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- EMDB-50938: Lysosomal transporting complex of beta-glucocerebrosidase (GCase)... -

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Entry
Database: EMDB / ID: EMD-50938
TitleLysosomal transporting complex of beta-glucocerebrosidase (GCase) and lysosomal integral membrane protein 2 (LIMP-2) with bound Pro-macrobodies (LIMP-2 local refinement)
Map dataFocused refinement of LIMP-2 in complex with GCase and two bound Pro-macrobodies. Sharpened map (b-factor:-130.5) obtained from local refinement.
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: Lysosomal membrane protein 2
    • Protein or peptide: Nanobody Nb1
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 / regulation of TOR signaling / glucosyltransferase activity / endosome to plasma membrane protein transport / regulation of lysosome organization / response to thyroid hormone / protein targeting to lysosome / ceramide biosynthetic process / microglia differentiation / Glycosphingolipid catabolism / lipid storage / positive regulation of type 2 mitophagy / phosphatidylcholine binding / 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 / scavenger receptor activity / negative regulation of protein metabolic process / lysosome organization / motor behavior / Transferases; Glycosyltransferases; Hexosyltransferases / neuromuscular process / cholesterol binding / phosphatidylserine binding / hematopoietic stem cell proliferation / antigen processing and presentation / response to dexamethasone / Association of TriC/CCT with target proteins during biosynthesis / response to testosterone / negative regulation of interleukin-6 production / homeostasis of number of cells / regulation of macroautophagy / establishment of skin barrier / negative regulation of protein-containing complex assembly / negative regulation of MAPK cascade / mitophagy / cell maturation / 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 / negative regulation of inflammatory response / autophagy / response to estrogen / transmembrane signaling receptor activity / endocytic vesicle membrane / late endosome membrane / Cargo recognition for clathrin-mediated endocytosis / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / 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 / lysosomal membrane / signaling receptor binding / focal adhesion / endoplasmic reticulum membrane / enzyme binding / Golgi apparatus / endoplasmic reticulum / 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) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 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
DepositionJul 8, 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_50938.png

Downloads & links

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Map

FileDownload / File: emd_50938.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFocused refinement of LIMP-2 in complex with GCase and two bound Pro-macrobodies. Sharpened map (b-factor:-130.5) obtained from local refinement.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (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: 0.35
Minimum - Maximum-2.6579278 - 3.9039097
Average (Standard dev.)-0.0001216178 (±0.044398133)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Half map: Half map B

Fileemd_50938_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_50938_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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: Lysosomal membrane protein 2
    • Protein or peptide: Nanobody Nb1

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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: all
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: Lysosomal acid glucosylceramidase

MacromoleculeName: Lysosomal acid glucosylceramidase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: glucosylceramidase
Source (natural)Organism: Homo sapiens (human)
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 #2: Nanobody Nb6

MacromoleculeName: Nanobody Nb6 / type: protein_or_peptide / ID: 2
Details: Pro-Macrobody; nanobody fused to maltose-binding protein (MBP) via Pro-Pro linker; MBP not resolved and not modeled.
Enantiomer: LEVO
Source (natural)Organism: unidentified (others)
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 #3: Lysosomal membrane protein 2

MacromoleculeName: Lysosomal membrane protein 2 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
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 DERFVSYLDF LAP NQEDHE TFVDINPLTG IILKAAKRFQ INIYVKKLDD FVETGDIRTM VFPVMYLNES VHIDKETASR LKSMI

UniProtKB: Lysosome membrane protein 2

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Macromolecule #4: Nanobody Nb1

MacromoleculeName: Nanobody Nb1 / 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.
Enantiomer: LEVO
Source (natural)Organism: unidentified (others)
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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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
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 reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2) / 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

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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: RIGID BODY FIT

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Atomic model buiding 3

Initial modelPDB ID:

9ena
PDB Unreleased entry


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

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