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- PDB-8awk: Structure of recombinant human beta-glucocerebrosidase in complex... -

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Basic information

Entry
Database: PDB / ID: 8awk
TitleStructure of recombinant human beta-glucocerebrosidase in complex with D-carbaxylosyl chloride
ComponentsLysosomal acid glucosylceramidase
KeywordsHYDROLASE / beta-glucocerebrosidase / glycoside hydrolase / inhibitor / carbasugar / pharmacological chaperone
Function / homology
Function and homology information


steryl-beta-glucosidase activity / positive regulation of neuronal action potential / beta-glucoside catabolic process / cerebellar Purkinje cell layer formation / termination of signal transduction / galactosylceramidase / galactosylceramidase activity / glucosylceramidase / scavenger receptor binding / lymphocyte migration ...steryl-beta-glucosidase activity / positive regulation of neuronal action potential / beta-glucoside catabolic process / cerebellar Purkinje cell layer formation / termination of signal transduction / galactosylceramidase / galactosylceramidase activity / glucosylceramidase / scavenger receptor binding / lymphocyte migration / glucosylceramide catabolic process / regulation of lysosomal protein catabolic process / response to thyroid hormone / glucosylceramidase activity / sphingosine biosynthetic process / autophagosome organization / lysosomal protein catabolic process / microglial cell proliferation / glucosyltransferase activity / regulation of TOR signaling / Glycosphingolipid catabolism / microglia differentiation / lipid storage / ceramide biosynthetic process / positive regulation of type 2 mitophagy / : / brain morphogenesis / Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds / response to pH / pyramidal neuron differentiation / negative regulation of protein metabolic process / Transferases; Glycosyltransferases; Hexosyltransferases / lysosome organization / neuromuscular process / response to dexamethasone / hematopoietic stem cell proliferation / antigen processing and presentation / response to testosterone / Association of TriC/CCT with target proteins during biosynthesis / motor behavior / 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 / cholesterol metabolic process / mitophagy / cell maturation / negative regulation of MAPK cascade / lysosomal lumen / cellular response to starvation / determination of adult lifespan / respiratory electron transport chain / trans-Golgi network / autophagy / negative regulation of inflammatory response / response to estrogen / cellular response to tumor necrosis factor / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / T cell differentiation in thymus / neuron apoptotic process / proteasome-mediated ubiquitin-dependent protein catabolic process / negative regulation of neuron apoptotic process / lysosome / signaling receptor binding / lysosomal membrane / endoplasmic reticulum / Golgi apparatus / extracellular exosome
Similarity search - Function
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
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.58 Å
AuthorsRowland, R.J. / Davies, G.J.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/M011151/1 United Kingdom
CitationJournal: To be published
Title: Single turnover covalent inhibitors for functional chaperoning of lysosomal glycoside hydrolases
Authors: Bhosale, S. / Kandalkar, S. / Gilormini, P.A. / Akintola, O. / Rowland, R.J. / Adabala, P.J.P. / King, D. / Deen, M.C. / Xi, K. / Davies, G.J. / Vocadlo, D.J. / Bennet, A.J.
History
DepositionAug 30, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
AAA: Lysosomal acid glucosylceramidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)58,61130
Polymers55,6591
Non-polymers2,95229
Water9,854547
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6060 Å2
ΔGint26 kcal/mol
Surface area18550 Å2
MethodPISA
Unit cell
Length a, b, c (Å)53.277, 76.465, 68.469
Angle α, β, γ (deg.)90.000, 102.047, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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Protein , 1 types, 1 molecules AAA

#1: Protein Lysosomal acid glucosylceramidase / Lysosomal acid GCase / Acid beta-glucosidase / Alglucerase / Beta-glucocerebrosidase / Beta-GC / ...Lysosomal acid GCase / Acid beta-glucosidase / Alglucerase / Beta-glucocerebrosidase / Beta-GC / Cholesterol glucosyltransferase / SGTase / Cholesteryl-beta-glucosidase / D-glucosyl-N-acylsphingosine glucohydrolase / Imiglucerase


Mass: 55659.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Recombinant human beta-glucocerebrosidase missing the N-terminal signalling sequence
Source: (gene. exp.) Homo sapiens (human) / Gene: GBA, GC, GLUC / Cell line (production host): HiFive / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P04062, glucosylceramidase, Transferases; Glycosyltransferases; Hexosyltransferases, EC: 3.2.1.104

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Sugars , 2 types, 3 molecules

#2: Polysaccharide alpha-D-mannopyranose-(1-6)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1- ...alpha-D-mannopyranose-(1-6)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-6DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3/a4-b1_b4-c1_c6-d1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(6+1)][a-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.: 2 / 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 , 5 types, 573 molecules

#3: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: SO4
#4: Chemical...
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 21 / Source method: obtained synthetically / Formula: C2H6O2
#6: Chemical ChemComp-OIW / (2~{S},3~{S},4~{R})-cyclohex-5-ene-1,2,3,4-tetrol / D-carbaxylosyl chloride


