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- PDB-7rph: Cryo-EM structure of murine Dispatched 'R' conformation -

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Basic information

Entry
Database: PDB / ID: 7rph
TitleCryo-EM structure of murine Dispatched 'R' conformation
ComponentsProtein dispatched homolog 1
KeywordsMEMBRANE PROTEIN / RND transporter / Hedgehog binding / Sterol sensing domain / sodium binding
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 / membrane
Similarity search - Function
: / Protein patched/dispatched / Patched family / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain
Similarity search - Domain/homology
Lauryl Maltose Neopentyl Glycol / CHOLESTEROL HEMISUCCINATE / Protein dispatched homolog 1
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 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

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Structure visualization

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Assembly

Deposited unit
A: Protein dispatched homolog 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)167,91137
Polymers152,0711
Non-polymers15,84036
Water1,54986
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1150 Å2
ΔGint-12 kcal/mol
Surface area45560 Å2

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Components

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Protein / Sugars , 2 types, 6 molecules A

#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
#5: 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 , 4 types, 117 molecules

#2: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
#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
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 86 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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 'R' conformation / Type: COMPLEX / Entity ID: #1 / 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 supportDetails: Pelco easiGlow, 15 mA, 30 seconds hold / Grid 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: 4487
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameCategory
2RELIONparticle selection
3SerialEMimage acquisition
5cryoSPARCCTF correction
6RELIONCTF correction
9ISOLDEmodel fitting
10Cootmodel fitting
12cryoSPARCinitial Euler assignment
13cryoSPARCfinal Euler assignment
14RELIONclassification
15cryoSPARC3D reconstruction
16PHENIXmodel refinement
17ISOLDEmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3717941
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 166203 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 69.9 / Protocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0078758
ELECTRON MICROSCOPYf_angle_d0.91412053
ELECTRON MICROSCOPYf_dihedral_angle_d15.2941401
ELECTRON MICROSCOPYf_chiral_restr0.0421440
ELECTRON MICROSCOPYf_plane_restr0.0041338

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