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- PDB-8cb6: Crystal structure of human lysosomal acid-alpha-glucosidase, GAA,... -

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Basic information

Entry
Database: PDB / ID: 8cb6
TitleCrystal structure of human lysosomal acid-alpha-glucosidase, GAA, in covalent complex with TAMRA tagged 1,6-Epi-cylcophellitol aziridine activity based probe
Components(Lysosomal alpha-glucosidase ...) x 2
KeywordsHYDROLASE / Gaa / Pompe disease / activity based probe
Function / homology
Function and homology information


vacuolar sequestering / autolysosome lumen / maltose metabolic process / alpha-glucosidase activity / alpha-1,4-glucosidase activity / sucrose metabolic process / Glycogen storage disease type II (GAA) / : / alpha-glucosidase / diaphragm contraction ...vacuolar sequestering / autolysosome lumen / maltose metabolic process / alpha-glucosidase activity / alpha-1,4-glucosidase activity / sucrose metabolic process / Glycogen storage disease type II (GAA) / : / alpha-glucosidase / diaphragm contraction / neuromuscular process controlling posture / glycophagy / tissue development / regulation of the force of heart contraction / glycogen catabolic process / aorta development / azurophil granule membrane / Glycogen breakdown (glycogenolysis) / muscle cell cellular homeostasis / lysosome organization / tertiary granule membrane / neuromuscular process controlling balance / ficolin-1-rich granule membrane / heart morphogenesis / cardiac muscle contraction / lysosomal lumen / locomotory behavior / glucose metabolic process / carbohydrate binding / lysosome / lysosomal membrane / intracellular membrane-bounded organelle / Neutrophil degranulation / extracellular exosome / membrane / plasma membrane
Similarity search - Function
P-type trefoil, conserved site / P-type 'Trefoil' domain signature. / Trefoil (P-type) domain / P-type trefoil domain / P-type trefoil domain superfamily / P-type 'Trefoil' domain profile. / P or trefoil or TFF domain / Glycosyl hydrolases family 31, conserved site / Glycosyl hydrolases family 31 signature 2. / Glycosyl hydrolases family 31, active site ...P-type trefoil, conserved site / P-type 'Trefoil' domain signature. / Trefoil (P-type) domain / P-type trefoil domain / P-type trefoil domain superfamily / P-type 'Trefoil' domain profile. / P or trefoil or TFF domain / Glycosyl hydrolases family 31, conserved site / Glycosyl hydrolases family 31 signature 2. / Glycosyl hydrolases family 31, active site / Glycosyl hydrolases family 31 active site. / Glycoside hydrolase family 31, N-terminal domain / Glycosyl hydrolase 31 N-terminal galactose mutarotase-like domain / : / Glycosyl hydrolase family 31 C-terminal domain / Glycoside hydrolase family 31 / Glycosyl hydrolases family 31 TIM-barrel domain / Galactose mutarotase-like domain superfamily / Glycosyl hydrolase, all-beta / Glycoside hydrolase superfamily
Similarity search - Domain/homology
TRIETHYLENE GLYCOL / Chem-U54 / Lysosomal alpha-glucosidase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 1.9 Å
AuthorsSulzenbacher, G. / Roig-Zamboni, V. / Overkleeft, H. / Artola, M.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Chem Sci / Year: 2023
Title: Fluorescence polarisation activity-based protein profiling for the identification of deoxynojirimycin-type inhibitors selective for lysosomal retaining alpha- and beta-glucosidases.
Authors: van der Gracht, D. / Rowland, R.J. / Roig-Zamboni, V. / Ferraz, M.J. / Louwerse, M. / Geurink, P.P. / Aerts, J.M.F.G. / Sulzenbacher, G. / Davies, G.J. / Overkleeft, H.S. / Artola, M.
History
DepositionJan 25, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 13, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
F: Lysosomal alpha-glucosidase (76 kDa)
A: Lysosomal alpha-glucosidase (70 kDa)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,52937
Polymers96,9972
Non-polymers4,53335
Water11,025612
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12240 Å2
ΔGint-91 kcal/mol
Surface area31600 Å2
MethodPISA
Unit cell
Length a, b, c (Å)97.340, 102.945, 129.916
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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Lysosomal alpha-glucosidase ... , 2 types, 2 molecules FA

#1: Protein Lysosomal alpha-glucosidase (76 kDa)


Mass: 14095.020 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: FULL-LENGTH RHGAA HAS BEEN TREATED WITH CHYMOTRYPSIN, LEADING TO A SAMPLE STARTING AT RESIDUE GLN81. MISSING SURFACE LOOPS HAVE EQUALLY BEEN REMOVED BY PROTEOLYTIC CLEAVAGE.
Source: (gene. exp.) Homo sapiens (human) / Gene: GAA / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P10253
#2: Protein Lysosomal alpha-glucosidase (70 kDa)


