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- EMDB-23836: Human Hedgehog acyltransferase (HHAT) in complex with palmitoyl-C... -

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Basic information

Entry
Database: EMDB / ID: EMD-23836
TitleHuman Hedgehog acyltransferase (HHAT) in complex with palmitoyl-CoA and two Fab antibody fragments
Map datafinal map
Sample
  • Complex: HHAT in complex with palmitoyl-CoA and two Fab antibody fragments
    • Protein or peptide: Protein-cysteine N-palmitoyltransferase HHAT
    • Protein or peptide: 1C06 Fab heavy chain
    • Protein or peptide: 1C06 Fab light chain
    • Protein or peptide: 3H02 Fab heavy chain
    • Protein or peptide: 3H02 Fab light chain
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: Palmitoyl-CoA
  • Ligand: Digitonin
  • Ligand: water
Function / homology
Function and homology information


N-terminal peptidyl-L-cysteine N-palmitoylation / O-acyltransferase activity / HHAT G278V doesn't palmitoylate Hh-Np / palmitoyltransferase activity / smoothened signaling pathway / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / Hedgehog ligand biogenesis / Golgi membrane / endoplasmic reticulum membrane / GTP binding ...N-terminal peptidyl-L-cysteine N-palmitoylation / O-acyltransferase activity / HHAT G278V doesn't palmitoylate Hh-Np / palmitoyltransferase activity / smoothened signaling pathway / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / Hedgehog ligand biogenesis / Golgi membrane / endoplasmic reticulum membrane / GTP binding / Golgi apparatus / endoplasmic reticulum
Similarity search - Function
Membrane bound O-acyl transferase, MBOAT / MBOAT, membrane-bound O-acyltransferase family
Similarity search - Domain/homology
Protein-cysteine N-palmitoyltransferase HHAT
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 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-
UpdateAug 25, 2021-
Current statusAug 25, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.65
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 1.65
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7mhy
  • Surface level: 1.65
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7mhy
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23836.png

Downloads & links

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Map

FileDownload / File: emd_23836.map.gz / Format: CCP4 / Size: 272.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfinal map
Voxel sizeX=Y=Z: 0.532 Å
Density
Contour LevelBy AUTHOR: 1.65 / Movie #1: 1.65
Minimum - Maximum-10.477143 - 18.682293
Average (Standard dev.)-1.1082734e-11 (±0.40181586)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin1019593
Dimensions415415415
Spacing415415415
CellA=B=C: 220.78 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.5320.5320.532
M x/y/z415415415
origin x/y/z0.0000.0000.000
length x/y/z220.780220.780220.780
α/β/γ90.00090.00090.000
start NX/NY/NZ1019593
NX/NY/NZ415415415
MAP C/R/S213
start NC/NR/NS9510193
NC/NR/NS415415415
D min/max/mean-10.47718.682-0.000

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Supplemental data

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Sample components

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Entire : HHAT in complex with palmitoyl-CoA and two Fab antibody fragments

EntireName: HHAT in complex with palmitoyl-CoA and two Fab antibody fragments
Components
  • Complex: HHAT in complex with palmitoyl-CoA and two Fab antibody fragments
    • Protein or peptide: Protein-cysteine N-palmitoyltransferase HHAT
    • Protein or peptide: 1C06 Fab heavy chain
    • Protein or peptide: 1C06 Fab light chain
    • Protein or peptide: 3H02 Fab heavy chain
    • Protein or peptide: 3H02 Fab light chain
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: Palmitoyl-CoA
  • Ligand: Digitonin
  • Ligand: water

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Supramolecule #1: HHAT in complex with palmitoyl-CoA and two Fab antibody fragments

SupramoleculeName: HHAT in complex with palmitoyl-CoA and two Fab antibody fragments
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Protein-cysteine N-palmitoyltransferase HHAT

MacromoleculeName: Protein-cysteine N-palmitoyltransferase HHAT / type: protein_or_peptide / ID: 1 / 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

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Macromolecule #2: 1C06 Fab heavy chain

MacromoleculeName: 1C06 Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 22.847662 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: MERHWIFLFL FSVTAGVHSQ VQLRQSGAEL AKPGASVKMS CKASGYTFTN YWLHWIKQRP GQGLEWIGYI SPNTGSTEYS QKFKDRATL TADKSSSTAY MQLSSLTSED SAVYYCAGSR ITGTWFAYWG QGTLVTVSAA KTTPPSVYPL APGSAAQTNS M VTLGCLVK ...String:
MERHWIFLFL FSVTAGVHSQ VQLRQSGAEL AKPGASVKMS CKASGYTFTN YWLHWIKQRP GQGLEWIGYI SPNTGSTEYS QKFKDRATL TADKSSSTAY MQLSSLTSED SAVYYCAGSR ITGTWFAYWG QGTLVTVSAA KTTPPSVYPL APGSAAQTNS M VTLGCLVK GYFPEPVTVT WNSGSLSSGV HTFPAVLQSD LYTLSSSVTV PSS

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Macromolecule #3: 1C06 Fab light chain

MacromoleculeName: 1C06 Fab light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 22.897879 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: MKLPVRLLVL MFWIPASRSD VLMTQTPLSL PVSLGDQVSI SCRSSQSIVH SNGNTYLEWY LQKPGQSPKL LIYWVSNRFS GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP WTFGGGTKLE IKRADAAPTV SIFPPSSEQL TSGGASVVCF L NNFYPKDI ...String:
MKLPVRLLVL MFWIPASRSD VLMTQTPLSL PVSLGDQVSI SCRSSQSIVH SNGNTYLEWY LQKPGQSPKL LIYWVSNRFS GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP WTFGGGTKLE IKRADAAPTV SIFPPSSEQL TSGGASVVCF L NNFYPKDI NVKWKIDGSE RQNGVLNSWT DQDSKDSTYS MSSTLTLTK

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Macromolecule #4: 3H02 Fab heavy chain

MacromoleculeName: 3H02 Fab heavy chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.229082 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: MDWLWNLLFL MAAAQSAQAQ IQLVQSGPEL KKPGETVKIS CKASGYTFTN YGMNWVRQAP GKGLKWMGWI NTYTGEPTYA DDFKGRFAF SLETSASTAY LQINNLKDED MATYFCARVW NYDYYFDYWG QGTTLTVSSA KTTPPSVYPL APGSAAQTNS M VTLGCLVK ...String:
MDWLWNLLFL MAAAQSAQAQ IQLVQSGPEL KKPGETVKIS CKASGYTFTN YGMNWVRQAP GKGLKWMGWI NTYTGEPTYA DDFKGRFAF SLETSASTAY LQINNLKDED MATYFCARVW NYDYYFDYWG QGTTLTVSSA KTTPPSVYPL APGSAAQTNS M VTLGCLVK GYFPEPVTVT WNSGSLSSGV HTFPAVLQSD LYTLSSSVTV PSS

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Macromolecule #5: 3H02 Fab light chain

MacromoleculeName: 3H02 Fab light chain / type: protein_or_peptide / ID: 5 / 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 #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 / Heme B

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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 / Palmitoyl-CoA

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Macromolecule #8: Digitonin

MacromoleculeName: Digitonin / type: ligand / ID: 8 / Number of copies: 14 / Formula: AJP
Molecular weightTheoretical: 1.229312 KDa
Chemical component information

ChemComp-AJP:
Digitonin / detergent*YM / Digitonin

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Macromolecule #9: water

MacromoleculeName: water / type: ligand / ID: 9 / Number of copies: 15 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 26.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 174058

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