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- PDB-1m04: Mutant Streptomyces plicatus beta-hexosaminidase (D313N) in compl... -

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Basic information

Entry
Database: PDB / ID: 1m04
TitleMutant Streptomyces plicatus beta-hexosaminidase (D313N) in complex with product (GlcNAc)
ComponentsBeta-N-acetylhexosaminidaseHexosaminidase
KeywordsHYDROLASE / substrate assisted catalysis / hexosaminidase / family 20 glycosidase
Function / homology
Function and homology information


beta-N-acetylhexosaminidase / beta-N-acetylhexosaminidase activity / N-acetyl-beta-D-galactosaminidase activity / carbohydrate metabolic process
Similarity search - Function
Beta-hexosaminidase / Glycoside hydrolase family 20, catalytic domain / Glycosyl hydrolase family 20, catalytic domain / Chitobiase/beta-hexosaminidase domain 2-like / Chitobiase; domain 2 / Beta-hexosaminidase, bacterial type, N-terminal / Glycosyl hydrolase family 20, domain 2 / Beta-hexosaminidase-like, domain 2 / Glycosidases / Glycoside hydrolase superfamily ...Beta-hexosaminidase / Glycoside hydrolase family 20, catalytic domain / Glycosyl hydrolase family 20, catalytic domain / Chitobiase/beta-hexosaminidase domain 2-like / Chitobiase; domain 2 / Beta-hexosaminidase, bacterial type, N-terminal / Glycosyl hydrolase family 20, domain 2 / Beta-hexosaminidase-like, domain 2 / Glycosidases / Glycoside hydrolase superfamily / TIM Barrel / Alpha-Beta Barrel / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
beta-N-acetylhexosaminidase
Similarity search - Component
Biological speciesStreptomyces plicatus (bacteria)
MethodX-RAY DIFFRACTION / FOURIER SYNTHESIS / Resolution: 1.95 Å
AuthorsWilliams, S.J. / Mark, B.L. / Vocadlo, D.J. / James, M.N.G. / Withers, S.G.
CitationJournal: J.Biol.Chem. / Year: 2002
Title: Aspartate 313 in the Streptomyces plicatus hexosaminidase plays a critical role in substrate-assisted catalysis by orienting the 2-acetamido group and stabilizing the transition state.
Authors: Williams, S.J. / Mark, B.L. / Vocadlo, D.J. / James, M.N. / Withers, S.G.
History
DepositionJun 11, 2002Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 11, 2002Provider: repository / Type: Initial release
Revision 1.1Apr 28, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Derived calculations / Version format compliance
Revision 1.3Jul 29, 2020Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_ref_seq_dif / struct_site / struct_site_gen
Item: _chem_comp.name / _chem_comp.type ..._chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_ref_seq_dif.details
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.4Oct 27, 2021Group: Database references / Structure summary / Category: chem_comp / database_2 / struct_ref_seq_dif
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Beta-N-acetylhexosaminidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)56,6427
Polymers56,1261
Non-polymers5166
Water5,170287
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
A: Beta-N-acetylhexosaminidase
hetero molecules

A: Beta-N-acetylhexosaminidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)113,28314
Polymers112,2522
Non-polymers1,03112
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation12_565x,x-y+1,-z+1/61
Buried area5150 Å2
ΔGint-88 kcal/mol
Surface area32800 Å2
MethodPISA, PQS
Unit cell
Length a, b, c (Å)133.704, 133.704, 176.142
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number178
Space group name H-MP6122
Components on special symmetry positions
IDModelComponents
11A-510-

CL

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Components

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Protein / Sugars , 2 types, 2 molecules A

#1: Protein Beta-N-acetylhexosaminidase / Hexosaminidase / beta-hexosaminidase / SpHEX


Mass: 56125.793 Da / Num. of mol.: 1 / Mutation: D319N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces plicatus (bacteria) / Plasmid: pET3A / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: O85361, beta-N-acetylhexosaminidase
#2: 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.: 1
Source method: isolated from a genetically manipulated source
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 , 4 types, 292 molecules

