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- EMDB-22830: Murine core lysosomal multienzyme complex (LMC) composed of acid ... -

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Basic information

Entry
Database: EMDB / ID: EMD-22830
TitleMurine core lysosomal multienzyme complex (LMC) composed of acid beta-galactosidase (GLB1) and protective protein cathepsin A (PPCA, CTSA)
Map dataunsharpened
Sample
  • Complex: core lysosomal multienzyme complex composed of CTSA and GLB1
    • Protein or peptide: Beta-galactosidase
    • Protein or peptide: Lysosomal protective protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Keywordsglycosidase / protease / lysosome / HYDROLASE
Function / homology
Function and homology information


Keratan sulfate degradation / HS-GAG degradation / keratan sulfate proteoglycan catabolic process / response to cortisone / Sialic acid metabolism / response to Thyroglobulin triiodothyronine / carboxypeptidase C / Glycosphingolipid catabolism / galactose catabolic process / serine-type carboxypeptidase activity ...Keratan sulfate degradation / HS-GAG degradation / keratan sulfate proteoglycan catabolic process / response to cortisone / Sialic acid metabolism / response to Thyroglobulin triiodothyronine / carboxypeptidase C / Glycosphingolipid catabolism / galactose catabolic process / serine-type carboxypeptidase activity / galactoside binding / glycoprotein catabolic process / beta-galactosidase / MHC class II antigen presentation / ganglioside catabolic process / beta-galactosidase activity / negative regulation of chaperone-mediated autophagy / Neutrophil degranulation / regulation of protein stability / lysosome / hydrolase activity / Golgi apparatus / protein homodimerization activity / mitochondrion / proteolysis / extracellular space / membrane / cytoplasm
Similarity search - Function
Beta-galactosidase 1-like / : / Beta-galactosidase, first all-beta domain / Serine carboxypeptidase, serine active site / : / Beta-galactosidase, galactose-binding domain / Serine carboxypeptidases, serine active site. / Peptidase S10, serine carboxypeptidase / Serine carboxypeptidases, histidine active site / Serine carboxypeptidase ...Beta-galactosidase 1-like / : / Beta-galactosidase, first all-beta domain / Serine carboxypeptidase, serine active site / : / Beta-galactosidase, galactose-binding domain / Serine carboxypeptidases, serine active site. / Peptidase S10, serine carboxypeptidase / Serine carboxypeptidases, histidine active site / Serine carboxypeptidase / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.59 Å
AuthorsGorelik A / Illes K / Hasan SMN / Nagar B / Mazhab-Jafari MT
Funding support Canada, 3 items
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, 2020-
Header (metadata) releaseMar 17, 2021-
Map releaseMar 17, 2021-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kdv
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7kdv
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22830.png

Downloads & links

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Map

FileDownload / File: emd_22830.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 320 pix.
= 329.6 Å		1.03 Å/pix.
x 320 pix.
= 329.6 Å		1.03 Å/pix.
x 320 pix.
= 329.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.03 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.055 / Movie #1: 0.2
Minimum - Maximum-0.20294254 - 0.67577523
Average (Standard dev.)0.002004645 (±0.052423134)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 329.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.031.031.03
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z329.600329.600329.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.2030.6760.002

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Supplemental data

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Additional map: density-modified with Phenix ResolveCryoEM

Fileemd_22830_additional_1.map
Annotationdensity-modified with Phenix ResolveCryoEM
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: sharpened with Phenix Autosharpen

Fileemd_22830_additional_2.map
Annotationsharpened with Phenix Autosharpen
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map A

Fileemd_22830_half_map_1.map
Annotationhalf-map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map B

Fileemd_22830_half_map_2.map
Annotationhalf-map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : core lysosomal multienzyme complex composed of CTSA and GLB1

EntireName: core lysosomal multienzyme complex composed of CTSA and GLB1
Components
  • Complex: core lysosomal multienzyme complex composed of CTSA and GLB1
    • Protein or peptide: Beta-galactosidase
    • Protein or peptide: Lysosomal protective protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: core lysosomal multienzyme complex composed of CTSA and GLB1

SupramoleculeName: core lysosomal multienzyme complex composed of CTSA and GLB1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Beta-galactosidase

