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- EMDB-50502: Lysosomal transporting complex of beta-glucocerebrosidase (GCase)... -

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Basic information

Entry
Database: EMDB / ID: EMD-50502
TitleLysosomal transporting complex of beta-glucocerebrosidase (GCase) and lysosomal integral membrane protein 2 (LIMP-2) with bound Pro-macrobodies (Combined focus map)
Map dataComposite Map of GCase/LIMP-2/PMbs tetramer, generated in Phenix. Obtained from Consensus map and focused local refinements of GCase and LIMP-2.
Sample
  • Complex: Lysosomal transporting complex of beta-glucocerebrosidase and lysosomal integral membrane protein 2 (LIMP-2) with two bound Pro-macrobodies.
    • Protein or peptide: Lysosomal acid glucosylceramidase
    • Protein or peptide: Nanobody Nb6
    • Protein or peptide: Lysosome membrane protein 2
    • Protein or peptide: Nanobody Nb1
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: 2-(N-MORPHOLINO)-ETHANESULFONIC ACID
  • Ligand: water
KeywordsParkinson / lysosome / Gaucher / GCase / SCARB2 / Pro-macrobody / Glucosylceramide / complex / TRANSPORT PROTEIN
Function / homology
Function and homology information


regulation of glucosylceramide catabolic process / regulation of carbohydrate catabolic process / regulation of endosome organization / steryl-beta-glucosidase activity / positive regulation of neuronal action potential / beta-glucoside catabolic process / cerebellar Purkinje cell layer formation / aminophospholipid transport / termination of signal transduction / galactosylceramidase ...regulation of glucosylceramide catabolic process / regulation of carbohydrate catabolic process / regulation of endosome organization / steryl-beta-glucosidase activity / positive regulation of neuronal action potential / beta-glucoside catabolic process / cerebellar Purkinje cell layer formation / aminophospholipid transport / termination of signal transduction / galactosylceramidase / galactosylceramidase activity / glucosylceramidase / scavenger receptor binding / positive regulation of protein lipidation / lymphocyte migration / glucosylceramide catabolic process / regulation of lysosomal protein catabolic process / glucosylceramidase activity / sphingosine biosynthetic process / autophagosome organization / microglial cell proliferation / glucosyltransferase activity / endosome to plasma membrane protein transport / regulation of TOR signaling / regulation of lysosome organization / protein targeting to lysosome / response to thyroid hormone / Glycosphingolipid catabolism / microglia differentiation / ceramide biosynthetic process / phosphatidylcholine binding / positive regulation of type 2 mitophagy / lipid storage / lipid glycosylation / brain morphogenesis / positive regulation of protein-containing complex disassembly / response to pH / Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds / pyramidal neuron differentiation / cargo receptor activity / negative regulation of protein metabolic process / scavenger receptor activity / motor behavior / lysosome organization / Transferases; Glycosyltransferases; Hexosyltransferases / cholesterol binding / neuromuscular process / phosphatidylserine binding / hematopoietic stem cell proliferation / antigen processing and presentation / Association of TriC/CCT with target proteins during biosynthesis / response to testosterone / response to dexamethasone / negative regulation of interleukin-6 production / homeostasis of number of cells / regulation of macroautophagy / establishment of skin barrier / negative regulation of MAPK cascade / negative regulation of protein-containing complex assembly / cell maturation / mitophagy / receptor-mediated endocytosis / cholesterol metabolic process / lysosomal lumen / cellular response to starvation / respiratory electron transport chain / determination of adult lifespan / sensory perception of sound / trans-Golgi network / clathrin-coated endocytic vesicle membrane / positive regulation of neuron projection development / autophagy / negative regulation of inflammatory response / response to estrogen / endocytic vesicle membrane / late endosome membrane / transmembrane signaling receptor activity / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / Cargo recognition for clathrin-mediated endocytosis / cellular response to tumor necrosis factor / Clathrin-mediated endocytosis / protein-folding chaperone binding / T cell differentiation in thymus / virus receptor activity / neuron apoptotic process / negative regulation of neuron apoptotic process / proteasome-mediated ubiquitin-dependent protein catabolic process / lysosome / endosome membrane / Golgi membrane / signaling receptor binding / lysosomal membrane / focal adhesion / endoplasmic reticulum membrane / enzyme binding / endoplasmic reticulum / Golgi apparatus / protein homodimerization activity / extracellular exosome / membrane
