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- EMDB-24616: Cryo-EM structure of murine Dispatched NNN mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-24616
TitleCryo-EM structure of murine Dispatched NNN mutant
Map dataDISP1-A-NNN - auto-sharpened map from cryoSPARC nonuniform refinement (paper version), units of standard deviation above mean density.
Sample
  • Complex: Dispatched protein NNN mutant
    • Protein or peptide: Protein dispatched homolog 1
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHOLESTEROL HEMISUCCINATE
Function / homology
Function and homology information


patched ligand maturation / diaphragm development / embryonic pattern specification / peptide transport / dorsal/ventral pattern formation / peptide transmembrane transporter activity / determination of left/right symmetry / smoothened signaling pathway / 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 speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsAsarnow D / Wang Q / Ding K / Cheng Y / Beachy PA
Funding support United States, 5 items
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, 2021-
Header (metadata) releaseOct 27, 2021-
Map releaseOct 27, 2021-
UpdateNov 24, 2021-
Current statusNov 24, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.5
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 3.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7rpj
  • Surface level: 3.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24616.png

Downloads & links

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Map

FileDownload / File: emd_24616.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDISP1-A-NNN - auto-sharpened map from cryoSPARC nonuniform refinement (paper version), units of standard deviation above mean density.
Voxel sizeX=Y=Z: 0.834 Å
Density
Contour LevelBy AUTHOR: 3.5 / Movie #1: 3.5
Minimum - Maximum-11.060163 - 20.326393
Average (Standard dev.)-0.13611251 (±0.58032787)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 240.192 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8340.8340.834
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z240.192240.192240.192
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-11.06020.326-0.136

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Supplemental data

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Mask #1

Fileemd_24616_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_24616_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: DISP1-A-NNN - raw map from cryoSPARC nonuniform refinement...

Fileemd_24616_additional_1.map
AnnotationDISP1-A-NNN - raw map from cryoSPARC nonuniform refinement (paper version).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: DISP1-A-NNN - auto-sharpened map from cryoSPARC nonuniform refinement...

Fileemd_24616_additional_2.map
AnnotationDISP1-A-NNN - auto-sharpened map from cryoSPARC nonuniform refinement (paper version).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: DISP1-A-NNN - half map 1 from cryoSPARC nonuniform...

Fileemd_24616_half_map_1.map
AnnotationDISP1-A-NNN - half map 1 from cryoSPARC nonuniform refinement (paper version).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: DISP1-A-NNN - half map 2 from cryoSPARC nonuniform...

Fileemd_24616_half_map_2.map
AnnotationDISP1-A-NNN - half map 2 from cryoSPARC nonuniform refinement (paper version).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dispatched protein NNN mutant

EntireName: Dispatched protein NNN mutant
Components
  • Complex: Dispatched protein NNN mutant
    • Protein or peptide: Protein dispatched homolog 1
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHOLESTEROL HEMISUCCINATE

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Supramolecule #1: Dispatched protein NNN mutant

