+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-43345 | |||||||||
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Title | Cryo-EM structure of human HGSNAT in inactive state | |||||||||
Map data | sharpened map | |||||||||
Sample |
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Keywords | Membrane protein / Lysosome / Transmembrane acetyltransferase / TRANSFERASE | |||||||||
Function / homology | : Function and homology information | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.49 Å | |||||||||
Authors | Li F | |||||||||
Funding support | United States, 2 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_43345.map.gz | 307.1 MB | EMDB map data format | |
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Header (meta data) | emd-43345-v30.xml emd-43345.xml | 17 KB 17 KB | Display Display | EMDB header |
Images | emd_43345.png | 96.7 KB | ||
Filedesc metadata | emd-43345.cif.gz | 6.1 KB | ||
Others | emd_43345_additional_1.map.gz emd_43345_half_map_1.map.gz emd_43345_half_map_2.map.gz | 161.3 MB 301.2 MB 301.2 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-43345 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-43345 | HTTPS FTP |
-Validation report
Summary document | emd_43345_validation.pdf.gz | 921.6 KB | Display | EMDB validaton report |
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Full document | emd_43345_full_validation.pdf.gz | 921.1 KB | Display | |
Data in XML | emd_43345_validation.xml.gz | 16.9 KB | Display | |
Data in CIF | emd_43345_validation.cif.gz | 20.1 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-43345 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-43345 | HTTPS FTP |
-Related structure data
Related structure data | 8vlvMC 8vkjC 8vlgC 8vliC 8vluC 8vlyC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_43345.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | sharpened map | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.834 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: unsharpened map used to build TM1 (residue 191-226)....
File | emd_43345_additional_1.map | ||||||||||||
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Annotation | unsharpened map used to build TM1 (residue 191-226). All other area is built based on the sharpened map. The entire model, including TM1, is refined against the sharpened map. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map
File | emd_43345_half_map_1.map | ||||||||||||
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Annotation | half map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map
File | emd_43345_half_map_2.map | ||||||||||||
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Annotation | half map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : purified human HGSNAT
Entire | Name: purified human HGSNAT |
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Components |
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-Supramolecule #1: purified human HGSNAT
Supramolecule | Name: purified human HGSNAT / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 73 KDa |
-Macromolecule #1: Heparan-alpha-glucosaminide N-acetyltransferase
Macromolecule | Name: Heparan-alpha-glucosaminide N-acetyltransferase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: ec: 2.3.1.78 |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 73.365039 KDa |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: 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: UNIPROTKB: Q68CP4 |
-Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose
Macromolecule | Name: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 7 / Formula: NAG |
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Molecular weight | Theoretical: 221.208 Da |
Chemical component information | ChemComp-NAG: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 2.9 mg/mL |
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Buffer | pH: 7.4 |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 54.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination 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 |
-Image processing
Startup model | Type of model: INSILICO MODEL |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 134353 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |