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- PDB-8ax3: Structure of recombinant human beta-glucocerebrosidase in complex... -

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Basic information

Entry
Database: PDB / ID: 8ax3
TitleStructure of recombinant human beta-glucocerebrosidase in complex with L-carbaxylosyl fluoride
ComponentsLysosomal acid glucosylceramidase
KeywordsHYDROLASE / beta-glucocerebrosidase / glycoside hydrolase / inhibitor / carbasugar / pharmacological chaperone
Function / homology
Function and homology information


positive regulation of protein lipidation / steryl-beta-glucosidase activity / beta-glucoside catabolic process / positive regulation of neuronal action potential / cerebellar Purkinje cell layer formation / galactosylceramidase / termination of signal transduction / galactosylceramidase activity / positive regulation of autophagy of mitochondrion in response to mitochondrial depolarization / lymphocyte migration ...positive regulation of protein lipidation / steryl-beta-glucosidase activity / beta-glucoside catabolic process / positive regulation of neuronal action potential / cerebellar Purkinje cell layer formation / galactosylceramidase / termination of signal transduction / galactosylceramidase activity / positive regulation of autophagy of mitochondrion in response to mitochondrial depolarization / lymphocyte migration / glucosylceramidase / glucosylceramide catabolic process / scavenger receptor binding / regulation of lysosomal protein catabolic process / sphingosine biosynthetic process / autophagosome organization / glucosylceramidase activity / microglial cell proliferation / regulation of TOR signaling / glucosyltransferase activity / ceramide biosynthetic process / lipid storage / response to thyroid hormone / microglia differentiation / Glycosphingolipid catabolism / pyramidal neuron differentiation / lipid glycosylation / brain morphogenesis / Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds / response to pH / positive regulation of protein-containing complex disassembly / motor behavior / neuromuscular process / Transferases; Glycosyltransferases; Hexosyltransferases / hematopoietic stem cell proliferation / lysosome organization / response to testosterone / response to dexamethasone / Association of TriC/CCT with target proteins during biosynthesis / negative regulation of interleukin-6 production / homeostasis of number of cells / antigen processing and presentation / regulation of macroautophagy / establishment of skin barrier / negative regulation of protein-containing complex assembly / positive regulation of protein dephosphorylation / cell maturation / respiratory electron transport chain / cellular response to starvation / cholesterol metabolic process / lysosomal lumen / negative regulation of MAP kinase activity / determination of adult lifespan / trans-Golgi network / autophagy / negative regulation of inflammatory response / response to estrogen / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / T cell differentiation in thymus / cellular response to tumor necrosis factor / proteasome-mediated ubiquitin-dependent protein catabolic process / neuron apoptotic process / negative regulation of neuron apoptotic process / lysosome / lysosomal membrane / signaling receptor binding / Golgi apparatus / endoplasmic reticulum / 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: 1.59 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Lysosomal acid glucosylceramidase
B: Lysosomal acid glucosylceramidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)117,60067
Polymers111,3182
Non-polymers6,28165
Water16,087893
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area15440 Å2
ΔGint46 kcal/mol
Surface area34860 Å2
Unit cell
Length a, b, c (Å)52.920, 155.984, 68.036
Angle α, β, γ (deg.)90.000, 101.964, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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Protein , 1 types, 2 molecules AB

#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.: 2
Source method: isolated from a genetically manipulated source
Details: Human beta-glucocerebrosidase with the N-terminal signalling sequence missing
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, steryl-beta-glucosidase

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Sugars , 3 types, 5 molecules

#2: 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.: 2
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
#3: 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.: 1
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
#6: 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 / N-Acetylglucosamine


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 , 7 types, 953 molecules

#4: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: SO4
#5: Chemical...
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 47 / Source method: obtained synthetically / Formula: C2H6O2
#7: Chemical
ChemComp-OD0 / (1~{S},2~{R},3~{S},6~{S})-6-fluoranylcyclohex-4-ene-1,2,3-triol


Mass: 148.132 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C6H9FO3 / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#9: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#10: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#11: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 893 / 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: 2.47 Å3/Da / Density % sol: 50.16 %
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: I03 / Wavelength: 0.97623 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Jun 23, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97623 Å / Relative weight: 1
ReflectionResolution: 1.59→51.99 Å / Num. obs: 994940 / % possible obs: 99.6 % / Redundancy: 6.9 % / CC1/2: 0.997 / Rmerge(I) obs: 0.135 / Net I/σ(I): 8.6
Reflection shellResolution: 1.59→1.62 Å / Rmerge(I) obs: 1.8 / Mean I/σ(I) obs: 1.1 / Num. unique obs: 49937 / CC1/2: 0.544

