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- EMDB-23837: Human Hedgehog acyltransferase (HHAT) in complex with a palmitoyl... -

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Basic information

Entry
Database: EMDB / ID: EMD-23837
TitleHuman Hedgehog acyltransferase (HHAT) in complex with a palmitoylated Hedgehog peptide product and a Fab antibody fragment
Map datafinal map
Sample
  • Complex: HHAT in complex with a palmitoylated Hedgehog peptide product and a Fab antibody fragment
    • Protein or peptide: Sonic hedgehog protein N-product peptide
    • Protein or peptide: Protein-cysteine N-palmitoyltransferase HHAT
    • Protein or peptide: 3H02 Fab heavy chain
    • Protein or peptide: 3H02 Fab light chain
  • Ligand: PALMITIC ACID
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: Palmitoyl-CoA
  • Ligand: Digitonin
KeywordsMBOAT / GOAT / porcupine / acyl transferase / membrane protein / membrane enzyme / ER / palmitoyl-CoA / TRANSFERASE-IMMUNE SYSTEM complex
Function / homology
Function and homology information


N-terminal peptidyl-L-cysteine N-palmitoylation / 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 ...N-terminal peptidyl-L-cysteine N-palmitoylation / 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 / morphogen activity / regulation of odontogenesis / positive regulation of mesenchymal cell proliferation involved in ureter development / O-acyltransferase activity / trunk neural crest cell migration / Formation of lateral plate mesoderm / 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 / metanephric mesenchymal cell proliferation involved in metanephros development / formation of anatomical boundary / lung epithelium development / positive regulation of striated muscle cell differentiation / ventral midline development / trachea morphogenesis / cholesterol-protein transferase activity / bud outgrowth involved in lung branching / HHAT G278V doesn't palmitoylate Hh-Np / telencephalon regionalization / epithelial-mesenchymal cell signaling / Ligand-receptor interactions / laminin-1 binding / negative regulation of cholesterol efflux / 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 / palmitoyltransferase activity / 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 / Activation of SMO / lung lobe morphogenesis / embryonic skeletal system development / establishment of epithelial cell polarity / skeletal muscle fiber differentiation / 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 / 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 / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / smooth muscle tissue development / negative thymic T cell selection / pattern specification process / artery development / oligodendrocyte development / positive regulation of astrocyte differentiation / male genitalia development / regulation of stem cell proliferation / self proteolysis / epithelial cell proliferation involved in prostate gland development / positive regulation of epithelial cell proliferation involved in prostate gland development / embryonic pattern specification / branching involved in salivary gland morphogenesis / Release of Hh-Np from the secreting cell / dopaminergic neuron differentiation / lung-associated mesenchyme development / glycosaminoglycan binding / Formation of axial mesoderm / regulation of proteolysis / intein-mediated protein splicing / metanephric collecting duct development / metanephros development
Similarity search - Function
: / Hedgehog, N-terminal signalling domain / Hedgehog protein / Hedgehog protein, Hint domain / : / Hint module / Hedgehog amino-terminal signalling domain / Membrane bound O-acyl transferase, MBOAT / MBOAT, membrane-bound O-acyltransferase family / Hedgehog signalling/DD-peptidase zinc-binding domain superfamily ...: / Hedgehog, N-terminal signalling domain / Hedgehog protein / Hedgehog protein, Hint domain / : / Hint module / Hedgehog amino-terminal signalling domain / Membrane bound O-acyl transferase, MBOAT / MBOAT, membrane-bound O-acyltransferase family / Hedgehog signalling/DD-peptidase zinc-binding domain superfamily / 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
Sonic hedgehog protein / Protein-cysteine N-palmitoyltransferase HHAT
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLong SB / Jiang Y
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM131921 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)P30CA008748 United States
CitationJournal: Science / Year: 2021
Title: Substrate and product complexes reveal mechanisms of Hedgehog acylation by HHAT.
Authors: Yiyang Jiang / Thomas L Benz / Stephen B Long /
Abstract: Hedgehog proteins govern crucial developmental steps in animals and drive certain human cancers. Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino- ...Hedgehog proteins govern crucial developmental steps in animals and drive certain human cancers. Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino-terminal palmitoylation by Hedgehog acyltransferase (HHAT). We present cryo-electron microscopy structures of human HHAT in complex with its palmitoyl-coenzyme A substrate and of a product complex with a palmitoylated Hedgehog peptide at resolutions of 2.7 and 3.2 angstroms, respectively. The structures reveal how HHAT overcomes the challenges of bringing together substrates that have different physiochemical properties from opposite sides of the endoplasmic reticulum membrane within a membrane-embedded active site for catalysis. These principles are relevant to related enzymes that catalyze the acylation of Wnt and of the appetite-stimulating hormone ghrelin. The structural and mechanistic insights may advance the development of inhibitors for cancer.
History
DepositionApr 16, 2021-
Header (metadata) releaseJun 16, 2021-
Map releaseJun 16, 2021-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.877
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.877
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7mhz
  • Surface level: 0.877
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7mhz
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23837.png

