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- EMDB-13860: Structure of Hedgehog acyltransferase (HHAT) in complex with mega... -

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Basic information

Entry
Database: EMDB / ID: EMD-13860
TitleStructure of Hedgehog acyltransferase (HHAT) in complex with megabody 177 bound to IMP-1575
Map dataLocally filtered, globally sharpened, NU-refinement focused on the HHAT-Nanobody part
Sample
  • Complex: Hedgehog acyltransferase (HHAT) bound to non hydrolysable palmitoyl-CoA in complex with megabody 177
    • Complex: Megabody 177
      • Protein or peptide: Megabody 177
    • Complex: Hedgehog acyltransferase (HHAT) bound to non hydrolysable palmitoyl-CoA
      • Protein or peptide: Protein-cysteine N-palmitoyltransferase HHAT
  • Ligand: CHOLESTEROL
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: 2-(2-methylpropylamino)-1-[(4R)-4-(6-methylpyridin-2-yl)-6,7-dihydro-4H-thieno[3,2-c]pyridin-5-yl]ethanone
  • Ligand: PALMITIC ACID
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) / Escherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.59 Å
AuthorsCoupland C / Carrique L / Siebold C
Funding supportEuropean Union, 4 items
OrganizationGrant numberCountry
Cancer Research UKC20724/A26752European Union
Cancer Research UKC20724/A14414European Union
Biotechnology and Biological Sciences Research Council (BBSRC)BB/T01508X/1European Union
European Research Council (ERC)647278European Union
CitationJournal: Mol Cell / Year: 2021
Title: Structure, mechanism, and inhibition of Hedgehog acyltransferase.
Authors: Claire E Coupland / Sebastian A Andrei / T Bertie Ansell / Loic Carrique / Pramod Kumar / Lea Sefer / Rebekka A Schwab / Eamon F X Byrne / Els Pardon / Jan Steyaert / Anthony I Magee / ...Authors: Claire E Coupland / Sebastian A Andrei / T Bertie Ansell / Loic Carrique / Pramod Kumar / Lea Sefer / Rebekka A Schwab / Eamon F X Byrne / Els Pardon / Jan Steyaert / Anthony I Magee / Thomas Lanyon-Hogg / Mark S P Sansom / Edward W Tate / Christian Siebold /
Abstract: The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate ...The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.
History
DepositionNov 9, 2021-
Header (metadata) releaseJan 26, 2022-
Map releaseJan 26, 2022-
UpdateJan 26, 2022-
Current statusJan 26, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7q6z
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13860.png

Downloads & links

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Map

FileDownload / File: emd_13860.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally filtered, globally sharpened, NU-refinement focused on the HHAT-Nanobody part
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.11 Å/pix.
x 300 pix.
= 332.4 Å		1.11 Å/pix.
x 300 pix.
= 332.4 Å		1.11 Å/pix.
x 300 pix.
= 332.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.108 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.714 / Movie #1: 0.8
Minimum - Maximum-5.676953 - 8.781211
Average (Standard dev.)0.0011193987 (±0.06758404)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 332.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1081.1081.108
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z332.400332.400332.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-5.6778.7810.001

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Supplemental data

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Mask #1

Fileemd_13860_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_13860_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_13860_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hedgehog acyltransferase (HHAT) bound to non hydrolysable palmito...

EntireName: Hedgehog acyltransferase (HHAT) bound to non hydrolysable palmitoyl-CoA in complex with megabody 177
Components
  • Complex: Hedgehog acyltransferase (HHAT) bound to non hydrolysable palmitoyl-CoA in complex with megabody 177
    • Complex: Megabody 177
      • Protein or peptide: Megabody 177
    • Complex: Hedgehog acyltransferase (HHAT) bound to non hydrolysable palmitoyl-CoA
      • Protein or peptide: Protein-cysteine N-palmitoyltransferase HHAT
  • Ligand: CHOLESTEROL
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: 2-(2-methylpropylamino)-1-[(4R)-4-(6-methylpyridin-2-yl)-6,7-dihydro-4H-thieno[3,2-c]pyridin-5-yl]ethanone
  • Ligand: PALMITIC ACID

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Supramolecule #1: Hedgehog acyltransferase (HHAT) bound to non hydrolysable palmito...

SupramoleculeName: Hedgehog acyltransferase (HHAT) bound to non hydrolysable palmitoyl-CoA in complex with megabody 177
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK293T

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Supramolecule #2: Megabody 177

SupramoleculeName: Megabody 177 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: WK6 / Recombinant plasmid: pMESP23E2

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Supramolecule #3: Hedgehog acyltransferase (HHAT) bound to non hydrolysable palmito...

