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- PDB-8tu9: Cryo-EM structure of HGSNAT-acetyl-CoA complex at pH 7.5 -

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Basic information

Entry
Database: PDB / ID: 8tu9
TitleCryo-EM structure of HGSNAT-acetyl-CoA complex at pH 7.5
ComponentsEnhanced green fluorescent protein,Heparan-alpha-glucosaminide N-acetyltransferase,Isoform 2 of Heparan-alpha-glucosaminide N-acetyltransferase
KeywordsTRANSFERASE / Heparan-alpha-glucosaminide N-acetyltransferase / Transmembrane protein 76 / membrane protein
Function / homology
Function and homology information


heparan-alpha-glucosaminide N-acetyltransferase / heparan-alpha-glucosaminide N-acetyltransferase activity / MPS IIIC - Sanfilippo syndrome C / heparan sulfate proteoglycan catabolic process / HS-GAG degradation / lysosomal transport / acyltransferase activity / protein complex oligomerization / tertiary granule membrane / specific granule membrane ...heparan-alpha-glucosaminide N-acetyltransferase / heparan-alpha-glucosaminide N-acetyltransferase activity / MPS IIIC - Sanfilippo syndrome C / heparan sulfate proteoglycan catabolic process / HS-GAG degradation / lysosomal transport / acyltransferase activity / protein complex oligomerization / tertiary granule membrane / specific granule membrane / lysosomal lumen / bioluminescence / generation of precursor metabolites and energy / lysosomal membrane / Neutrophil degranulation / plasma membrane
Similarity search - Function
Heparan-alpha-glucosaminide N-acetyltransferase, catalytic domain / Heparan-alpha-glucosaminide N-acetyltransferase, catalytic / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein
Similarity search - Domain/homology
ACETYL COENZYME *A / Green fluorescent protein / Heparan-alpha-glucosaminide N-acetyltransferase
Similarity search - Component
Biological speciesAequorea victoria (jellyfish)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.26 Å
AuthorsNavratna, V. / Kumar, A. / Mosalaganti, S.
Funding support1items
OrganizationGrant numberCountry
Other government
Citation
Journal: Elife / Year: 2024
Title: Structure of the human heparan-α-glucosaminide -acetyltransferase (HGSNAT).
Authors: Vikas Navratna / Arvind Kumar / Jaimin K Rana / Shyamal Mosalaganti /
Abstract: Degradation of heparan sulfate (HS), a glycosaminoglycan (GAG) comprised of repeating units of -acetylglucosamine and glucuronic acid, begins in the cytosol and is completed in the lysosomes. ...Degradation of heparan sulfate (HS), a glycosaminoglycan (GAG) comprised of repeating units of -acetylglucosamine and glucuronic acid, begins in the cytosol and is completed in the lysosomes. Acetylation of the terminal non-reducing amino group of α-D-glucosamine of HS is essential for its complete breakdown into monosaccharides and free sulfate. Heparan-α-glucosaminide -acetyltransferase (HGSNAT), a resident of the lysosomal membrane, catalyzes this essential acetylation reaction by accepting and transferring the acetyl group from cytosolic acetyl-CoA to terminal α-D-glucosamine of HS in the lysosomal lumen. Mutation-induced dysfunction in HGSNAT causes abnormal accumulation of HS within the lysosomes and leads to an autosomal recessive neurodegenerative lysosomal storage disorder called mucopolysaccharidosis IIIC (MPS IIIC). There are no approved drugs or treatment strategies to cure or manage the symptoms of, MPS IIIC. Here, we use cryo-electron microscopy (cryo-EM) to determine a high-resolution structure of the HGSNAT-acetyl-CoA complex, the first step in the HGSNAT-catalyzed acetyltransferase reaction. In addition, we map the known MPS IIIC mutations onto the structure and elucidate the molecular basis for mutation-induced HGSNAT dysfunction.
#1: Journal: eLife / Year: 2024
Title: Structure of the human heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT)
Authors: Navratna, V. / Kumar, A. / Mosalaganti, S.
#2: Journal: bioRxiv / Year: 2024
Title: Structure of the human heparan-α-glucosaminide -acetyltransferase (HGSNAT).
Authors: Vikas Navratna / Arvind Kumar / Jaimin K Rana / Shyamal Mosalaganti /
Abstract: Degradation of heparan sulfate (HS), a glycosaminoglycan (GAG) comprised of repeating units of -acetylglucosamine and glucuronic acid, begins in the cytosol and is completed in the lysosomes. ...Degradation of heparan sulfate (HS), a glycosaminoglycan (GAG) comprised of repeating units of -acetylglucosamine and glucuronic acid, begins in the cytosol and is completed in the lysosomes. Acetylation of the terminal non-reducing amino group of a-D-glucosamine of HS is essential for its complete breakdown into monosaccharides and free sulfate. Heparan-a-glucosaminide -acetyltransferase (HGSNAT), a resident of the lysosomal membrane, catalyzes this essential acetylation reaction by accepting and transferring the acetyl group from cytosolic acetyl-CoA to terminal a-D-glucosamine of HS in the lysosomal lumen. Mutation-induced dysfunction in HGSNAT causes abnormal accumulation of HS within the lysosomes and leads to an autosomal recessive neurodegenerative lysosomal storage disorder called mucopolysaccharidosis IIIC (MPS IIIC). There are no approved drugs or treatment strategies to cure or manage the symptoms of, MPS IIIC. Here, we use cryo-electron microscopy (cryo-EM) to determine a high-resolution structure of the HGSNAT-acetyl-CoA complex, the first step in HGSNAT catalyzed acetyltransferase reaction. In addition, we map the known MPS IIIC mutations onto the structure and elucidate the molecular basis for mutation-induced HGSNAT dysfunction.
History
DepositionAug 15, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 7, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 11, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Enhanced green fluorescent protein,Heparan-alpha-glucosaminide N-acetyltransferase,Isoform 2 of Heparan-alpha-glucosaminide N-acetyltransferase
B: Enhanced green fluorescent protein,Heparan-alpha-glucosaminide N-acetyltransferase,Isoform 2 of Heparan-alpha-glucosaminide N-acetyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)204,58110
Polymers201,6342
Non-polymers2,9468
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "B"
d_2ens_1chain "A"