Downloads & links

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Map

FileDownload / File: emd_23837.map.gz / Format: CCP4 / Size: 204.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfinal map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
0.53 Å/pix.
x 377 pix.
= 200.564 Å		0.53 Å/pix.
x 377 pix.
= 200.564 Å		0.53 Å/pix.
x 377 pix.
= 200.564 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.532 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.877 / Movie #1: 0.877
Minimum - Maximum-1.9214307 - 3.9598546
Average (Standard dev.)0.07945373 (±0.15568584)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin1229869
Dimensions377377377
Spacing377377377
CellA=B=C: 200.564 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.5320.5320.532
M x/y/z377377377
origin x/y/z0.0000.0000.000
length x/y/z200.564200.564200.564
α/β/γ90.00090.00090.000
start NX/NY/NZ1229869
NX/NY/NZ377377377
MAP C/R/S213
start NC/NR/NS9812269
NC/NR/NS377377377
D min/max/mean-1.9213.9600.079

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Supplemental data

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Sample components

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Entire : HHAT in complex with a palmitoylated Hedgehog peptide product and...

EntireName: HHAT in complex with a palmitoylated Hedgehog peptide product and a Fab antibody fragment
Components
  • Complex: HHAT in complex with a palmitoylated Hedgehog peptide product and a Fab antibody fragment
    • Protein or peptide: Sonic hedgehog protein N-product peptide
    • Protein or peptide: Protein-cysteine N-palmitoyltransferase HHAT
    • Protein or peptide: 3H02 Fab heavy chain
    • Protein or peptide: 3H02 Fab light chain
  • Ligand: PALMITIC ACID
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: Palmitoyl-CoA
  • Ligand: Digitonin

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Supramolecule #1: HHAT in complex with a palmitoylated Hedgehog peptide product and...

SupramoleculeName: HHAT in complex with a palmitoylated Hedgehog peptide product and a Fab antibody fragment
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Sonic hedgehog protein N-product peptide

MacromoleculeName: Sonic hedgehog protein N-product peptide / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 750.848 Da
SequenceString:
CGPGRGFG

UniProtKB: Sonic hedgehog protein

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Macromolecule #2: Protein-cysteine N-palmitoyltransferase HHAT

MacromoleculeName: Protein-cysteine N-palmitoyltransferase HHAT / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: Transferases; Acyltransferases; Transferring groups other than aminoacyl groups
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 57.358742 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MLPRWELALY LLASLGFHFY SFYEVYKVSR EHEEELDQEF ELETDTLFGG LKKDATDFEW SFWMEWGKQW LVWLLLGHMV VSQMATLLA RKHRPWILML YGMWACWCVL GTPGVAMVLL HTTISFCVAQ FRSQLLTWLC SLLLLSTLRL QGVEEVKRRW Y KTENEYYL ...String:
MLPRWELALY LLASLGFHFY SFYEVYKVSR EHEEELDQEF ELETDTLFGG LKKDATDFEW SFWMEWGKQW LVWLLLGHMV VSQMATLLA RKHRPWILML YGMWACWCVL GTPGVAMVLL HTTISFCVAQ FRSQLLTWLC SLLLLSTLRL QGVEEVKRRW Y KTENEYYL LQFTLTVRCL YYTSFSLELC WQQLPAASTS YSFPWMLAYV FYYPVLHNGP ILSFSEFIKQ MQQQEHDSLK AS LCVLALG LGRLLCWWWL AELMAHLMYM HAIYSSIPLL ETVSCWTLGG LALAQVLFFY VKYLVLFGVP ALLMRLDGLT PPA LPRCVS TMFSFTGMWR YFDVGLHNFL IRYVYIPVGG SQHGLLGTLF STAMTFAFVS YWHGGYDYLW CWAALNWLGV TVEN GVRRL VETPCIQDSL ARYFSPQARR RFHAALASCS TSMLILSNLV FLGGNEVGKT YWNRIFIQGW PWVTLSVLGF LYCYS HVGI AWAQTYATD