Mass: 146.141 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H10O4 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 547 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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: 2.45 Å3/Da / Density % sol: 49.81 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7
Details: 0.2 M Na2SO4, 14% (v/v) PEG3350, 0.25 M HEPES pH 7.0

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.979499 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Dec 8, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979499 Å / Relative weight: 1
ReflectionResolution: 1.58→52.158 Å / Num. obs: 73542 / % possible obs: 100 % / Redundancy: 6.7 % / CC1/2: 0.998 / Rmerge(I) obs: 0.143 / Net I/σ(I): 8.8
Reflection shellResolution: 1.58→1.61 Å / Rmerge(I) obs: 1.569 / Mean I/σ(I) obs: 1.1 / Num. unique obs: 22723 / CC1/2: 0.563

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
xia2data reduction
Aimlessdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6TN1

6tn1


Resolution: 1.58→52.158 Å / Cor.coef. Fo:Fc: 0.977 / Cor.coef. Fo:Fc free: 0.965 / SU B: 1.998 / SU ML: 0.065 / Cross valid method: FREE R-VALUE / ESU R: 0.076 / ESU R Free: 0.078
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.1849 3644 4.957 %
Rwork0.1515 69863 -
all0.153 --
obs-73507 99.929 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 19.369 Å2
Baniso -1Baniso -2Baniso -3
1-0.979 Å20 Å20.81 Å2
2--0.23 Å20 Å2
3----1.425 Å2
Refinement stepCycle: LAST / Resolution: 1.58→52.158 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3926 0 187 547 4660
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.020.0134450
X-RAY DIFFRACTIONr_bond_other_d0.0010.0174051
X-RAY DIFFRACTIONr_angle_refined_deg1.5511.6796056
X-RAY DIFFRACTIONr_angle_other_deg1.3921.5949416
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.4225544
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.48221.674215
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.16315680
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.4141526
X-RAY DIFFRACTIONr_chiral_restr0.0780.2572
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.024962
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02965
X-RAY DIFFRACTIONr_nbd_refined0.2140.2841
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1820.23781
X-RAY DIFFRACTIONr_nbtor_refined0.1690.22029
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0920.21845
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1710.2427
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_other0.1560.22
X-RAY DIFFRACTIONr_metal_ion_refined0.110.25
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.1840.215
X-RAY DIFFRACTIONr_nbd_other0.1950.255
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.1830.238
X-RAY DIFFRACTIONr_mcbond_it1.1711.7712110
X-RAY DIFFRACTIONr_mcbond_other1.171.7712108
X-RAY DIFFRACTIONr_mcangle_it1.7172.6552676
X-RAY DIFFRACTIONr_mcangle_other1.7172.6552677
X-RAY DIFFRACTIONr_scbond_it1.9132.1362339
X-RAY DIFFRACTIONr_scbond_other1.9122.1362340
X-RAY DIFFRACTIONr_scangle_it2.8143.0873380
X-RAY DIFFRACTIONr_scangle_other2.8143.0873381
X-RAY DIFFRACTIONr_lrange_it5.17923.0455027
X-RAY DIFFRACTIONr_lrange_other5.17823.0445028
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.58-1.6210.2993010.2955092X-RAY DIFFRACTION99.778
1.621-1.6650.2652400.2655054X-RAY DIFFRACTION100
1.665-1.7140.2722450.2534857X-RAY DIFFRACTION99.9608
1.714-1.7660.2572720.2254742X-RAY DIFFRACTION99.9601
1.766-1.8240.2332610.2054559X-RAY DIFFRACTION99.9585
1.824-1.8880.2052300.1784423X-RAY DIFFRACTION100
1.888-1.9590.1992180.1744317X-RAY DIFFRACTION100
1.959-2.0390.1961990.1584140X-RAY DIFFRACTION100
2.039-2.130.2081790.1534013X-RAY DIFFRACTION100
2.13-2.2340.172070.1323772X-RAY DIFFRACTION100
2.234-2.3550.1691870.1233608X-RAY DIFFRACTION100
2.355-2.4970.1741940.1243405X-RAY DIFFRACTION99.9722
2.497-2.670.171830.1273203X-RAY DIFFRACTION100
2.67-2.8830.1711570.1222984X-RAY DIFFRACTION99.9364
2.883-3.1580.1551410.1282764X-RAY DIFFRACTION100
3.158-3.530.1771300.1372514X-RAY DIFFRACTION100
3.53-4.0750.1491030.1222227X-RAY DIFFRACTION100
4.075-4.9870.117770.1081884X-RAY DIFFRACTION100
4.987-7.0370.183800.1491465X-RAY DIFFRACTION100
7.037-52.1580.158400.15840X-RAY DIFFRACTION99.8865

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