MacromoleculeName: Beta-galactosidase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: beta-galactosidase
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 71.45018 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DRHHHHHHGS VTQRTFKLDY SRDRFLKDGQ PFRYISGSIH YFRIPRFYWE DRLLKMKMAG LNAIQMYVPW NFHEPQPGQY EFSGDRDVE HFIQLAHELG LLVILRPGPY ICAEWDMGGL PAWLLEKQSI VLRSSDPDYL VAVDKWLAVL LPKMKPLLYQ N GGPIITVQ ...String:
DRHHHHHHGS VTQRTFKLDY SRDRFLKDGQ PFRYISGSIH YFRIPRFYWE DRLLKMKMAG LNAIQMYVPW NFHEPQPGQY EFSGDRDVE HFIQLAHELG LLVILRPGPY ICAEWDMGGL PAWLLEKQSI VLRSSDPDYL VAVDKWLAVL LPKMKPLLYQ N GGPIITVQ VENEYGSYFA CDYDYLRFLV HRFRYHLGND VILFTTDGAS EKMLKCGTLQ DLYATVDFGT GNNITQAFLV QR KFEPKGP LINSEFYTGW LDHWGKPHST VKTKTLATSL YNLLARGANV NLYMFIGGTN FAYWNGANTP YEPQPTSYDY DAP LSEAGD LTKKYFALRE VIQMFKEVPE GPIPPSTPKF AYGKVALRKF KTVAEALGIL CPNGPVKSLY PLTFTQVKQY FGYV LYRTT LPQDCSNPKP IFSSPFNGVR DRAYVSVDGV PQGILDRNLM TALNIQGKAG ATLDILVENM GRVNYGRFIN DFKGL ISNM TINSTVLTNW TVFPLDTEAM VRNHLWGREA SDGGHLDGRS TSNSSDLILP TFYVGNFSIP SGIPDLPQDT FIQFPG WSK GQVWINGFNL GRYWPTMGPQ KTLFVPRNIL TTSAPNNITV LELEFAPCSE GTPELCTVEF VDTPVIS

UniProtKB: Beta-galactosidase

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Macromolecule #2: Lysosomal protective protein

MacromoleculeName: Lysosomal protective protein / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO / EC number: carboxypeptidase C
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 52.657141 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DRHHHHHHGS APDQDEIDAL PGLAKQPSFR QYSGYLRASD SKHFHYWFVE SQNDPKNSPV VLWLNGGPGC SSLDGLLTEH GPFLIQPDG VTLEYNPYAW NLIANVLYIE SPAGVGFSYS DDKMYVTNDT EVAENNYEAL KDFFRLFPEY KDNKLFLTGE S YAGIYIPT ...String:
DRHHHHHHGS APDQDEIDAL PGLAKQPSFR QYSGYLRASD SKHFHYWFVE SQNDPKNSPV VLWLNGGPGC SSLDGLLTEH GPFLIQPDG VTLEYNPYAW NLIANVLYIE SPAGVGFSYS DDKMYVTNDT EVAENNYEAL KDFFRLFPEY KDNKLFLTGE S YAGIYIPT LAVLVMQDPS MNLQGLAVGN GLASYEQNDN SLVYFAYYHG LLGNRLWTSL QTHCCAQNKC NFYDNKDPEC VN NLLEVSR IVGKSGLNIY NLYAPCAGGV PGRHRYEDTL VVQDFGNIFT RLPLKRRFPE ALMRSGDKVR LDPPCTNTTA PSN YLNNPY VRKALHIPES LPRWDMCNFL VNLQYRRLYQ SMNSQYLKLL SSQKYQILLY NGDVDMACNF MGDEWFVDSL NQKM EVQRR PWLVDYGESG EQVAGFVKEC SHITFLTIKG AGHMVPTDKP RAAFTMFSRF LNKEPY

UniProtKB: Lysosomal protective protein

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Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 30 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

Concentration0.5 mg/mL
BufferpH: 4.5 / Details: 50 mM sodium acetate pH 4.5, 50 mM NaCl
GridModel: Homemade / Material: COPPER/RHODIUM / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Detailstwo datasets: untilted and 40 degrees tilted
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 315 / Average electron dose: 41.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 75000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE / Details: Ab initio reconstruction in cryoSPARC V2
Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 4.59 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 17591
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 2)
Details: Stochastic gradient descent (SGD) implemented in cryoSPARC V2 ab-initio reconstruction.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Details: Branch-and-bound algorithm implemented in cryoSPARC V2
Final 3D classificationSoftware - Name: cryoSPARC

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Atomic model buiding 1

Initial model
PDB IDChain

3thc

source_name: PDB, initial_model_type: experimental model

1ivy

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 116
Output model

PDB-7kdv:
Murine core lysosomal multienzyme complex (LMC) composed of acid beta-galactosidase (GLB1) and protective protein cathepsin A (PPCA, CTSA)

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