UniProtKB: Protein-cysteine N-palmitoyltransferase HHAT

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Macromolecule #3: 3H02 Fab heavy chain

MacromoleculeName: 3H02 Fab heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 51.307891 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: MGWSCIILFL VATATGVHSQ IQLVQSGPEL KKPGETVKIS CKASGYTFTN YGMNWVRQAP GKGLKWMGWI NTYTGEPTYA DDFKGRFAF SLETSASTAY LQINNLKDED MATYFCARVW NYDYYFDYWG QGTTLTVSSA KTTPPSVYPL APGSAAQTNS M VTLGCLVK ...String:
MGWSCIILFL VATATGVHSQ IQLVQSGPEL KKPGETVKIS CKASGYTFTN YGMNWVRQAP GKGLKWMGWI NTYTGEPTYA DDFKGRFAF SLETSASTAY LQINNLKDED MATYFCARVW NYDYYFDYWG QGTTLTVSSA KTTPPSVYPL APGSAAQTNS M VTLGCLVK GYFPEPVTVT WNSGSLSSGV HTFPAVLQSD LYTLSSSVTV PSSPRPSETV TCNVAHPASS TKVDKKIVPR DC GCKPCIC TVPEVSSVFI FPPKPKDVLT ITLTPKVTCV VVDISKDDPE VQFSWFVDDV EVHTAQTQPR EEQFNSTFRS VSE LPIMHQ DWLNGKEFKC RVNSAAFPAP IEKTISKTKG RPKAPQVYTI PPPKEQMAKD KVSLTCMITD FFPEDITVEW QWNG QPAEN YKNTQPIMNT NGSYFVYSKL NVQKSNWEAG NTFTCSVLHE GLHNHHTEKS LSHSPGK

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Macromolecule #4: 3H02 Fab light chain

MacromoleculeName: 3H02 Fab light chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 22.868863 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: MKLPVRLLVL MFWIPASRSD VLMTQTPLSL PVSLGDQVSI SCRSSQSIVH SNGNTYLEWY LQKPGQSPKL LIYRVSNRFS GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP WTFGGGTKLE IKRADAAPTV SIFPPSSEQL TSGGASVVCF L NNFYPKDI ...String:
MKLPVRLLVL MFWIPASRSD VLMTQTPLSL PVSLGDQVSI SCRSSQSIVH SNGNTYLEWY LQKPGQSPKL LIYRVSNRFS GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP WTFGGGTKLE IKRADAAPTV SIFPPSSEQL TSGGASVVCF L NNFYPKDI NVKWKIDGSE RQNGVLNSWT DQDSKDSTYS MSSTLTLTK

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Macromolecule #5: PALMITIC ACID

MacromoleculeName: PALMITIC ACID / type: ligand / ID: 5 / Number of copies: 1 / Formula: PLM
Molecular weightTheoretical: 256.424 Da
Chemical component information

ChemComp-PLM:
PALMITIC ACID

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Macromolecule #6: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 6 / Number of copies: 1 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE

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Macromolecule #7: Palmitoyl-CoA

MacromoleculeName: Palmitoyl-CoA / type: ligand / ID: 7 / Number of copies: 3 / Formula: PKZ
Molecular weightTheoretical: 1.005943 KDa
Chemical component information

ChemComp-PKZ:
Palmitoyl-CoA

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Macromolecule #8: Digitonin

MacromoleculeName: Digitonin / type: ligand / ID: 8 / Number of copies: 10 / Formula: AJP
Molecular weightTheoretical: 1.229312 KDa
Chemical component information

ChemComp-AJP:
Digitonin / detergent*YM

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 43.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 142121
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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