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- EMDB-13520: Cryo-EM structures of human fucosidase FucA1 reveal insight into ... -

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Basic information

Entry
Database: EMDB / ID: EMD-13520
TitleCryo-EM structures of human fucosidase FucA1 reveal insight into substate recognition and catalysis.
Map data
Sample
  • Complex: FucA1 homotetramer in complex with deoxyfuconojirimycin
    • Protein or peptide: Tissue alpha-L-fucosidase
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (2S,3R,4S,5R)-2-METHYLPIPERIDINE-3,4,5-TRIOL
  • Ligand: water
Function / homology
Function and homology information


alpha-L-fucosidase / glycolipid catabolic process / glycosaminoglycan catabolic process / Reactions specific to the complex N-glycan synthesis pathway / alpha-L-fucosidase activity / fucose metabolic process / glycoside catabolic process / lysosomal lumen / azurophil granule lumen / lysosome ...alpha-L-fucosidase / glycolipid catabolic process / glycosaminoglycan catabolic process / Reactions specific to the complex N-glycan synthesis pathway / alpha-L-fucosidase activity / fucose metabolic process / glycoside catabolic process / lysosomal lumen / azurophil granule lumen / lysosome / intracellular membrane-bounded organelle / Neutrophil degranulation / extracellular exosome / extracellular region / cytoplasm
Similarity search - Function
Glycoside hydrolase, family 29, conserved site / Tissue alpha-L-fucosidase / Alpha-L-fucosidase putative active site. / Alpha-L-fucosidase, C-terminal / Alpha-L-fucosidase C-terminal domain / Alpha-L-fucosidase, metazoa-type / Glycoside hydrolase, family 29 / Alpha-L-fucosidase / Alpha-L-fucosidase / Glycosyl hydrolase, all-beta / Glycoside hydrolase superfamily
Similarity search - Domain/homology
Tissue alpha-L-fucosidase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.49 Å
AuthorsArmstrong Z / Meek RW / Wu L / Blaza JN / Davies GJ
Funding support United Kingdom, 4 items
OrganizationGrant numberCountry
Royal Society180016 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)R001162 United Kingdom
UK Research and Innovation (UKRI)T040742 United Kingdom
Wellcome Trust218579 United Kingdom
CitationJournal: Structure / Year: 2022
Title: Cryo-EM structures of human fucosidase FucA1 reveal insight into substrate recognition and catalysis.
Authors: Zachary Armstrong / Richard W Meek / Liang Wu / James N Blaza / Gideon J Davies /
Abstract: Enzymatic hydrolysis of α-L-fucose from fucosylated glycoconjugates is consequential in bacterial infections and the neurodegenerative lysosomal storage disorder fucosidosis. Understanding human α- ...Enzymatic hydrolysis of α-L-fucose from fucosylated glycoconjugates is consequential in bacterial infections and the neurodegenerative lysosomal storage disorder fucosidosis. Understanding human α-L-fucosidase catalysis, in an effort toward drug design, has been hindered by the absence of three-dimensional structural data for any animal fucosidase. Here, we have used cryoelectron microscopy (cryo-EM) to determine the structure of human lysosomal α-L-fucosidase (FucA1) in both an unliganded state and in complex with the inhibitor deoxyfuconojirimycin. These structures, determined at 2.49 Å resolution, reveal the homotetrameric structure of FucA1, the architecture of the catalytic center, and the location of both natural population variations and disease-causing mutations. Furthermore, this work has conclusively identified the hitherto contentious identity of the catalytic acid/base as aspartate-276, representing a shift from both the canonical glutamate acid/base residue and a previously proposed glutamate residue. These findings have furthered our understanding of how FucA1 functions in both health and disease.
History
DepositionSep 1, 2021-
Header (metadata) releaseAug 10, 2022-
Map releaseAug 10, 2022-
UpdateOct 19, 2022-
Current statusOct 19, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13520.png

Downloads & links

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Map

FileDownload / File: emd_13520.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.656 Å
Density
Contour LevelBy AUTHOR: 0.018
Minimum - Maximum-0.040441856 - 0.08772386
Average (Standard dev.)7.474136e-05 (±0.0017539314)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 293.888 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : FucA1 homotetramer in complex with deoxyfuconojirimycin

EntireName: FucA1 homotetramer in complex with deoxyfuconojirimycin
Components
  • Complex: FucA1 homotetramer in complex with deoxyfuconojirimycin
    • Protein or peptide: Tissue alpha-L-fucosidase
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (2S,3R,4S,5R)-2-METHYLPIPERIDINE-3,4,5-TRIOL
  • Ligand: water

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Supramolecule #1: FucA1 homotetramer in complex with deoxyfuconojirimycin

SupramoleculeName: FucA1 homotetramer in complex with deoxyfuconojirimycin
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
Molecular weightExperimental: 225 kDa/nm

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Macromolecule #1: Tissue alpha-L-fucosidase

MacromoleculeName: Tissue alpha-L-fucosidase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: alpha-L-fucosidase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 50.525625 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GQPPRRYTPD WPSLDSRPLP AWFDEAKFGV FIHWGVFSVP AWGSEWFWWH WQGEGRPQYQ RFMRDNYPPG FSYADFGPQF TARFFHPEE WADLFQAAGA KYVVLTTKHH EGFTNWPSPV SWNWNSKDVG PHRDLVGELG TALRKRNIRY GLYHSLLEWF H PLYLLDKK ...String:
GQPPRRYTPD WPSLDSRPLP AWFDEAKFGV FIHWGVFSVP AWGSEWFWWH WQGEGRPQYQ RFMRDNYPPG FSYADFGPQF TARFFHPEE WADLFQAAGA KYVVLTTKHH EGFTNWPSPV SWNWNSKDVG PHRDLVGELG TALRKRNIRY GLYHSLLEWF H PLYLLDKK NGFKTQHFVS AKTMPELYDL VNSYKPDLIW SDGEWECPDT YWNSTNFLSW LYNDSPVKDE VVVNDRWGQN CS CHHGGYY NCEDKFKPQS LPDHKWEMCT SIDKFSWGYR RDMALSDVTE ESEIISELVQ TVSLGGNYLL NIGPTKDGLI VPI FQERLL AVGKWLSING EAIYASKPWR VQWEKNTTSV WYTSKGSAVY AIFLHWPENG VLNLESPITT STTKITMLGI QGDL KWSTD PDKGLFISLP QLPPSAVPAE FAWTIKLTGV K

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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: 4 / 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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Macromolecule #3: (2S,3R,4S,5R)-2-METHYLPIPERIDINE-3,4,5-TRIOL

MacromoleculeName: (2S,3R,4S,5R)-2-METHYLPIPERIDINE-3,4,5-TRIOL / type: ligand / ID: 3 / Number of copies: 4 / Formula: DFU
Molecular weightTheoretical: 147.172 Da
Chemical component information

ChemComp-DFU:
(2S,3R,4S,5R)-2-METHYLPIPERIDINE-3,4,5-TRIOL

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 60 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration2 mg/mL
BufferpH: 4.5
Component:
ConcentrationFormulaName
15.0 mMC8H18N2O4S4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
150.0 mMNaClSodium chlorideSodium chloride
1.0 mMC4H10O2S2Dithiothreitol
60.0 mMNa3C6H5O7Sodium Citrate
60.0 mMNa2HPO4Sodium hydrogen phosphate
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 2 seconds before plunging.

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.49 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.2) / Number images used: 98815

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