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- PDB-7pm4: Cryo-EM structures of human fucosidase FucA1 reveal insight into ... -

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Basic information

Entry
Database: PDB / ID: 7pm4
TitleCryo-EM structures of human fucosidase FucA1 reveal insight into substate recognition and catalysis.
ComponentsTissue alpha-L-fucosidase
KeywordsHYDROLASE / Fucosidase
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
(2S,3R,4S,5R)-2-METHYLPIPERIDINE-3,4,5-TRIOL / Tissue alpha-L-fucosidase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.49 Å
AuthorsArmstrong, Z. / Meek, R.W. / Wu, L. / Blaza, J.N. / Davies, G.J.
Funding support United Kingdom, 4items
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 10, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 19, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Tissue alpha-L-fucosidase
B: Tissue alpha-L-fucosidase
C: Tissue alpha-L-fucosidase
D: Tissue alpha-L-fucosidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)203,57612
Polymers202,1034
Non-polymers1,4748
Water1,08160
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Tissue alpha-L-fucosidase / Alpha-L-fucosidase I / Alpha-L-fucoside fucohydrolase 1 / Alpha-L-fucosidase 1


Mass: 50525.625 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FUCA1, Nbla10230 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P04066, alpha-L-fucosidase
#2: 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 / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H15NO6
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
#3: Chemical
ChemComp-DFU / (2S,3R,4S,5R)-2-METHYLPIPERIDINE-3,4,5-TRIOL / DEOXYFUCONOJIRIMYCIN


Mass: 147.172 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C6H13NO3 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 60 / Source method: isolated from a natural source / Formula: H2O
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: FucA1 homotetramer in complex with deoxyfuconojirimycin
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 225 kDa/nm / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 4.5
Buffer component
IDConc.NameFormulaBuffer-ID
115 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)C8H18N2O4S1
2150 mMSodium chlorideNaClSodium chloride1
31 mMDithiothreitolC4H10O2S21
460 mMSodium CitrateNa3C6H5O71
560 mMSodium hydrogen phosphateNa2HPO41
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K / Details: blot for 2 seconds before plunging

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
7Coot0.8.9.2model fitting
9PHENIX1.19model refinement
13RELION3.1.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.49 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 98815 / Symmetry type: POINT

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