NCS domain segments:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11LEULEUILEILEBB49 - 635317 - 903
d_12ACOACOACOACOBG701
d_21LEULEUILEILEAA49 - 635317 - 903
d_22ACOACOACOACOAC701

NCS oper: (Code: givenMatrix: (-0.999999634014, -0.000836221116112, 0.000180850914369), (0.00083627082141, -0.999999612529, 0.000274940524232), (0.000180620933222, 0.00027509166395, 0.99999994585) ...NCS oper: (Code: given
Matrix: (-0.999999634014, -0.000836221116112, 0.000180850914369), (0.00083627082141, -0.999999612529, 0.000274940524232), (0.000180620933222, 0.00027509166395, 0.99999994585)
Vector: 306.088850156, 305.90454485, -0.228578359587)

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Components

#1: Protein Enhanced green fluorescent protein,Heparan-alpha-glucosaminide N-acetyltransferase,Isoform 2 of Heparan-alpha-glucosaminide N-acetyltransferase / Transmembrane protein 76


Mass: 100817.172 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aequorea victoria (jellyfish), (gene. exp.) Homo sapiens (human)
Gene: MMB69_26960, HGSNAT, TMEM76 / Plasmid: pEG BacMam N term StrepII eGFP 3C / Details (production host): Addgene # 160683 / Cell line (production host): HEK293S GnTI- / Production host: Homo sapiens (human)
References: UniProt: A0A9X4KGN5, UniProt: Q68CP4, heparan-alpha-glucosaminide N-acetyltransferase
#2: Chemical ChemComp-ACO / ACETYL COENZYME *A


Mass: 809.571 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C23H38N7O17P3S / Feature type: SUBJECT OF INVESTIGATION
#3: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Heparan acetyl-CoA: alpha-glucosaminide N-acetyltransferase (HGSNAT)
Type: COMPLEX
Details: Full length (Isoform 2) human HGSNAT expressed as a recombinant fusion protein with N-terminal GFP, in mammalian cells.
Entity ID: #1 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.1007 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular locationOrganelle
21Homo sapiens (human)9606Lysosomal membraneLysosomes
31Aequorea victoria (Water jellyfish) (Mesonema victoria)6100
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293S GnTI- / Plasmid: pEG BacMam N term StrepII eGFP 3C
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
10.5 %DigitoninC56H92O291
225 mMTrizma baseNH2C(CH2OH)31
3200 mMSodium ChlorideNaCl1
SpecimenConc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: HGSNAT in digitonin micelle, purified by Strep-Tactin affinity chromatography.
Specimen supportDetails: 15 mA current / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 10000
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4particle selection
2SerialEM9.03image acquisition
4cryoSPARC4CTF correction
12cryoSPARC43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 15000000
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 57739 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingDetails: Made using Model angelo / Source name: Other / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 20.21 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01268518
ELECTRON MICROSCOPYf_angle_d1.912811652
ELECTRON MICROSCOPYf_chiral_restr0.09181336
ELECTRON MICROSCOPYf_plane_restr0.01951426
ELECTRON MICROSCOPYf_dihedral_angle_d6.67781160
Refine LS restraints NCSType: NCS constraints / Rms dev position: 8.33142807632E-11 Å

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