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- EMDB-13841: Focused refinement of Hedgehog acyltransferase (HHAT) in complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-13841
TitleFocused refinement of Hedgehog acyltransferase (HHAT) in complex with megabody 177 bound to non-hydrolysable palmitoyl-CoA
Map dataLocally filtered, globally sharpened map of focused NU-refinement of the HHAT-Nanobody part.
Sample
  • Complex: Hedgehog acyltransferase (HHAT) bound to non hydrolysable palmitoyl-CoA in complex with megabody 177
    • Complex: Megabody 177
    • Complex: Hedgehog acyltransferase (HHAT) bound to non hydrolysable palmitoyl-CoA
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 / endoplasmic reticulum / Golgi apparatus
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 speciesEscherichia coli K-12 (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.69 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 3, 2021-
Header (metadata) releaseJan 19, 2022-
Map releaseJan 19, 2022-
UpdateJan 19, 2022-
Current statusJan 19, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.98
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 1.98
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13841.png

Downloads & links

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Map

FileDownload / File: emd_13841.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally filtered, globally sharpened map of focused NU-refinement of 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.
= 331.599 Å		1.11 Å/pix.
x 300 pix.
= 331.599 Å		1.11 Å/pix.
x 300 pix.
= 331.599 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.10533 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.98 / Movie #1: 1.98
Minimum - Maximum-6.835238 - 10.617896
Average (Standard dev.)0.0012989199 (±0.096313715)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 331.599 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.105331.105331.10533
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z331.599331.599331.599
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-6.83510.6180.001

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Supplemental data

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Mask #1

Fileemd_13841_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_13841_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_13841_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
    • Complex: Hedgehog acyltransferase (HHAT) bound to non hydrolysable palmitoyl-CoA

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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

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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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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 - Pressure: 101.325 kPa
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: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 3637 / Average electron dose: 54.8 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: 977000
CTF correctionSoftware - Name: cryoSPARC (ver. V3.1.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.69 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. V3.1.1) / Number images used: 238000
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)
FSC plot (resolution estimation)

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Atomic model buiding 1

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

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