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- EMDB-22689: Hedgehog receptor Patched (PTCH1) in complex with conformation se... -

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Basic information

Entry
Database: EMDB / ID: EMD-22689
TitleHedgehog receptor Patched (PTCH1) in complex with conformation selective nanobody TI23
Map dataSharpened map of Patched in complex with nanobody TI23
Sample
  • Complex: Complex between Hedgehog receptor PTCH1 and nanobody TI23
    • Complex: Hedgehog receptor Patched PTCH1
      • Protein or peptide: Protein patched homolog 1
    • Complex: nanobody TI23
      • Protein or peptide: nanobody TI23
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: 2-{[(4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranosyl)oxy]methyl}-4-{[(3beta,9beta,14beta,17beta,25R)-spirost-5-en-3-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranoside
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 / neural tube patterning ...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 / neural tube patterning / hedgehog family protein binding / hindlimb morphogenesis / Hedgehog 'on' state / epidermal cell fate specification / spinal cord motor neuron differentiation / prostate gland development / negative regulation of cell division / Hedgehog 'off' state / patched binding / limb morphogenesis / somite development / dorsal/ventral neural tube patterning / cellular response to cholesterol / smooth muscle tissue development / pattern specification process / pharyngeal system development / mammary gland duct morphogenesis / mammary gland epithelial cell differentiation / cell fate determination / mammary gland development / commissural neuron axon guidance / metanephric collecting duct development / dorsal/ventral pattern formation / embryonic limb morphogenesis / regulation of growth / cholesterol binding / negative regulation of multicellular organism growth / branching involved in ureteric bud morphogenesis / positive regulation of epidermal cell differentiation / spermatid development / dendritic growth cone / positive regulation of cholesterol efflux / keratinocyte proliferation / negative regulation of keratinocyte proliferation / epidermis development / negative regulation of osteoblast differentiation / embryonic organ development / negative regulation of smoothened signaling pathway / axonal growth cone / response to mechanical stimulus / heart morphogenesis / response to retinoic acid / negative regulation of stem cell proliferation / regulation of mitotic cell cycle / cyclin binding / epithelial cell proliferation / stem cell proliferation / neural tube closure / liver regeneration / caveola / protein localization to plasma membrane / animal organ morphogenesis / brain development / cilium / protein processing / regulation of protein localization / negative regulation of epithelial cell proliferation / glucose homeostasis / response to estradiol / apical part of cell / heparin binding / regulation of cell population proliferation / midbody / in utero embryonic development / response to xenobiotic stimulus / negative regulation of cell population proliferation / intracellular membrane-bounded organelle / negative regulation of DNA-templated transcription / protein-containing complex binding / perinuclear region of cytoplasm / positive regulation of DNA-templated transcription / 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) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsZhang Y / Bulkley DP / Liang J / Manglik A / Cheng Y / Beachy PA
Funding support United States, 3 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)R01GM098672 United States
National Institutes of Health/Office of the DirectorDP5OD023048 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Hedgehog pathway activation through nanobody-mediated conformational blockade of the Patched sterol conduit.
Authors: Yunxiao Zhang / Wan-Jin Lu / David P Bulkley / Jiahao Liang / Arthur Ralko / Shuo Han / Kelsey J Roberts / Anping Li / Wonhwa Cho / Yifan Cheng / Aashish Manglik / Philip A Beachy /
Abstract: Activation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, ...Activation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, however, may be detrimental, and agents amenable to tissue targeting for therapeutic application have been lacking. We have developed an agonist, a conformation-specific nanobody against the Hedgehog receptor Patched1 (PTCH1). This nanobody potently activates the Hedgehog pathway in vitro and in vivo by stabilizing an alternative conformation of a Patched1 "switch helix," as revealed by our cryogenic electron microscopy structure. Nanobody-binding likely traps Patched in one stage of its transport cycle, thus preventing substrate movement through the Patched1 sterol conduit. Unlike the native Hedgehog ligand, this nanobody does not require lipid modifications for its activity, facilitating mechanistic studies of Hedgehog pathway activation and the engineering of pathway activating agents for therapeutic use. Our conformation-selective nanobody approach may be generally applicable to the study of other PTCH1 homologs.
History
DepositionSep 18, 2020-
Header (metadata) releaseMar 17, 2021-
Map releaseMar 17, 2021-
UpdateMar 17, 2021-
Current statusMar 17, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 4.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7k65
  • Surface level: 4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22689.png

Downloads & links

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Map

FileDownload / File: emd_22689.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of Patched in complex with nanobody TI23
Voxel sizeX=Y=Z: 1.059 Å
Density
Contour LevelBy AUTHOR: 4.8 / Movie #1: 4.8
Minimum - Maximum-14.967468 - 25.975851
Average (Standard dev.)-0.021284537 (±0.87775165)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.104 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0591.0591.059
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z271.104271.104271.104
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-14.96725.976-0.021

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Supplemental data

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Half map: half map 1

Fileemd_22689_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_22689_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex between Hedgehog receptor PTCH1 and nanobody TI23

EntireName: Complex between Hedgehog receptor PTCH1 and nanobody TI23
Components
  • Complex: Complex between Hedgehog receptor PTCH1 and nanobody TI23
    • Complex: Hedgehog receptor Patched PTCH1
      • Protein or peptide: Protein patched homolog 1
    • Complex: nanobody TI23
      • Protein or peptide: nanobody TI23
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: 2-{[(4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranosyl)oxy]methyl}-4-{[(3beta,9beta,14beta,17beta,25R)-spirost-5-en-3-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranoside

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Supramolecule #1: Complex between Hedgehog receptor PTCH1 and nanobody TI23