Similarity search - Function
Lysosome membrane protein II / CD36 family / CD36 family / Glycosyl hydrolase family 30, TIM-barrel domain / Glycosyl hydrolase family 30 TIM-barrel domain / Glycosyl hydrolase family 30, beta sandwich domain / Glycosyl hydrolase family 30 beta sandwich domain / Glycoside hydrolase family 30 / Glycoside hydrolase superfamily
Similarity search - Domain/homology
Lysosomal acid glucosylceramidase / Lysosome membrane protein 2
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsDobert JP / Schaefer JHS / Dal Maso T / Socher E / Versees W / Moeller A / Zunke F / Arnold P
Funding support United States, 5 items
OrganizationGrant numberCountry
Michael J. Fox FoundationMJFF-17240; MJFF-020706; MJFF-022745 United States
Other governmentInterdisciplinary Center for Clinical Research (IZKF); Jochen-Kalden funding programme N8
Other governmentFonds voor Wetenschappelijk Onderzoek; G031324N
Other governmentVUB Strategic Research Program Financing; SRP95
Other privateFriedrich-Ebert Foundation; no grant number
CitationJournal: Nat Commun / Year: 2025
Title: Cryo-TEM structure of β-glucocerebrosidase in complex with its transporter LIMP-2.
Authors: Jan Philipp Dobert / Jan-Hannes Schäfer / Thomas Dal Maso / Priyadarshini Ravindran / Dustin J E Huard / Eileen Socher / Lisa A Schildmeyer / Raquel L Lieberman / Wim Versées / Arne ...Authors: Jan Philipp Dobert / Jan-Hannes Schäfer / Thomas Dal Maso / Priyadarshini Ravindran / Dustin J E Huard / Eileen Socher / Lisa A Schildmeyer / Raquel L Lieberman / Wim Versées / Arne Moeller / Friederike Zunke / Philipp Arnold /
Abstract: Targeting proteins to their final cellular destination requires transport mechanisms and nearly all lysosomal enzymes reach the lysosome via the mannose-6-phosphate receptor pathway. One of the few ...Targeting proteins to their final cellular destination requires transport mechanisms and nearly all lysosomal enzymes reach the lysosome via the mannose-6-phosphate receptor pathway. One of the few known exceptions is the enzyme β-glucocerebrosidase (GCase) that requires the lysosomal integral membrane protein type-2 (LIMP-2) as a proprietary lysosomal transporter. Genetic variations in the GCase encoding gene GBA1 cause Gaucher's disease (GD) and present the highest genetic risk factor to develop Parkinson's disease (PD). Activators targeting GCase emerge as a promising therapeutic approach to treat GD and PD, with pre-clinical and clinical trials ongoing. In this study, we resolve the complex of GCase and LIMP-2 using cryo-electron microscopy with the aid of an engineered LIMP-2 shuttle and two GCase-targeted pro-macrobodies. We identify helix 5 and helix 7 of LIMP-2 to interact with a binding pocket in GCase, forming a mostly hydrophobic interaction interface supported by one essential salt bridge. Understanding the interplay of GCase and LIMP-2 on a structural level is crucial to identify potential activation sites and conceptualizing novel therapeutic approaches targeting GCase. Here, we unveil the protein structure of a mannose-6-phosphate-independent lysosomal transport complex and provide fundamental knowledge for translational clinical research to overcome GD and PD.
History
DepositionMay 31, 2024-
Header (metadata) releaseApr 9, 2025-
Map releaseApr 9, 2025-
UpdateApr 9, 2025-
Current statusApr 9, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50502.png

Downloads & links

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Map

FileDownload / File: emd_50502.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite Map of GCase/LIMP-2/PMbs tetramer, generated in Phenix. Obtained from Consensus map and focused local refinements of GCase and LIMP-2.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.93 Å/pix.
x 320 pix.
= 296. Å		0.93 Å/pix.
x 320 pix.
= 296. Å		0.93 Å/pix.
x 320 pix.
= 296. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.925 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.5
Minimum - Maximum-47.509205000000001 - 79.621444999999994
Average (Standard dev.)-0.0077985683 (±1.0409676)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 296.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Lysosomal transporting complex of beta-glucocerebrosidase and lys...