SupramoleculeName: Hedgehog acyltransferase (HHAT) bound to non hydrolysable palmitoyl-CoA
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293S GnTI-/- / Recombinant plasmid: pHR-CMV-TetO2

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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: 58.327754 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 YYTNFSLELC 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 AWAQTYATDG TETSQVAPA

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Macromolecule #2: Megabody 177

MacromoleculeName: Megabody 177 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 102.601586 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKYLLPTAAA GLLLLAAQPA MAQVQLVESG GGLVKEETQS GLNNYARVVE KGQYDSLEIP AQVAASWESG RDDAAVFGFI DKEQLDKYV ANGGKRSDWT VKFAENRSQD GTLLGYSLLQ ESVDQASYMY SDNHYLAEMA TILGKPEEAK RYRQLAQQLA D YINTCMFD ...String:
MKYLLPTAAA GLLLLAAQPA MAQVQLVESG GGLVKEETQS GLNNYARVVE KGQYDSLEIP AQVAASWESG RDDAAVFGFI DKEQLDKYV ANGGKRSDWT VKFAENRSQD GTLLGYSLLQ ESVDQASYMY SDNHYLAEMA TILGKPEEAK RYRQLAQQLA D YINTCMFD PTTQFYYDVR IEDKPLANGC AGKPIVERGK GPEGWSPLFN GAATQANADA VVKVMLDPKE FNTFVPLGTA AL TNPAFGA DIYWRGRVWV DQFWFGLKGM ERYGYRDDAL KLADTFFRHA KGLTADGPIQ ENYNPLTGAQ QGAPNFSWSA AHL YMLYND FFRKQASGGG SGGGGSGGGG SGNADNYKNV INRTGAPQYM KDYDYDDHQR FNPFFDLGAW HGHLLPDGPN TMGG FPGVA LLTEEYINFM ASNFDRLTVW QDGKKVDFTL EAYSIPGALV QKLTAKDVQV EMTLRFATPR TSLLETKITS NKPLD LVWD GELLEKLEAK EGKPLSDKTI AGEYPDYQRK ISATRDGLKV TFGKVRATWD LLTSGESEYQ VHKSLPVQTE INGNRF TSK AHINGSTTLY TTYSHLLTAQ EVSKEQMQIR DILARPAFYL TASQQRWEEY LKKGLTNPDA TPEQTRVAVK AIETLNG NW RSPGGAVKFN TVTPSVTGRW FSGNQTWPWD TWKQAFAMAH FNPDIAKENI RAVFSWQIQP GDSVRPQDVG FVPDLIAW N LSPERGGDGG NWNERNTKPS LAAWSVMEVY NVTQDKTWVA EMYPKLVAYH DWWLRNRDHN GNGVPEYGAT RDKAHNTES GEMLFTVKKS LRLSCTASGA IFSTYDVSWY RQAPEKPREL VAIITRGGNT HYADTVKGRF TISRDNAKKT VNLQMNSLKP EDTAVYYCH AGVQGAMLGP RNYWGQGTQV TVSSHHHHHH

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Macromolecule #3: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 3 / Number of copies: 1 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #4: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 4 / 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 #5: 2-(2-methylpropylamino)-1-[(4R)-4-(6-methylpyridin-2-yl)-6,7-dihy...

MacromoleculeName: 2-(2-methylpropylamino)-1-[(4R)-4-(6-methylpyridin-2-yl)-6,7-dihydro-4H-thieno[3,2-c]pyridin-5-yl]ethanone
type: ligand / ID: 5 / Number of copies: 1 / Formula: 9V3
Molecular weightTheoretical: 343.486 Da
Chemical component information

ChemComp-9V3:
2-(2-methylpropylamino)-1-[(4R)-4-(6-methylpyridin-2-yl)-6,7-dihydro-4H-thieno[3,2-c]pyridin-5-yl]ethanone

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Macromolecule #6: PALMITIC ACID

MacromoleculeName: PALMITIC ACID / type: ligand / ID: 6 / Number of copies: 1 / Formula: PLM
Molecular weightTheoretical: 256.424 Da
Chemical component information

ChemComp-PLM:
PALMITIC ACID

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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 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
20.0 mMTris-HCl
150.0 mMsodium chlorideNaCl
5.0 mM16:0 Ether Coenzyme AC37H77N10O16P3S
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 10683 / Average electron dose: 53.48 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
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: 3000000
CTF correctionSoftware - Name: cryoSPARC (ver. V3.1.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.59 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. V3.1.1) / Number images used: 319000
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. V3.1.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. V3.1.1)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 3.1.1)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 56
Output model

PDB-7q6z:
Structure of Hedgehog acyltransferase (HHAT) in complex with megabody 177 bound to IMP-1575

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