SupramoleculeName: Complex between Hedgehog receptor PTCH1 and nanobody TI23
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: Hedgehog receptor Patched PTCH1

SupramoleculeName: Hedgehog receptor Patched PTCH1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293

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Supramolecule #3: nanobody TI23

SupramoleculeName: nanobody TI23 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: Protein patched homolog 1

MacromoleculeName: 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: 142.619938 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MASAGNAAGA LGRQAGGGRR RRTGGPHRAA PDRDYLHRPS YCDAAFALEQ ISKGKATGRK APLWLRAKFQ RLLFKLGCYI QKNCGKFLV VGLLIFGAFA VGLKAANLET NVEELWVEVG GRVSRELNYT RQKIGEEAMF NPQLMIQTPK EEGANVLTTE A LLQHLDSA ...String:
MASAGNAAGA LGRQAGGGRR RRTGGPHRAA PDRDYLHRPS YCDAAFALEQ ISKGKATGRK APLWLRAKFQ RLLFKLGCYI QKNCGKFLV VGLLIFGAFA VGLKAANLET NVEELWVEVG GRVSRELNYT RQKIGEEAMF NPQLMIQTPK EEGANVLTTE A LLQHLDSA LQASRVHVYM YNRQWKLEHL CYKSGELITE TGYMDQIIEY LYPCLIITPL DCFWEGAKLQ SGTAYLLGKP PL RWTNFDP LEFLEELKKI NYQVDSWEEM LNKAEVGHGY MDRPCLNPAD PDCPATAPNK NSTKPLDVAL VLNGGCQGLS RKY MHWQEE LIVGGTVKNA TGKLVSAHAL QTMFQLMTPK QMYEHFRGYD YVSHINWNED RAAAILEAWQ RTYVEVVHQS VAPN STQKV LPFTTTTLDD ILKSFSDVSV IRVASGYLLM LAYACLTMLR WDCSKSQGAV GLAGVLLVAL SVAAGLGLCS LIGIS FNAA TTQVLPFLAL GVGVDDVFLL AHAFSETGQN KRIPFEDRTG ECLKRTGASV ALTSISNVTA FFMAALIPIP ALRAFS LQA AVVVVFNFAM VLLIFPAILS MDLYRREDRR LDIFCCFTSP CVSRVIQVEP QAYTEPMQST VQLRTEYDPH THVYYTT AE PRSEISVQPV TVTQDNLSCQ SPESTSSTRD LLSQFSDSSL HCLEPPCTKW TLSSFAEKHY APFLLKPKAK VVVILLFL G LLGVSLYGTT RVRDGLDLTD IVPRETREYD FIAAQFKYFS FYNMYIVTQK ADYPNIQHLL YDLHKSFSNV KYVMLEENK QLPQMWLHYF RDWLQGLQDA FDSDWETGRI MPNNYKNGSD DGVLAYKLLV QTGSRDKPID ISQLTKQRLV DADGIINPSA FYIYLTAWV SNDPVAYAAS QANIRPHRPE WVHDKADYMP ETRLRIPAAE PIEYAQFPFY LNGLRDTSDF VEAIEKVRVI C NNYTSLGL SSYPNGYPFL FWEQYISLRH WLLLSISVVL ACTFLVCAVF LLNPWTAGII VMVLALMTVE LFGMMGLIGI KL SAVPVVI LIASVGIGVE FTVHVALAFL TAIGDKNHRA MLALEHMFAP VLDGAVSTLL GVLMLAGSEF DFIVRYFFAV LAI LTVLGV LNGLVLLPVL LSFFGPCPEV SPANGLNRLP TPSPEPPPSV VRFAVPPGHT NNGSDSSDSE YSSQTTVSGI SEEL RQYEA QQGAGGPAHQ VIVEATENPV FARSTVVHPD SRHQPPLTPR QQPHLDSGSL SPGRQGQQPR RDMDEKTTGW RGGHV V

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Macromolecule #2: nanobody TI23

MacromoleculeName: nanobody TI23 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 13.821417 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
QVQLQESGGG LVQAGGSLRL SCAASGNIFA YYIMGWYRQA PGKERELVAT IDIGGNTNYA DSVKGRFTIS RDNAKNNVYL QMNSLKPED TAVYYCAVQA VPIRYRRYWG QGTQVTVSSH HHHHH

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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: 5 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #5: 2-{[(4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranosyl)oxy]methyl...

MacromoleculeName: 2-{[(4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranosyl)oxy]methyl}-4-{[(3beta,9beta,14beta,17beta,25R)-spirost-5-en-3-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranoside
type: ligand / ID: 5 / Number of copies: 4 / Formula: Q7G
Molecular weightTheoretical: 1.165315 KDa
Chemical component information

ChemComp-Q7G:
2-{[(4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranosyl)oxy]methyl}-4-{[(3beta,9beta,14beta,17beta,25R)-spirost-5-en-3-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranoside

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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

Concentration4.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
20.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
0.02 %GDNglycol-diosgenin
GridModel: Quantifoil
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number real images: 7046 / Average exposure time: 12.0 sec. / Average electron dose: 85.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3621265
Details: Particles were picked first using a Gaussian blob as the template and then were re-picked using the 2D classes generated from the first round.
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final 3D classificationNumber classes: 2 / Avg.num./class: 307652 / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final reconstructionNumber classes used: 1 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1) / Number images used: 307652

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Atomic model buiding 1

Initial modelPDB ID:

6mg8


Chain - Chain ID: A
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7k65:
Hedgehog receptor Patched (PTCH1) in complex with conformation selective nanobody TI23

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