EntireName: Lysosomal transporting complex of beta-glucocerebrosidase and lysosomal integral membrane protein 2 (LIMP-2) with two bound Pro-macrobodies.
Components
  • Complex: Lysosomal transporting complex of beta-glucocerebrosidase and lysosomal integral membrane protein 2 (LIMP-2) with two bound Pro-macrobodies.
    • Protein or peptide: Lysosomal acid glucosylceramidase
    • Protein or peptide: Nanobody Nb6
    • Protein or peptide: Lysosome membrane protein 2
    • Protein or peptide: Nanobody Nb1
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: 2-(N-MORPHOLINO)-ETHANESULFONIC ACID
  • Ligand: water

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Supramolecule #1: Lysosomal transporting complex of beta-glucocerebrosidase and lys...

SupramoleculeName: Lysosomal transporting complex of beta-glucocerebrosidase and lysosomal integral membrane protein 2 (LIMP-2) with two bound Pro-macrobodies.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2, #4, #1, #3
Details: Maltose-binding protein (MBP) domains of Pro-macrobodies were not resolved, only nanobody domains are included in the model.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 250 KDa

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Macromolecule #1: Lysosome membrane protein 2

MacromoleculeName: Lysosome membrane protein 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.862691 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: KKIVLRNGTE AFDSWEKPPL PVYTQFYFFN VTNPEEILRG ETPRVEEVGP YTYRELRNKA NIQFGDNGTT ISAVSNKAYV FERDQSVGD PKIDLIRTLN IPVLTVIEWS QVHFLREIIE AMLKAYQQKL FVTHTVDELL WGYKDEILSL IHVFRPDISP Y FGLFYEKN ...String:
KKIVLRNGTE AFDSWEKPPL PVYTQFYFFN VTNPEEILRG ETPRVEEVGP YTYRELRNKA NIQFGDNGTT ISAVSNKAYV FERDQSVGD PKIDLIRTLN IPVLTVIEWS QVHFLREIIE AMLKAYQQKL FVTHTVDELL WGYKDEILSL IHVFRPDISP Y FGLFYEKN GTNDGDYVFL TGEDSYLNFT KIVEWNGKTS LDWWITDKCN MINGTDGDSF HPLITKDEVL YVFPSDFCRS VY ITFSDYE SVQGLPAFRY KVPAEILANT SDNAGFCIPE GNCLGSGVLN VSICKNGAPI IMSFPHFYQA DERFVSAIEG MHP NQEDHE TFVDINPLTG IILKAAKRFQ INIYVKKLDD FVETGDIRTM VFPVMYLNES VHIDKETASR LKSMI

UniProtKB: Lysosome membrane protein 2

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Macromolecule #2: Lysosomal acid glucosylceramidase

MacromoleculeName: Lysosomal acid glucosylceramidase / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: glucosylceramidase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 55.659219 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: ARPCIPKSFG YSSVVCVCNA TYCDSFDPPT FPALGTFSRY ESTRSGRRME LSMGPIQANH TGTGLLLTLQ PEQKFQKVKG FGGAMTDAA ALNILALSPP AQNLLLKSYF SEEGIGYNII RVPMASCDFS IRTYTYADTP DDFQLHNFSL PEEDTKLKIP L IHRALQLA ...String:
ARPCIPKSFG YSSVVCVCNA TYCDSFDPPT FPALGTFSRY ESTRSGRRME LSMGPIQANH TGTGLLLTLQ PEQKFQKVKG FGGAMTDAA ALNILALSPP AQNLLLKSYF SEEGIGYNII RVPMASCDFS IRTYTYADTP DDFQLHNFSL PEEDTKLKIP L IHRALQLA QRPVSLLASP WTSPTWLKTN GAVNGKGSLK GQPGDIYHQT WARYFVKFLD AYAEHKLQFW AVTAENEPSA GL LSGYPFQ CLGFTPEHQR DFIARDLGPT LANSTHHNVR LLMLDDQRLL LPHWAKVVLT DPEAAKYVHG IAVHWYLDFL APA KATLGE THRLFPNTML FASEACVGSK FWEQSVRLGS WDRGMQYSHS IITNLLYHVV GWTDWNLALN PEGGPNWVRN FVDS PIIVD ITKDTFYKQP MFYHLGHFSK FIPEGSQRVG LVASQKNDLD AVALMHPDGS AVVVVLNRSS KDVPLTIKDP AVGFL ETIS PGYSIHTYLW RRQ

