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- PDB-7kdv: Murine core lysosomal multienzyme complex (LMC) composed of acid ... -

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Basic information

Entry
Database: PDB / ID: 7kdv
TitleMurine core lysosomal multienzyme complex (LMC) composed of acid beta-galactosidase (GLB1) and protective protein cathepsin A (PPCA, CTSA)
Components
  • Beta-galactosidase
  • Lysosomal protective protein
KeywordsHYDROLASE / glycosidase / protease / lysosome
Function / homology
Function and homology information


Keratan sulfate degradation / Glycosphingolipid metabolism / HS-GAG degradation / response to cortisone / Sialic acid metabolism / : / response to Thyroglobulin triiodothyronine / carboxypeptidase C / serine-type carboxypeptidase activity / galactose catabolic process ...Keratan sulfate degradation / Glycosphingolipid metabolism / HS-GAG degradation / response to cortisone / Sialic acid metabolism / : / response to Thyroglobulin triiodothyronine / carboxypeptidase C / serine-type carboxypeptidase activity / galactose catabolic process / galactoside binding / MHC class II antigen presentation / beta-galactosidase / negative regulation of chaperone-mediated autophagy / vacuole / beta-galactosidase activity / Neutrophil degranulation / regulation of protein stability / lysosome / hydrolase activity / lysosomal membrane / intracellular membrane-bounded organelle / Golgi apparatus / protein homodimerization activity / mitochondrion / proteolysis / extracellular space / cytoplasm
Similarity search - Function
Beta-galactosidase 1-like / Beta-galactosidase jelly roll domain / Serine carboxypeptidase, serine active site / Serine carboxypeptidases, serine active site. / Peptidase S10, serine carboxypeptidase / Serine carboxypeptidases, histidine active site / Serine carboxypeptidase / Beta-galactosidase second all-beta domain / Serine carboxypeptidases, histidine active site. / Glycoside hydrolase, family 35, conserved site ...Beta-galactosidase 1-like / Beta-galactosidase jelly roll domain / Serine carboxypeptidase, serine active site / Serine carboxypeptidases, serine active site. / Peptidase S10, serine carboxypeptidase / Serine carboxypeptidases, histidine active site / Serine carboxypeptidase / Beta-galactosidase second all-beta domain / Serine carboxypeptidases, histidine active site. / Glycoside hydrolase, family 35, conserved site / Glycosyl hydrolases family 35 putative active site. / Glycoside hydrolase 35, catalytic domain / Glycosyl hydrolases family 35 / Glycoside hydrolase, family 35 / Galactose-binding-like domain superfamily / Alpha/Beta hydrolase fold / Glycoside hydrolase superfamily
Similarity search - Domain/homology
Lysosomal protective protein / Beta-galactosidase
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.59 Å
AuthorsGorelik, A. / Illes, K. / Hasan, S.M.N. / Nagar, B. / Mazhab-Jafari, M.T.
Funding support Canada, 3items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)MFE-164773 Canada
Canadian Institutes of Health Research (CIHR)MOP-133535 Canada
Canadian Institutes of Health Research (CIHR)419240 Canada
CitationJournal: Sci Adv / Year: 2021
Title: Structure of the murine lysosomal multienzyme complex core.
Authors: Alexei Gorelik / Katalin Illes / S M Naimul Hasan / Bhushan Nagar / Mohammad T Mazhab-Jafari /