#3: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#5: 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
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 287 / Source method: isolated from a natural source / Formula: H2O

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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: 4.05 Å3/Da / Density % sol: 69.6 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 6
Details: AMMONIUM SULPHATE, TRI-SODIUM CITRATE, SODIUM CHLORIDE, GLYCEROL, NAG, pH 6.0, VAPOR DIFFUSION, HANGING DROP, temperature 298.0K
Crystal grow
*PLUS
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formulaDetails
150 mMtrisodium citrate1drop
2300 mM1dropNaCl
310 mg/mlprotein1drop
42.1 Mammonium sulfate1reservoir
5100 mMtrisodium citrate1reservoirpH6.0
620 %glycerol1reservoir

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU / Wavelength: 1.54 Å
DetectorType: RIGAKU RAXIS IV / Detector: IMAGE PLATE / Date: Aug 14, 2001 / Details: OSMIC optics
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 1.95→40 Å / Num. all: 65848 / Num. obs: 65848 / % possible obs: 96.9 % / Observed criterion σ(I): -3 / Redundancy: 19 % / Biso Wilson estimate: 15.7 Å2 / Rsym value: 0.056 / Net I/σ(I): 30.5
Reflection shellResolution: 1.95→2.02 Å / Redundancy: 19 % / Mean I/σ(I) obs: 5.5 / Rsym value: 0.256 / % possible all: 98.5
Reflection
*PLUS
Num. measured all: 1300022 / Rmerge(I) obs: 0.056
Reflection shell
*PLUS
% possible obs: 98.5 % / Num. unique obs: 6556 / Rmerge(I) obs: 0.256

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Processing

Software
NameClassification
DENZOdata reduction
SCALEPACKdata scaling
CNSrefinement
CNSphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: PDB ENTRY 1HP4

1hp4


Resolution: 1.95→39.19 Å / Rfactor Rfree error: 0.003 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.218 6659 10.1 %RANDOM
Rwork0.198 ---
obs0.198 65848 96.8 %-
all-65848 --
Solvent computationSolvent model: FLAT MODEL / Bsol: 43.3872 Å2 / ksol: 0.377555 e/Å3
Displacement parametersBiso mean: 24.6 Å2
Baniso -1Baniso -2Baniso -3
1--0.07 Å20.76 Å20 Å2
2---0.07 Å20 Å2
3---0.15 Å2
Refine analyzeLuzzati coordinate error free: 0.24 Å / Luzzati sigma a free: 0.16 Å
Refinement stepCycle: LAST / Resolution: 1.95→39.19 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3864 0 29 287 4180
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.005
X-RAY DIFFRACTIONc_bond_d_na
X-RAY DIFFRACTIONc_bond_d_prot
X-RAY DIFFRACTIONc_angle_d
X-RAY DIFFRACTIONc_angle_d_na
X-RAY DIFFRACTIONc_angle_d_prot
X-RAY DIFFRACTIONc_angle_deg1.3
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d22.9
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d0.83
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it1.141.5
X-RAY DIFFRACTIONc_mcangle_it1.682
X-RAY DIFFRACTIONc_scbond_it1.792
X-RAY DIFFRACTIONc_scangle_it2.642.5
LS refinement shellResolution: 1.95→2.02 Å / Rfactor Rfree error: 0.01 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.246 663 10.4 %
Rwork0.226 5735 -
obs--53.1 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2IFG_GOL.PARIFG_GOL.TOP
X-RAY DIFFRACTION3WATER_REP.PARAMWATER.TOP
X-RAY DIFFRACTION4ION.PARAMION.TOP
X-RAY DIFFRACTION5CARBOHYDRATE.PARAMCARBONAG.TOP
Refinement
*PLUS
Lowest resolution: 39.2 Å / % reflection Rfree: 10 %
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg22.9
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.83

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