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- EMDB-31754: Cryo-EM structure of Patched1 (V1084A mutant) in lipid nanodisc, ... -

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Basic information

Entry
Database: EMDB / ID: EMD-31754
TitleCryo-EM structure of Patched1 (V1084A mutant) in lipid nanodisc, 3.64 angstrom (reprocessed with the dataset of 7dzp)
Map data
Sample
  • Complex: Ptc1 in lipid nanodisc
    • Protein or peptide: Protein patched homolog 1,Protein patched homolog 1
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHOLESTEROL
Function / homology
Function and homology information


Ligand-receptor interactions / Activation of SMO / neural plate axis specification / cell differentiation involved in kidney development / hedgehog receptor activity / response to chlorate / cell proliferation involved in metanephros development / smoothened binding / neural tube formation / hedgehog family protein binding ...Ligand-receptor interactions / Activation of SMO / neural plate axis specification / cell differentiation involved in kidney development / hedgehog receptor activity / response to chlorate / cell proliferation involved in metanephros development / smoothened binding / neural tube formation / hedgehog family protein binding / hindlimb morphogenesis / Hedgehog 'on' state / epidermal cell fate specification / spinal cord motor neuron differentiation / prostate gland development / Hedgehog 'off' state / somite development / patched binding / negative regulation of cell division / cellular response to cholesterol / dorsal/ventral neural tube patterning / smooth muscle tissue development / pattern specification process / pharyngeal system development / mammary gland epithelial cell differentiation / mammary gland duct morphogenesis / mammary gland development / cell fate determination / commissural neuron axon guidance / metanephric collecting duct development / dorsal/ventral pattern formation / regulation of growth / embryonic limb morphogenesis / negative regulation of multicellular organism growth / branching involved in ureteric bud morphogenesis / cholesterol binding / positive regulation of epidermal cell differentiation / dendritic growth cone / keratinocyte proliferation / spermatid development / positive regulation of cholesterol efflux / epidermis development / negative regulation of keratinocyte proliferation / negative regulation of osteoblast differentiation / embryonic organ development / axonal growth cone / negative regulation of smoothened signaling pathway / heart morphogenesis / response to mechanical stimulus / response to retinoic acid / negative regulation of stem cell proliferation / regulation of mitotic cell cycle / cyclin binding / epithelial cell proliferation / stem cell proliferation / caveola / neural tube closure / protein localization to plasma membrane / liver regeneration / animal organ morphogenesis / brain development / protein processing / cilium / negative regulation of epithelial cell proliferation / regulation of protein localization / apical part of cell / response to estradiol / glucose homeostasis / heparin binding / regulation of cell population proliferation / midbody / in utero embryonic development / response to xenobiotic stimulus / negative regulation of cell population proliferation / negative regulation of DNA-templated transcription / protein-containing complex binding / positive regulation of DNA-templated transcription / perinuclear region of cytoplasm / Golgi apparatus / negative regulation of transcription by RNA polymerase II / signal transduction / zinc ion binding / extracellular region / plasma membrane
Similarity search - Function
Transmembrane receptor, patched / Sterol-sensing domain of SREBP cleavage-activation / Protein patched/dispatched / Patched family / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain
Similarity search - Domain/homology
Protein patched homolog 1
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.64 Å
AuthorsLuo Y / Zhao Y / Qu Q / Li D
Funding support China, 4 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31570748 China
National Natural Science Foundation of China (NSFC)U1632127 China
Chinese Academy of SciencesXDB37020204 China
Chinese Academy of SciencesQYZDB-SSW-SMC037 China
CitationJournal: Structure / Year: 2021
Title: Cryo-EM study of patched in lipid nanodisc suggests a structural basis for its clustering in caveolae.
Authors: Yitian Luo / Guoyue Wan / Xiang Zhang / Xuan Zhou / Qiuwen Wang / Jialin Fan / Hongmin Cai / Liya Ma / Hailong Wu / Qianhui Qu / Yao Cong / Yun Zhao / Dianfan Li /
Abstract: The 12-transmembrane protein Patched (Ptc1) acts as a suppressor for Hedgehog (Hh) signaling by depleting sterols in the cytoplasmic membrane leaflet that are required for the activation of ...The 12-transmembrane protein Patched (Ptc1) acts as a suppressor for Hedgehog (Hh) signaling by depleting sterols in the cytoplasmic membrane leaflet that are required for the activation of downstream regulators. The positive modulator Hh inhibits Ptc1's transporter function by binding to Ptc1 and its co-receptors, which are locally concentrated in invaginated microdomains known as caveolae. Here, we reconstitute the mouse Ptc1 into lipid nanodiscs and determine its structure using single-particle cryoelectron microscopy. The structure is overall similar to those in amphipol and detergents but displays various conformational differences in the transmembrane region. Although most particles show monomers, we observe Ptc1 dimers with distinct interaction patterns and different membrane curvatures, some of which are reminiscent of caveolae. We find that an extramembranous "hand-shake" region rich in hydrophobic and aromatic residues mediates inter-Ptc1 interactions under different membrane curvatures. Our data provide a plausible framework for Ptc1 clustering in the highly curved caveolae.
History
DepositionAug 20, 2021-
SupersessionSep 22, 2021ID: EMD-30927
Header (metadata) releaseSep 22, 2021-
Map releaseSep 22, 2021-
UpdateFeb 16, 2022-
Current statusFeb 16, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.23
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.23
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7v6z
  • Surface level: 0.23
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7v6z
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31754.png