Abstract: The enzymes β-galactosidase (GLB1) and neuraminidase 1 (NEU1; sialidase 1) participate in the degradation of glycoproteins and glycolipids in the lysosome. To remain active and stable, they ...The enzymes β-galactosidase (GLB1) and neuraminidase 1 (NEU1; sialidase 1) participate in the degradation of glycoproteins and glycolipids in the lysosome. To remain active and stable, they associate with PPCA [protective protein cathepsin A (CTSA)] into a high-molecular weight lysosomal multienzyme complex (LMC), of which several forms exist. Genetic defects in these three proteins cause the lysosomal storage diseases GM1-gangliosidosis/mucopolysaccharidosis IV type B, sialidosis, and galactosialidosis, respectively. To better understand the interactions between these enzymes, we determined the three-dimensional structure of the murine LMC core. This 0.8-MDa complex is composed of three GLB1 dimers and three CTSA dimers, adopting a triangular architecture maintained through six copies of a unique GLB1-CTSA polar interface. Mutations in this contact surface that occur in GM1-gangliosidosis prevent formation of the LMC in vitro. These findings may facilitate development of therapies for lysosomal storage disorders.
History
DepositionOct 9, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 17, 2021Provider: repository / Type: Initial release
Revision 1.1May 26, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Beta-galactosidase
B: Lysosomal protective protein
C: Beta-galactosidase
D: Lysosomal protective protein
E: Beta-galactosidase
F: Lysosomal protective protein
G: Beta-galactosidase
H: Lysosomal protective protein
I: Beta-galactosidase
J: Lysosomal protective protein
K: Beta-galactosidase
L: Lysosomal protective protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)759,89260
Polymers744,64412
Non-polymers15,24848
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, complex observed by gel filtration and dynamic light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area39720 Å2
ΔGint104 kcal/mol
Surface area248350 Å2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "K"
d_2ens_1chain "A"
d_3ens_1chain "E"
d_4ens_1chain "G"
d_5ens_1chain "I"
d_6ens_1chain "C"
d_1ens_2chain "L"
d_2ens_2chain "D"
d_3ens_2chain "F"
d_4ens_2chain "H"
d_5ens_2chain "J"
d_6ens_2chain "B"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1THRSERK1 - 600
d_12ens_1NAGNAGK
d_13ens_1NAGNAGK
d_14ens_1NAGNAGK
d_15ens_1NAGNAGK
d_16ens_1NAGNAGK
d_17ens_1NAGNAGK
d_18ens_1NAGNAGK
d_19ens_1NAGNAGK
d_21ens_1THRSERA1 - 600
d_22ens_1NAGNAGA
d_23ens_1NAGNAGA
d_24ens_1NAGNAGA
d_25ens_1NAGNAGA
d_26ens_1NAGNAGA
d_27ens_1NAGNAGA
d_28ens_1NAGNAGA
d_29ens_1NAGNAGA
d_31ens_1THRSERE1 - 600
d_32ens_1NAGNAGE
d_33ens_1NAGNAGE
d_34ens_1NAGNAGE
d_35ens_1NAGNAGE
d_36ens_1NAGNAGE
d_37ens_1NAGNAGE
d_38ens_1NAGNAGE
d_39ens_1NAGNAGE
d_41ens_1THRSERG1 - 600
d_42ens_1NAGNAGG
d_43ens_1NAGNAGG
d_44ens_1NAGNAGG
d_45ens_1NAGNAGG
d_46ens_1NAGNAGG
d_47ens_1NAGNAGG
d_48ens_1NAGNAGG
d_49ens_1NAGNAGG
d_51ens_1THRSERI1 - 600
d_52ens_1NAGNAGI
d_53ens_1NAGNAGI
d_54ens_1NAGNAGI
d_55ens_1NAGNAGI
d_56ens_1NAGNAGI
d_57ens_1NAGNAGI
d_58ens_1NAGNAGI
d_59ens_1NAGNAGI
d_61ens_1THRSERC1 - 600
d_62ens_1NAGNAGC
d_63ens_1NAGNAGC
d_64ens_1NAGNAGC
d_65ens_1NAGNAGC
d_66ens_1NAGNAGC
d_67ens_1NAGNAGC
d_68ens_1NAGNAGC
d_69ens_1NAGNAGC
d_11ens_2SERTYRL1 - 452
d_12ens_2NAGNAGL
d_13ens_2NAGNAGL
d_14ens_2NAGNAGL
d_15ens_2BMABMAL
d_21ens_2SERTYRD1 - 452
d_22ens_2NAGNAGD
d_23ens_2NAGNAGD
d_24ens_2NAGNAGD
d_25ens_2BMABMAD
d_31ens_2SERTYRF1 - 452
d_32ens_2NAGNAGF
d_33ens_2NAGNAGF
d_34ens_2NAGNAGF
d_35ens_2BMABMAF
d_41ens_2SERTYRH1 - 452
d_42ens_2NAGNAGH
d_43ens_2NAGNAGH
d_44ens_2NAGNAGH
d_45ens_2BMABMAH
d_51ens_2SERTYRJ1 - 452
d_52ens_2NAGNAGJ
d_53ens_2NAGNAGJ
d_54ens_2NAGNAGJ
d_55ens_2BMABMAJ
d_61ens_2SERTYRB1 - 452
d_62ens_2NAGNAGB
d_63ens_2NAGNAGB
d_64ens_2NAGNAGB
d_65ens_2BMABMAB