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Processing

Software
NameVersionClassification
REFMAC5.8.0267refinement
xia2data reduction
Aimlessdata scaling
REFMACphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 8AWK

8awk


Resolution: 1.59→51.824 Å / Cor.coef. Fo:Fc: 0.964 / Cor.coef. Fo:Fc free: 0.95 / SU B: 2.59 / SU ML: 0.084 / Cross valid method: FREE R-VALUE / ESU R: 0.09 / ESU R Free: 0.09
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.2125 7124 4.965 %
Rwork0.1819 136365 -
all0.183 --
obs-143489 99.434 %
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: 20.388 Å2
Baniso -1Baniso -2Baniso -3
1-1.471 Å20 Å20.406 Å2
2---0.003 Å2-0 Å2
3----1.504 Å2
Refinement stepCycle: LAST / Resolution: 1.59→51.824 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7845 0 400 893 9138
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0070.0138575
X-RAY DIFFRACTIONr_bond_other_d0.0010.0157956
X-RAY DIFFRACTIONr_angle_refined_deg1.4171.67511613
X-RAY DIFFRACTIONr_angle_other_deg1.3021.5918332
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.45351066
X-RAY DIFFRACTIONr_dihedral_angle_other_1_deg40.0932013
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.28421.607417
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.14215.1071309
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.0041548
X-RAY DIFFRACTIONr_chiral_restr0.0710.21099
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.029464
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021981
X-RAY DIFFRACTIONr_nbd_refined0.2040.21764
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1810.27635
X-RAY DIFFRACTIONr_nbtor_refined0.1690.23984
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0870.23747
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1750.2741
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_other0.0740.23
X-RAY DIFFRACTIONr_metal_ion_refined0.2050.21
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.1670.223
X-RAY DIFFRACTIONr_nbd_other0.2320.2112
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.1950.232
X-RAY DIFFRACTIONr_mcbond_it1.1381.9434034
X-RAY DIFFRACTIONr_mcbond_other1.1371.9434033
X-RAY DIFFRACTIONr_mcangle_it1.6932.9145060
X-RAY DIFFRACTIONr_mcangle_other1.6932.9155061
X-RAY DIFFRACTIONr_scbond_it1.5572.224541
X-RAY DIFFRACTIONr_scbond_other1.5572.224542
X-RAY DIFFRACTIONr_scangle_it2.3253.2256553
X-RAY DIFFRACTIONr_scangle_other2.3253.2266554
X-RAY DIFFRACTIONr_lrange_it4.28124.2739765
X-RAY DIFFRACTIONr_lrange_other4.13523.7689555
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.59-1.6310.3255020.3110031X-RAY DIFFRACTION98.8828
1.631-1.6760.2895340.2869772X-RAY DIFFRACTION99.0295
1.676-1.7250.2855070.2739491X-RAY DIFFRACTION99.3047
1.725-1.7780.2565020.2549240X-RAY DIFFRACTION99.2461
1.778-1.8360.2494570.2279032X-RAY DIFFRACTION99.4029
1.836-1.90.2434670.218657X-RAY DIFFRACTION99.4984
1.9-1.9720.2644440.2398383X-RAY DIFFRACTION99.3696
1.972-2.0530.2223870.1798119X-RAY DIFFRACTION99.6369
2.053-2.1440.234070.1887784X-RAY DIFFRACTION99.7079
2.144-2.2480.2273770.1877368X-RAY DIFFRACTION99.5118
2.248-2.370.2083960.1687019X-RAY DIFFRACTION99.4768
2.37-2.5140.2013400.1516729X-RAY DIFFRACTION99.8446
2.514-2.6870.1893280.1496265X-RAY DIFFRACTION99.773
2.687-2.9020.2033130.1545848X-RAY DIFFRACTION99.9351
2.902-3.1790.1922700.1615405X-RAY DIFFRACTION99.8417
3.179-3.5540.1962830.1664877X-RAY DIFFRACTION99.7487
3.554-4.1040.1552440.1454281X-RAY DIFFRACTION99.5381
4.104-5.0260.1611710.1373627X-RAY DIFFRACTION99.4762
5.026-7.1030.2321300.1772859X-RAY DIFFRACTION99.7664
7.103-51.8240.221650.2011580X-RAY DIFFRACTION99.1561

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