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- EMDB-41620: Cryo-EM structure of HGSNAT-acetyl-CoA complex at pH 7.5 -

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Basic information

Entry
Database: EMDB / ID: EMD-41620
TitleCryo-EM structure of HGSNAT-acetyl-CoA complex at pH 7.5
Map dataSharpened map resulting from Non-Uniform refinement (C2) in cryoSPARC. This is the primary map used for model building and PDB validation.
Sample
  • Complex: Heparan acetyl-CoA: alpha-glucosaminide N-acetyltransferase (HGSNAT)
    • Protein or peptide: Enhanced green fluorescent protein,Heparan-alpha-glucosaminide N-acetyltransferase,Isoform 2 of Heparan-alpha-glucosaminide N-acetyltransferase
  • Ligand: ACETYL COENZYME *A
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsHeparan-alpha-glucosaminide N-acetyltransferase / Transmembrane protein 76 / membrane protein / TRANSFERASE
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 / tertiary granule membrane / specific granule membrane / lysosomal lumen ...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 / tertiary granule membrane / specific granule membrane / lysosomal lumen / protein complex oligomerization / 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
Green fluorescent protein / Heparan-alpha-glucosaminide N-acetyltransferase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.26 Å
AuthorsNavratna V / Kumar A / Mosalaganti S
Funding support1 items
OrganizationGrant numberCountry
Other government
CitationJournal: bioRxiv / Year: 2023
Title: Structure of the human heparan-α-glucosaminide -acetyltransferase (HGSNAT).
Authors: Vikas Navratna / Arvind Kumar / 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 in an open-to-lumen conformation, 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, 2023-
Header (metadata) releaseFeb 7, 2024-
Map releaseFeb 7, 2024-
UpdateFeb 7, 2024-
Current statusFeb 7, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41620.png

Downloads & links

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Map

FileDownload / File: emd_41620.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map resulting from Non-Uniform refinement (C2) in cryoSPARC. This is the primary map used for model building and PDB validation.
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.207
Minimum - Maximum-1.2754602 - 1.8943442
Average (Standard dev.)0.0059850314 (±0.04655288)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 306.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Mask (auto-tightening) used for Non-Uniform refinement in cryoSPARC.

Fileemd_41620_additional_1.map
AnnotationMask (auto-tightening) used for Non-Uniform refinement in cryoSPARC.
Projections & Slices
AxesZYX

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Additional map: Half map (half-map A) resulting from local refinement...

Fileemd_41620_additional_10.map
AnnotationHalf map (half-map A) resulting from local refinement (C1) in cryoSPARC after symmetry expansion.
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

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Additional map: Half map (half-map B) resulting from local refinement...

Fileemd_41620_additional_11.map
AnnotationHalf map (half-map B) resulting from local refinement (C1) in cryoSPARC after symmetry expansion.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Mask used for Non-uniform refinement (C2) in cryoSPARC.

Fileemd_41620_additional_2.map
AnnotationMask used for Non-uniform refinement (C2) in cryoSPARC.
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AxesZYX

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Histogram

Histogram (log scale)

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Additional map: Mask used for Non-Uniform refinement (C2) in cryoSPARC.

Fileemd_41620_additional_3.map
AnnotationMask used for Non-Uniform refinement (C2) in cryoSPARC.
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AxesZYX

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Additional map: Mask Used for refinement (C1) during local refinement...

Fileemd_41620_additional_4.map
AnnotationMask Used for refinement (C1) during local refinement of symmetry expanded particles in cryoSPARC
Projections & Slices
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Additional map: Unsharpened map resulting from Non-Uniform refinement (C2) in...

Fileemd_41620_additional_5.map
AnnotationUnsharpened map resulting from Non-Uniform refinement (C2) in cryoSPARC.
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Additional map: Composite map for model validation generated from focused...

Fileemd_41620_additional_6.map
AnnotationComposite map for model validation generated from focused refinement of the extra-membranous domain and intra-membranous domain separately.
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Additional map: Mask (auto-tightening) used for refinement during local refinement...

Fileemd_41620_additional_7.map
AnnotationMask (auto-tightening) used for refinement during local refinement (C1) of symmetry expanded particles in cryoSPARC.
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Mask used for refinement during local refinement (C1)...

Fileemd_41620_additional_8.map
AnnotationMask used for refinement during local refinement (C1) of symmetry expanded particles in cryoSPARC.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map resulting from local refinement (C1) in...

Fileemd_41620_additional_9.map
AnnotationUnsharpened map resulting from local refinement (C1) in cryoSPARC after symmetry expansion.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map (half-map A) resulting from Non-Uniform refinement...

Fileemd_41620_half_map_1.map
AnnotationHalf map (half-map A) resulting from Non-Uniform refinement (C2) in cryoSPARC.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map (half-map B) resulting from Non-Uniform refinement...

