regulation of kidney smooth muscle cell differentiation / regulation of odontogenesis / positive regulation of mesenchymal cell proliferation involved in ureter development / mesenchymal-epithelial cell signaling involved in prostate gland development / trunk neural crest cell migration / hindgut morphogenesis / polarity specification of anterior/posterior axis / striated muscle tissue development / negative regulation of alpha-beta T cell differentiation / regulation of glial cell proliferation / regulation of prostatic bud formation / anatomical structure formation involved in morphogenesis / metanephric mesenchymal cell proliferation involved in metanephros development / formation of anatomical boundary / lung epithelium development / positive regulation of striated muscle cell differentiation / neural tube formation / trachea morphogenesis / cholesterol-protein transferase activity / positive regulation of penile erection / bud outgrowth involved in lung branching / diaphragm development / regulation of epithelial cell proliferation involved in prostate gland development / myotube differentiation / telencephalon regionalization / epithelial-mesenchymal cell signaling / laminin-1 binding / vasculogenesis involved in coronary vascular morphogenesis / negative regulation of cholesterol efflux / Hedgehog ligand biogenesis / salivary gland cavitation / spinal cord dorsal/ventral patterning / negative regulation of mesenchymal cell apoptotic process / determination of left/right asymmetry in lateral mesoderm / positive regulation of cerebellar granule cell precursor proliferation / negative regulation of T cell differentiation in thymus / spinal cord motor neuron differentiation / positive regulation of T cell differentiation in thymus / cell development / intermediate filament organization / prostate gland development / cerebellar granule cell precursor proliferation / limb bud formation / male genitalia morphogenesis / lung lobe morphogenesis / embryonic skeletal system development / establishment of epithelial cell polarity / skeletal muscle fiber differentiation / fungiform papilla formation / mesenchymal cell apoptotic process / patched binding / embryonic digestive tract morphogenesis / somite development / epithelial cell proliferation involved in salivary gland morphogenesis / animal organ formation / embryonic foregut morphogenesis / hindbrain development / neuron fate commitment / ectoderm development / positive regulation of skeletal muscle tissue development / branching involved in prostate gland morphogenesis / thalamus development / stem cell development / mesenchymal cell proliferation involved in lung development / negative regulation of dopaminergic neuron differentiation / skeletal muscle cell proliferation / lymphoid progenitor cell differentiation / dorsal/ventral neural tube patterning / positive regulation of immature T cell proliferation in thymus / mesenchymal cell proliferation / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment
Similarity search - Function
: / Hedgehog, N-terminal signalling domain / Hedgehog protein / Hedgehog protein, Hint domain / : / Hint module / Hedgehog amino-terminal signalling domain / Hedgehog signalling/DD-peptidase zinc-binding domain superfamily / Protein patched/dispatched / Patched family ...: / Hedgehog, N-terminal signalling domain / Hedgehog protein / Hedgehog protein, Hint domain / : / Hint module / Hedgehog amino-terminal signalling domain / Hedgehog signalling/DD-peptidase zinc-binding domain superfamily / Protein patched/dispatched / Patched family / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain / Hint domain C-terminal / Hint (Hedgehog/Intein) domain C-terminal region / Intein N-terminal splicing region / Intein N-terminal splicing motif profile. / Hint domain N-terminal / Hint (Hedgehog/Intein) domain N-terminal region / Hint domain superfamily
Similarity search - Domain/homology
Chem-6OE / Lauryl Maltose Neopentyl Glycol / CHOLESTEROL HEMISUCCINATE / Protein dispatched homolog 1 / Sonic hedgehog protein
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsAsarnow, D. / Wang, Q. / Ding, K. / Cheng, Y. / Beachy, P.A.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM102498 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM140847 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD020054 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD021741 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2021
Title: Dispatched uses Na flux to power release of lipid-modified Hedgehog.
Authors: Qianqian Wang / Daniel E Asarnow / Ke Ding / Randall K Mann / Jason Hatakeyama / Yunxiao Zhang / Yong Ma / Yifan Cheng / Philip A Beachy /
Abstract: The Dispatched protein, which is related to the NPC1 and PTCH1 cholesterol transporters and to H-driven transporters of the RND family, enables tissue-patterning activity of the lipid-modified ...The Dispatched protein, which is related to the NPC1 and PTCH1 cholesterol transporters and to H-driven transporters of the RND family, enables tissue-patterning activity of the lipid-modified Hedgehog protein by releasing it from tightly -localized sites of embryonic expression. Here we determine a cryo-electron microscopy structure of the mouse protein Dispatched homologue 1 (DISP1), revealing three Na ions coordinated within a channel that traverses its transmembrane domain. We find that the rate of Hedgehog export is dependent on the Na gradient across the plasma membrane. The transmembrane channel and Na binding are disrupted in DISP1-NNN, a variant with asparagine substitutions for three intramembrane aspartate residues that each coordinate and neutralize the charge of one of the three Na ions. DISP1-NNN and variants that disrupt single Na sites retain binding to, but are impaired in export of the lipid-modified Hedgehog protein to the SCUBE2 acceptor. Interaction of the amino-terminal signalling domain of the Sonic hedgehog protein (ShhN) with DISP1 occurs via an extensive buried surface area and contacts with an extended furin-cleaved DISP1 arm. Variability analysis reveals that ShhN binding is restricted to one extreme of a continuous series of DISP1 conformations. The bound and unbound DISP1 conformations display distinct Na-site occupancies, which suggests a mechanism by which transmembrane Na flux may power extraction of the lipid-linked Hedgehog signal from the membrane. Na-coordinating residues in DISP1 are conserved in PTCH1 and other metazoan RND family members, suggesting that Na flux powers their conformationally driven activities.
History
DepositionAug 3, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 27, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 10, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 24, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 13, 2023Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / entity / pdbx_entity_nonpoly
Item: _chem_comp.name / _chem_comp.pdbx_synonyms ..._chem_comp.name / _chem_comp.pdbx_synonyms / _entity.pdbx_description / _pdbx_entity_nonpoly.name
Revision 1.4Nov 13, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
A: Protein dispatched homolog 1
H: Sonic hedgehog protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)187,27442
Polymers170,8032
Non-polymers16,47040
Water75742
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, immunoprecipitation
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3460 Å2
ΔGint-53 kcal/mol
Surface area53210 Å2

