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- EMDB-14578: Structure of Hedgehog acyltransferase (HHAT) in complex with mega... -

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Basic information

Entry
Database: EMDB / ID: EMD-14578
TitleStructure of Hedgehog acyltransferase (HHAT) in complex with megabody 177 bound to non-hydrolysable palmitoyl-CoA (Consensus Map)
Map dataConsensus map used to generate a composite map.
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
KeywordsHHAT / inhibitor / palmitoyl-CoA / co enzyme A / Hedgehog acyl transferase / Sonic Hedgehog / SHH / MBOAT / morphogen / palmitoylation / signalling / endoplasmic reticulum / membrane protein / heme / small molecule binding / drug target
Biological speciesHomo sapiens (human) / Escherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.12 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
DepositionMar 22, 2022-
Header (metadata) releaseMar 30, 2022-
Map releaseMar 30, 2022-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14578.png

Downloads & links

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Map

FileDownload / File: emd_14578.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationConsensus map used to generate a composite map.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.11 Å/pix.
x 300 pix.
= 331.59 Å		1.11 Å/pix.
x 300 pix.
= 331.59 Å		1.11 Å/pix.
x 300 pix.
= 331.59 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1053 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.869
Minimum - Maximum-2.6911705 - 4.6501107
Average (Standard dev.)0.0022792607 (±0.080247104)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 331.59 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14578_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_14578_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_14578_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
Source (natural)Organism: Homo sapiens (human)

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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)

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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)

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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
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 / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
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
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.12 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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