SupramoleculeName: Dispatched protein NNN mutant / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK293F
Molecular weightTheoretical: 151.91198 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: Mus musculus (house mouse)
Molecular weightTheoretical: 152.068188 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MARPFKFPRS YAALLADWPV VVLGMCTLLI VVCALVGVLV PELPDFSDPL LGFEPRGTTI GQRLVTWNNM MRNTGYKATL ANYPYKYAE EQARSHRDDR WSDDHHERER REVDWNFQKD SFFCDVPSDG YSRVVFASAG GETLWNLPAI KSMCDVDNSR I RSHPQFSD ...String:
MARPFKFPRS YAALLADWPV VVLGMCTLLI VVCALVGVLV PELPDFSDPL LGFEPRGTTI GQRLVTWNNM MRNTGYKATL ANYPYKYAE EQARSHRDDR WSDDHHERER REVDWNFQKD SFFCDVPSDG YSRVVFASAG GETLWNLPAI KSMCDVDNSR I RSHPQFSD LCQRTTAVSC CPSWTLGNYI AILNNRSSCQ KIVERDVSHT LKLLRTCAKH YQNGTLGPDC WDKAARRKDQ LK CTNVPRK CTKYNAVYQI LHYLVDKDFM TPKTADYAVP ALKYSMLFSP TEKGESMMNI YLDNFENWNS SDGITTVTGI EFG IKHSLF QDYLLMDTVY PAIAIAIVLL IMCVYTKSMF ITLMTMFAII SSLIVSYFLY RVVFNFEFFP FMNLTALIIL VGIG ANNAF VLCDVWNYTK FDKPRAETSE AVSVTLQHAA LSMFVTSFTT AAAFYANYVS NITAIRCFGV YAGTAILVNY VLMVT WLPA VIVLHERYLL NIFTCFRKPQ PQAYDKSCWA VLCQKCRRVL FAVSEASRIF FEKVLPCIVI KFRYLWLIWF LALTVG GAY IVCVNPKMKL PSLELSEFQV FRSSHPFERY DAEFKKLFMF ERVHHGEELH MPITVIWGVS PEDSGDPLNP KSKGELT LD STFNIASPAS QAWILHFCQK LRNQTFFHQT EQQDFTSCFI ETFKQWMENQ DCDEPALYPC CSHCSFPYKQ EVFELCIK K AIMELDRSTG YHLNNKTPGP RFDINDTIRA VVLEFQSTFL FTLAYEKMQQ FYKEVDSWIS HELSSAPEGL SRGWFVSNL EFYDLQDSLS DGTLIAMGLS VAVAFSVMLL TTWNIIISLY AIVSIAGTIF VTVGSLVLLG WELNVLESVT ISVAVGLSVN FAVHYGVAY RLAPDPDREG KVIFSLSRMG SAIAMAALTT FVAGAMMMPS TVLAYTQLGT FMMLVMCVSW AFATFFFQCL C RCLGPQGT CGQIPFPTKL QCSPFSHTLS ARPGDRGPSK THAASAYSVD ARGQKSQLEH EFYELQPLAS HSCTSSEKTT YE EPHTCSE FFNGQAKNLR MPVPAAYSSE LTKSPSSEPG SALLQSCLEQ DTVCHFSLNP RCNCRDAYTH LQYGLPEIHC QQM GDSLCH KCASTAGGFV QIQSSVAPLK ASHQAAEGLL HPAQHMLPPG MQNSRPRNFF LHSVQHFQAQ ENLGRTSTHS TDER LPRTA ELSPPPSDSR STESFQRACC HPENNQRRLC KSRDPGDTEG SGGTKSKVSG LPNQTDKEEK QVEPSLLQTD ETVNS EHLN HNESNFTFSH LPGEAGCRSC PNSPQSCRSI MRSKCGTEDC QTPNLEANVP AVPTHSDLSG ESLLIKTL

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Macromolecule #2: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 5 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #3: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 3 / Number of copies: 14 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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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

Concentration0.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
300.0 mMNaClsodium chloride
0.012 % (w/v)C47H88O22lauryl maltoside neopentyl glycol
0.0025 % (w/v)C56H92O25glyo-diosgenin
0.0024 % (w/v)C31H50O4cholesteryl hemisuccinate
5.0 mMCaCl2calcium chloride
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Details: Wait time 20 seconds, blot time 4 seconds, blotting force 0
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV
Details: Wait time 20 seconds, blot time 4 seconds, blotting force 0.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
DetailsAutomated data collection in SerialEM using 3x3 image shift pattern (one shot per hole), using beam tilt compensation. Low particle concentration necessitated targeting of hole edges.
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 4467 / Average electron dose: 65.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 59880 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3489403
CTF correctionSoftware: (Name: cryoSPARC, RELION)
Startup modelType of model: OTHER / Details: Ab initio reconstruction from cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 55982
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 8 / Software - Name: RELION

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 116.7
Output model

PDB-7rpj:
Cryo-EM structure of murine Dispatched NNN mutant

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