UniProtKB: Lysosomal acid glucosylceramidase

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Macromolecule #3: Nanobody Nb1

MacromoleculeName: Nanobody Nb1 / type: protein_or_peptide / ID: 3
Details: Pro-Macrobody; nanobody fused to maltose-binding protein (MBP) via Pro-Pro linker; MBP not resolved and not modeled.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.409816 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString:
QVQLVESGGG LVQPGGSLRL SCAASGFTLD YYAIGWFRQA PGKEREGVSC ISSSDGSTYY ADSAKGRFTI SRDNAKNTVY LQMNSLKPE DTAVYYCATD RGQCTYYSSG YYRDLRWYDY WGQGTQVTVP P

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Macromolecule #4: Nanobody Nb6

MacromoleculeName: Nanobody Nb6 / type: protein_or_peptide / ID: 4
Details: Pro-Macrobody; nanobody fused to maltose-binding protein (MBP) via Pro-Pro linker; MBP not resolved and not modeled.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.145612 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString:
QVQLVESGGG LVQPGGSLRL SCAASGSIFS INTMGWYRQA PGKEREMVAY IITFGSTNYA DSVKGRFTIS GDNANNTMWL QMNSLKPED TAVYYCYAAI RPTDSSTYTS YWGQGTQVTV PP

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Macromolecule #8: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 8 / Number of copies: 7 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

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

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Macromolecule #9: 2-(N-MORPHOLINO)-ETHANESULFONIC ACID

MacromoleculeName: 2-(N-MORPHOLINO)-ETHANESULFONIC ACID / type: ligand / ID: 9 / Number of copies: 4 / Formula: MES
Molecular weightTheoretical: 195.237 Da
Chemical component information

ChemComp-MES:
2-(N-MORPHOLINO)-ETHANESULFONIC ACID / pH buffer*YM

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Macromolecule #10: water

MacromoleculeName: water / type: ligand / ID: 10 / Number of copies: 27 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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

Concentration0.6 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC6H13NO4SMES; 2-(N-morpholino)ethanesulfonic acid
150.0 mMNaClsodium chloride

Details: 20 mM MES; 150 mM NaCl, pH: 7.4
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Details: Protochips; CF-1.2/1.3-3Cu-50
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsMonodisperse, complex seperated from monomers via size exclusion chhromatography.

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Electron microscopy

MicroscopeTFS GLACIOS
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 10550 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 130000
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: PHENIX (ver. 1.20.1_4487) / Number images used: 397385
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6tn1


Chain - Chain ID: AAA / Chain - Residue range: 1-497 / Chain - Source name: PDB / Chain - Initial model type: experimental model / Details: GCase
DetailsInitial fitting with ChimeraX, manual flexible fitting with Coot, refinement with Phenix
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9fjf:
Lysosomal transporting complex of beta-glucocerebrosidase (GCase) and lysosomal integral membrane protein 2 (LIMP-2) with bound Pro-macrobodies (Combined focus map)

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Atomic model buiding 2

Initial modelPDB ID:

4q4f


Chain - Chain ID: A / Chain - Residue range: 37-430 / Chain - Source name: PDB / Chain - Initial model type: experimental model / Details: LIMP-2
DetailsInitial fitting with ChimeraX, manual flexible fitting with Coot, refinement with Phenix
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9fjf:
Lysosomal transporting complex of beta-glucocerebrosidase (GCase) and lysosomal integral membrane protein 2 (LIMP-2) with bound Pro-macrobodies (Combined focus map)

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Atomic model buiding 3

Initial modelPDB ID:

9ena


Chain - Source name: PDB / Chain - Initial model type: experimental model / Details: Nanobody Nb1; Unpublished
DetailsInitial fitting with ChimeraX, manual flexible fitting with Coot, refinement with Phenix
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9fjf:
Lysosomal transporting complex of beta-glucocerebrosidase (GCase) and lysosomal integral membrane protein 2 (LIMP-2) with bound Pro-macrobodies (Combined focus map)

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