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- EMDB-43344: Cryo-EM structure of human HGSNAT bound with CoA -

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Basic information

Entry
Database: EMDB / ID: EMD-43344
TitleCryo-EM structure of human HGSNAT bound with CoA
Map datasharpened map
Sample
  • Complex: Purified human HGSNAT
    • Protein or peptide: Heparan-alpha-glucosaminide N-acetyltransferase
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: COENZYME A
KeywordsMembrane protein / Lysosome / Transmembrane acetyltransferase
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 / lysosomal membrane / Neutrophil degranulation / plasma membrane
Similarity search - Function
Heparan-alpha-glucosaminide N-acetyltransferase, catalytic domain / Heparan-alpha-glucosaminide N-acetyltransferase, catalytic
Similarity search - Domain/homology
Heparan-alpha-glucosaminide N-acetyltransferase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsLi F / Zhao B
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)K99MH119591 United States
Other private United States
CitationJournal: Nat Commun / Year: 2024
Title: Structural and mechanistic insights into a lysosomal membrane enzyme HGSNAT involved in Sanfilippo syndrome.
Authors: Boyang Zhao / Zhongzheng Cao / Yi Zheng / Phuong Nguyen / Alisa Bowen / Robert H Edwards / Robert M Stroud / Yi Zhou / Menno Van Lookeren Campagne / Fei Li /
Abstract: Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the integral lysosomal membrane enzyme ...Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the integral lysosomal membrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT). Mutations of HGSNAT cause HS accumulation and consequently mucopolysaccharidosis IIIC, a devastating lysosomal storage disease characterized by progressive neurological deterioration and early death where no treatment is available. HGSNAT catalyzes a unique transmembrane acetylation reaction where the acetyl group of cytosolic acetyl-CoA is transported across the lysosomal membrane and attached to HS in one reaction. However, the reaction mechanism remains elusive. Here we report six cryo-EM structures of HGSNAT along the reaction pathway. These structures reveal a dimer arrangement and a unique structural fold, which enables the elucidation of the reaction mechanism. We find that a central pore within each monomer traverses the membrane and controls access of cytosolic acetyl-CoA to the active site at its luminal mouth where glucosamine binds. A histidine-aspartic acid catalytic dyad catalyzes the transfer reaction via a ternary complex mechanism. Furthermore, the structures allow the mapping of disease-causing variants and reveal their potential impact on the function, thus creating a framework to guide structure-based drug discovery efforts.
History
DepositionJan 12, 2024-
Header (metadata) releaseJun 26, 2024-
Map releaseJun 26, 2024-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43344.png

Downloads & links

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Map

FileDownload / File: emd_43344.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.81 Å/pix.
x 440 pix.
= 357.72 Å		0.81 Å/pix.
x 440 pix.
= 357.72 Å		0.81 Å/pix.
x 440 pix.
= 357.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.813 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-1.1773709 - 1.6937262
Average (Standard dev.)0.00001761598 (±0.023746483)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 357.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: unsharpened map used to build TM1 (residue 191-226)....

Fileemd_43344_additional_1.map
Annotationunsharpened map used to build TM1 (residue 191-226). All other areas are built based on the sharpened map. The entire model is refined against the sharpened map.
Projections & Slices
AxesZYX

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Half map: half-map

Fileemd_43344_half_map_1.map
Annotationhalf-map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map

Fileemd_43344_half_map_2.map
Annotationhalf-map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Purified human HGSNAT

EntireName: Purified human HGSNAT
Components
  • Complex: Purified human HGSNAT
    • Protein or peptide: Heparan-alpha-glucosaminide N-acetyltransferase
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: COENZYME A

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Supramolecule #1: Purified human HGSNAT

SupramoleculeName: Purified human HGSNAT / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 73 KDa

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Macromolecule #1: Heparan-alpha-glucosaminide N-acetyltransferase

MacromoleculeName: Heparan-alpha-glucosaminide N-acetyltransferase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: heparan-alpha-glucosaminide N-acetyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 73.365039 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTGARASAAE QRRAGRSGQA RAAERAAGMS GAGRALAALL LAASVLSAAL LAPGGSSGRD AQAAPPRDLD KKRHAELKMD QALLLIHNE LLWTNLTVYW KSECCYHCLF QVLVNVPQSP KAGKPSAAAA SVSTQHGSIL QLNDTLEEKE VCRLEYRFGE F GNYSLLVK ...String:
MTGARASAAE QRRAGRSGQA RAAERAAGMS GAGRALAALL LAASVLSAAL LAPGGSSGRD AQAAPPRDLD KKRHAELKMD QALLLIHNE LLWTNLTVYW KSECCYHCLF QVLVNVPQSP KAGKPSAAAA SVSTQHGSIL QLNDTLEEKE VCRLEYRFGE F GNYSLLVK NIHNGVSEIA CDLAVNEDPV DSNLPVSIAF LIGLAVIIVI SFLRLLLSLD DFNNWISKAI SSRETDRLIN SE LGSPSRT DPLDGDVQPA TWRLSALPPR LRSVDTFRGI ALILMVFVNY GGGKYWYFKH ASWNGLTVAD LVFPWFVFIM GSS IFLSMT SILQRGCSKF RLLGKIAWRS FLLICIGIII VNPNYCLGPL SWDKVRIPGV LQRLGVTYFV VAVLELLFAK PVPE HCASE RSCLSLRDIT SSWPQWLLIL VLEGLWLGLT FLLPVPGCPT GYLGPGGIGD FGKYPNCTGG AAGYIDRLLL GDDHL YQHP SSAVLYHTEV AYDPEGILGT INSIVMAFLG VQAGKILLYY KARTKDILIR FTAWCCILGL ISVALTKVSE NEGFIP VNK NLWSLSYVTT LSSFAFFILL VLYPVVDVKG LWTGTPFFYP GMNSILVYVG HEVFENYFPF QWKLKDNQSH KEHLTQN IV ATALWVLIAY ILYRKKIFWK I

UniProtKB: Heparan-alpha-glucosaminide N-acetyltransferase

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Macromolecule #2: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 7 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #3: COENZYME A

MacromoleculeName: COENZYME A / type: ligand / ID: 3 / Number of copies: 2 / Formula: COA
Molecular weightTheoretical: 767.534 Da
Chemical component information

ChemComp-COA:
COENZYME A

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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

Concentration3.1 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 22.0 µm / Nominal defocus min: 10.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 256492
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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