Fileemd_41620_half_map_2.map
AnnotationHalf map (half-map B) resulting from Non-Uniform refinement (C2) in cryoSPARC.
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

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Sample components

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Entire : Heparan acetyl-CoA: alpha-glucosaminide N-acetyltransferase (HGSNAT)

EntireName: Heparan acetyl-CoA: alpha-glucosaminide N-acetyltransferase (HGSNAT)
Components
  • Complex: Heparan acetyl-CoA: alpha-glucosaminide N-acetyltransferase (HGSNAT)
    • Protein or peptide: Enhanced green fluorescent protein,Heparan-alpha-glucosaminide N-acetyltransferase,Isoform 2 of Heparan-alpha-glucosaminide N-acetyltransferase
  • Ligand: ACETYL COENZYME *A
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Heparan acetyl-CoA: alpha-glucosaminide N-acetyltransferase (HGSNAT)

SupramoleculeName: Heparan acetyl-CoA: alpha-glucosaminide N-acetyltransferase (HGSNAT)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Full length (Isoform 2) human HGSNAT expressed as a recombinant fusion protein with N-terminal GFP, in mammalian cells.
Source (natural)Organism: Homo sapiens (human) / Organelle: Lysosomes / Location in cell: Lysosomal membrane
Molecular weightTheoretical: 100.7 KDa

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Macromolecule #1: Enhanced green fluorescent protein,Heparan-alpha-glucosaminide N-...

MacromoleculeName: Enhanced green fluorescent protein,Heparan-alpha-glucosaminide N-acetyltransferase,Isoform 2 of 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: 100.817172 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MWSHPQFEKG SGVSKGEELF TGVVPILVEL DGDVNGHKFS VSGEGEGDAT YGKLTLKFIC TTGKLPVPWP TLVTTLTYGV QCFSRYPDH MKQHDFFKSA MPEGYVQERT IFFKDDGNYK TRAEVKFEGD TLVNRIELKG IDFKEDGNIL GHKLEYNYNS H NVYIMADK ...String:
MWSHPQFEKG SGVSKGEELF TGVVPILVEL DGDVNGHKFS VSGEGEGDAT YGKLTLKFIC TTGKLPVPWP TLVTTLTYGV QCFSRYPDH MKQHDFFKSA MPEGYVQERT IFFKDDGNYK TRAEVKFEGD TLVNRIELKG IDFKEDGNIL GHKLEYNYNS H NVYIMADK QKNGIKVNFK IRHNIEDGSV QLADHYQQNT PIGDGPVLLP DNHYLSTQSK LSKDPNEKRD HMVLLEFVTA AG ITLGMDE LYKSGLRSGL EVLFQGPEFE FMSGAGRALA ALLLAASVLS AALLAPGGSS GRDAQAAPPR DLDKKRHAEL KMD QALLLI HNELLWTNLT VYWKSECCYH CLFQVLVNVP QSPKAGKPSA AAASVSTQHG SILQLNDTLE EKEVCRLEYR FGEF GNYSL LVKNIHNGVS EIACDLAVNE DPVDSNLPVS IAFLIGLAVI IVISFLRLLL SLDDFNNWIS KAISSRETDR LINSE LGSP SRTDPLDGDV QPATWRLSAL PPRLRSVDTF RGIALILMVF VNYGGGKYWY FKHASWNGLT VADLVFPWFV FIMGSS IFL SMTSILQRGC SKFRLLGKIA WRSFLLICIG IIIVNPNYCL GPLSWDKVRI PGVLQRLGVT YFVVAVLELL FAKPVPE HC ASERSCLSLR DITSSWPQWL LILVLEGLWL GLTFLLPVPG CPTGYLGPGG IGDFGKYPNC TGGAAGYIDR LLLGDDHL Y QHPSSAVLYH TEVAYDPEGI LGTINSIVMA FLGVQAGKIL LYYKARTKDI LIRFTAWCCI LGLISVALTK VSENEGFIP VNKNLWSLSY VTTLSSFAFF ILLVLYPVVD VKGLWTGTPF FYPGMNSILV YVGHEVFENY FPFQWKLKDN QSHKEHLTQN IVATALWVL IAYILYRKKI FWKI

UniProtKB: Green fluorescent protein, Heparan-alpha-glucosaminide N-acetyltransferase

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Macromolecule #2: ACETYL COENZYME *A

MacromoleculeName: ACETYL COENZYME *A / type: ligand / ID: 2 / Number of copies: 2 / Formula: ACO
Molecular weightTheoretical: 809.571 Da
Chemical component information

ChemComp-ACO:
ACETYL COENZYME *A / Acetyl-CoA

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 6 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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

Concentration0.9 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
0.5 %C56H92O29Digitonin
25.0 mMNH2C(CH2OH)3Trizma base
200.0 mMNaClSodium chlorideSodium Chloride
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Pressure: 0.026000000000000002 kPa / Details: 15 mA current
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV
DetailsHGSNAT in digitonin micelle, purified by Strep-Tactin affinity chromatography.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 2 / Number real images: 10000 / Average exposure time: 2.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 15000000
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0) / Number images used: 57739
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: in silico model / Details: Made using Model angelo
RefinementProtocol: RIGID BODY FIT
Output model

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

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