Downloads & links

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Map

FileDownload / File: emd_31754.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.23 / Movie #1: 0.23
Minimum - Maximum-1.3665602 - 1.8991281
Average (Standard dev.)0.0015832123 (±0.04826722)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions264264264
Spacing264264264
CellA=B=C: 216.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z264264264
origin x/y/z0.0000.0000.000
length x/y/z216.480216.480216.480
α/β/γ90.00090.00090.000
start NX/NY/NZ-140-140-140
NX/NY/NZ280280280
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS264264264
D min/max/mean-1.3671.8990.002

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Supplemental data

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Sample components

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Entire : Ptc1 in lipid nanodisc

EntireName: Ptc1 in lipid nanodisc
Components
  • Complex: Ptc1 in lipid nanodisc
    • Protein or peptide: Protein patched homolog 1,Protein patched homolog 1
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHOLESTEROL

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Supramolecule #1: Ptc1 in lipid nanodisc

SupramoleculeName: Ptc1 in lipid nanodisc / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightExperimental: 160 kDa/nm

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Macromolecule #1: Protein patched homolog 1,Protein patched homolog 1

MacromoleculeName: Protein patched homolog 1,Protein patched homolog 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 121.609555 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGSASAGNAA GALGRQAGGG RRRRTGGPHR AAPDRDYLHR PSYCDAAFAL EQISKGKATG RKAPLWLRAK FQRLLFKLGC YIQKNCGKF LVVGLLIFGA FAVGLKAANL ETNVEELWVE VGGRVSRELN YTRQKIGEEA MFNPQLMIQT PKEEGANVLT T EALLQHLD ...String:
MGSASAGNAA GALGRQAGGG RRRRTGGPHR AAPDRDYLHR PSYCDAAFAL EQISKGKATG RKAPLWLRAK FQRLLFKLGC YIQKNCGKF LVVGLLIFGA FAVGLKAANL ETNVEELWVE VGGRVSRELN YTRQKIGEEA MFNPQLMIQT PKEEGANVLT T EALLQHLD SALQASRVHV YMYNRQWKLE HLCYKSGELI TETGYMDQII EYLYPCLIIT PLDCFWEGAK LQSGTAYLLG KP PLRWTNF DPLEFLEELK KINYQVDSWE EMLNKAEVGH GYMDRPCLNP ADPDCPATAP NKNSTKPLDV ALVLNGGCQG LSR KYMHWQ EELIVGGTVK NATGKLVSAH ALQTMFQLMT PKQMYEHFRG YDYVSHINWN EDRAAAILEA WQRTYVEVVH QSVA PNSTQ KVLPFTTTTL DDILKSFSDV SVIRVASGYL LMLAYACLTM LRWDCSKSQG AVGLAGVLLV ALSVAAGLGL CSLIG ISFN AATTQVLPFL ALGVGVDDVF LLAHAFSETG QNKRIPFEDR TGECLKRTGA SVALTSISNV TAFFMAALIP IPALRA FSL QAAVVVVFNF AMVLLIFPAI LSMDLYRRED RRLDIFCCFT SPCVSRVIQV EPQEPPCTKW TLSSFAEKHY APFLLKP KA KVVVILLFLG LLGVSLYGTT RVRDGLDLTD IVPRETREYD FIAAQFKYFS FYNMYIVTQK ADYPNIQHLL YDLHKSFS N VKYVMLEENK QLPQMWLHYF RDWLQGLQDA FDSDWETGRI MPNNYKNGSD DGVLAYKLLV QTGSRDKPID ISQLTKQRL VDADGIINPS AFYIYLTAWV SNDPVAYAAS QANIRPHRPE WVHDKADYMP ETRLRIPAAE PIEYAQFPFY LNGLRDTSDF VEAIEKVRV ICNNYTSLGL SSYPNGYPFL FWEQYISLRH WLLLSISVVL ACTFLVCAVF LLNPWTAGII VMVLALMTVE L FGMMGLIG IKLSAVPVVI LIASVGIGVE FTAHVALAFL TAIGDKNHRA MLALEHMFAP VLDGAVSTLL GVLMLAGSEF DF IVRYFFA VLAILTVLGV LNGLVLLPVL LSFFGPCPEV SPANGTLEVL FQG

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 3 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

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

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Macromolecule #4: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 4 / Number of copies: 1 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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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

Concentration8.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMHEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
0.5 mMCaCl2calsium chloride
0.1 mMEDTAEthylenediaminetetraacetic acid
0.2 mMTCEPtris(2-carboxyethyl)phosphine
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 5140 / Average exposure time: 2.23 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 3.0 µm / Calibrated defocus min: 1.1 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 120000
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: 3311691
CTF correctionSoftware - Name: Gctf (ver. 1.18)
Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.64 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 328720
Initial angle assignmentType: PROJECTION MATCHING
Projection matching processing - Number reference projections: 4
Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: PROJECTION MATCHING
Projection matching processing - Number reference projections: 1
Software - Name: cryoSPARC (ver. 3.2)
Final 3D classificationNumber classes: 4 / Avg.num./class: 63736 / Software - Name: cryoSPARC (ver. 3.2)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER / Overall B value: 57.8
Output model

PDB-7v6z:
Cryo-EM structure of Patched1 (V1084A mutant) in lipid nanodisc, 3.64 angstrom (reprocessed with the dataset of 7dzp)

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