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Components

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Protein , 2 types, 2 molecules AH

#1: Protein Protein dispatched homolog 1 / Mdispa


Mass: 152071.141 Da / Num. of mol.: 1 / Fragment: UNP residues 172-1521
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Disp1, Disp, Dispa, Icb, Icbins / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q3TDN0
#2: Protein Sonic hedgehog protein / ShhN / Shh N-terminal processed signaling domains / ShhNp / Sonic hedgehog protein 19 kDa product


Mass: 18732.090 Da / Num. of mol.: 1 / Fragment: UNP residues 26-189
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Shh, Hhg1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q62226

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Sugars , 1 types, 5 molecules

#7: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 8 types, 77 molecules

#3: Chemical...
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 26 / Source method: obtained synthetically / Formula: C31H50O4 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-AV0 / Lauryl Maltose Neopentyl Glycol / 2,2-didecylpropane-1,3-bis-b-D-maltopyranoside / 2-decyl-2-{[(4-O-alpha-D-glucopyranosyl-beta-D-glucopyranosyl)oxy]methyl}dodecyl4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside


Mass: 1005.188 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C47H88O22 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-6OE / (2S)-3-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-2-(hexanoyloxy)propyl hexanoate


Mass: 411.428 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H34NO8P
#8: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#10: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#11: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 42 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Dispatched protein complexed with Sonic hedgehog 26-189
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.15191493 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293F
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2300 mMsodium chlorideNaCl1
30.012 % (w/v)lauryl maltoside neopentyl glycolC47H88O221
40.0025 % (w/v)glyo-diosgeninC56H92O251
50.0024 % (w/v)cholesteryl hemisuccinateC31H50O41
65 mMcalcium chlorideCaCl21
SpecimenConc.: 1.56 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K
Details: Wait time 20 seconds, blot time 4 seconds, blotting force 0

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Automated data collection in SerialEM using 3x3 image shift pattern (one shot per hole), using beam tilt compensation.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 59880 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 66.7 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1687
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
2SerialEMimage acquisition
4cryoSPARCCTF correction
7ISOLDEmodel fitting
8Cootmodel fitting
10PHENIXmodel refinement
11ISOLDEmodel refinement
12cryoSPARCinitial Euler assignment
13cryoSPARCfinal Euler assignment
14cryoSPARCclassification
15cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1406648
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 204786 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 69.9 / Protocol: FLEXIBLE FIT / Space: REAL

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