Mass: 82901.570 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: FULL-LENGTH RHGAA HAS BEEN TREATED WITH CHYMOTRYPSIN, LEADING TO A SAMPLE STARTING AT RESIDUE GLN81. MISSING SURFACE LOOPS HAVE EQUALLY BEEN REMOVED BY PROTEOLYTIC CLEAVAGE.
Source: (gene. exp.) Homo sapiens (human) / Gene: GAA / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P10253

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Sugars , 4 types, 5 molecules

#3: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta- ...2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 570.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1221m-1a_1-5]/1-1-2/a4-b1_a6-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}[(6+1)][a-L-Fucp]{}}LINUCSPDB-CARE
#4: 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.: 2
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
#5: 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.: 1
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
#10: 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.: 1 / 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

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Non-polymers , 7 types, 642 molecules

#6: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Cl
#7: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C2H6O2
#8: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C3H8O3
#9: Chemical ChemComp-U54 / (1S,2R,3R,4R,5R)-5-[8-[4-(4-azanylbutyl)-1,2,3-triazol-1-yl]octylamino]-4-(hydroxymethyl)cyclohexane-1,2,3-triol


Mass: 427.581 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H41N5O4 / Feature type: SUBJECT OF INVESTIGATION
#11: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO4
#12: Chemical ChemComp-PGE / TRIETHYLENE GLYCOL


Mass: 150.173 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14O4
#13: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 612 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.35 Å3/Da / Density % sol: 63 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7
Details: 1.9 M AMMONIUM SULPHATE, 0.1 M HEPES, 2% V/V PEG400

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 2 / Wavelength: 0.980112 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Sep 15, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.980112 Å / Relative weight: 1
ReflectionResolution: 1.9→48.67 Å / Num. obs: 103337 / % possible obs: 100 % / Redundancy: 13.7 % / CC1/2: 0.999 / Rmerge(I) obs: 0.13 / Rpim(I) all: 0.036 / Rrim(I) all: 0.135 / Χ2: 0.99 / Net I/σ(I): 14.5 / Num. measured all: 1418173
Reflection shellResolution: 1.9→1.93 Å / % possible obs: 100 % / Redundancy: 13.9 % / Rmerge(I) obs: 1.675 / Num. measured all: 70368 / Num. unique obs: 5053 / CC1/2: 0.695 / Rpim(I) all: 0.465 / Rrim(I) all: 1.739 / Χ2: 1.08 / Net I/σ(I) obs: 1.9

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Processing

Software
NameVersionClassification
REFMAC7.1.015refinement
Aimless0.7.7data scaling
XDSdata reduction
REFMACphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: 5NN3

5nn3


Resolution: 1.9→48.67 Å / Cor.coef. Fo:Fc: 0.973 / Cor.coef. Fo:Fc free: 0.963 / SU B: 2.413 / SU ML: 0.069 / Cross valid method: FREE R-VALUE / ESU R: 0.097 / ESU R Free: 0.096 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.17786 5146 5 %RANDOM
Rwork0.14871 ---
obs0.15015 98106 99.95 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 41.148 Å2
Baniso -1Baniso -2Baniso -3
1-0.78 Å2-0 Å20 Å2
2--0.02 Å2-0 Å2
3----0.8 Å2
Refinement stepCycle: 1 / Resolution: 1.9→48.67 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6691 0 281 612 7584
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0137219
X-RAY DIFFRACTIONr_bond_other_d0.0010.0176587
X-RAY DIFFRACTIONr_angle_refined_deg1.5721.6849868
X-RAY DIFFRACTIONr_angle_other_deg1.3441.60115220
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.045866
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.02322.151358
X-RAY DIFFRACTIONr_dihedral_angle_3_deg11.977151050
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.5061541
X-RAY DIFFRACTIONr_chiral_restr0.0820.2943
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.028050
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021662
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it2.6714.0123423
X-RAY DIFFRACTIONr_mcbond_other2.6664.0123422
X-RAY DIFFRACTIONr_mcangle_it3.3215.9944278
X-RAY DIFFRACTIONr_mcangle_other3.3215.9954279
X-RAY DIFFRACTIONr_scbond_it4.3274.5393796
X-RAY DIFFRACTIONr_scbond_other4.3264.5393797
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other6.2636.6355582
X-RAY DIFFRACTIONr_long_range_B_refined6.93748.1027802
X-RAY DIFFRACTIONr_long_range_B_other6.84947.5977679
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.9→1.949 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.263 361 -
Rwork0.244 7212 -
obs--99.85 %

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