NCS ensembles :
ID
ens_1
ens_2

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Components

#1: Protein
Beta-galactosidase / Acid beta-galactosidase / Lactase


Mass: 71450.180 Da / Num. of mol.: 6 / Mutation: R468Q, N517D, E534G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Glb1, Bgl, Glb-1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P23780, beta-galactosidase
#2: Protein
Lysosomal protective protein / Carboxypeptidase C / Carboxypeptidase L / Cathepsin A / Protective protein cathepsin A / PPCA / ...Carboxypeptidase C / Carboxypeptidase L / Cathepsin A / Protective protein cathepsin A / PPCA / Protective protein for beta-galactosidase


Mass: 52657.141 Da / Num. of mol.: 6 / Mutation: C32A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ctsa, Ppgb / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P16675, carboxypeptidase C
#3: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#4: Polysaccharide
beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#5: 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


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 30 / 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
Has ligand of interestN

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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: core lysosomal multienzyme complex composed of CTSA and GLB1
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 4.5 / Details: 50 mM sodium acetate pH 4.5, 50 mM NaCl
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER/RHODIUM / Grid type: Homemade
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: two datasets: untilted and 40 degrees tilted
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 75000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 41 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 315

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19rc6_4061refinement
PHENIX1.19rc6_4061refinement
EM software
IDNameVersionCategory
4cryoSPARC2CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cryoSPARC2initial Euler assignment
11cryoSPARC2final Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionDetails: Patch CTF implemented in cryoSPARC V2 / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 4.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 17591 / Symmetry type: POINT
Atomic model buildingB value: 116 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
13THC

3thc

1
21IVY

1ivy

1
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 116.52 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004253184
ELECTRON MICROSCOPYf_angle_d0.838672420
ELECTRON MICROSCOPYf_chiral_restr0.0487902
ELECTRON MICROSCOPYf_plane_restr0.00669312
ELECTRON MICROSCOPYf_dihedral_angle_d12.346219506
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.00070461484779
ens_1d_3EELECTRON MICROSCOPYNCS constraints0.000712040365276
ens_1d_4GELECTRON MICROSCOPYNCS constraints0.000709758902306
ens_1d_5IELECTRON MICROSCOPYNCS constraints0.00070447071257
ens_1d_6CELECTRON MICROSCOPYNCS constraints0.000712657601518
ens_2d_2DELECTRON MICROSCOPYNCS constraints0.000711383251419
ens_2d_3FELECTRON MICROSCOPYNCS constraints0.0007039782168
ens_2d_4HELECTRON MICROSCOPYNCS constraints0.000707792476645
ens_2d_5JELECTRON MICROSCOPYNCS constraints0.000711143676864
ens_2d_6BELECTRON MICROSCOPYNCS constraints0.000712922505645

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