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- PDB-6m4f: Crystal structure of the E496A mutant of HsBglA -

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Basic information

Entry
Database: PDB / ID: 6m4f
TitleCrystal structure of the E496A mutant of HsBglA
ComponentsBeta-galactosidase-like enzyme
KeywordsHYDROLASE / galactooligosaccharide / transgalactosylation / beta-galactosidase / beta-glucosidase / Hamamotoa singularis / STRUCTURAL PROTEIN
Function / homologyGlycosyl hydrolase family 1 / Glycoside hydrolase family 1 / beta-glucosidase activity / Glycoside hydrolase superfamily / carbohydrate metabolic process / metal ion binding / alpha-D-mannopyranose / Beta-galactosidase-like enzyme
Function and homology information
Biological speciesHamamotoa singularis (fungus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.2 Å
AuthorsUehara, R. / Iwamoto, R. / Aoki, S. / Yoshizawa, T. / Takano, K. / Matsumura, H. / Tanaka, S.-i.
Funding support Japan, 2items
OrganizationGrant numberCountry
Ministry of Education, Culture, Sports, Science and Technology (Japan)18K05445 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)19H05401 Japan
CitationJournal: Protein Sci. / Year: 2020
Title: Crystal structure of a GH1 beta-glucosidase from Hamamotoa singularis.
Authors: Uehara, R. / Iwamoto, R. / Aoki, S. / Yoshizawa, T. / Takano, K. / Matsumura, H. / Tanaka, S.I.
History
DepositionMar 6, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 2, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 16, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Nov 29, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Beta-galactosidase-like enzyme
C: Beta-galactosidase-like enzyme
E: Beta-galactosidase-like enzyme
G: Beta-galactosidase-like enzyme
hetero molecules


Theoretical massNumber of molelcules
Total (without water)263,21825
Polymers257,1534
Non-polymers6,06621
Water19,0961060
1
A: Beta-galactosidase-like enzyme
C: Beta-galactosidase-like enzyme
hetero molecules


Theoretical massNumber of molelcules
Total (without water)131,90213
Polymers128,5762
Non-polymers3,32611
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7100 Å2
ΔGint30 kcal/mol
Surface area38230 Å2
MethodPISA
2
E: Beta-galactosidase-like enzyme
G: Beta-galactosidase-like enzyme
hetero molecules


Theoretical massNumber of molelcules
Total (without water)131,31612
Polymers128,5762
Non-polymers2,74010
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6460 Å2
ΔGint26 kcal/mol
Surface area38130 Å2
MethodPISA
Unit cell
Length a, b, c (Å)192.751, 67.617, 200.867
Angle α, β, γ (deg.)90.000, 104.070, 90.000
Int Tables number5
Space group name H-MC121
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z

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Components

#1: Protein
Beta-galactosidase-like enzyme


Mass: 64288.199 Da / Num. of mol.: 4 / Mutation: E496A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hamamotoa singularis (fungus) / Gene: bglA / Plasmid: pPICZalphaA / Production host: Komagataella pastoris (fungus) / Strain (production host): X-33 / References: UniProt: Q564N5
#2: 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.: 8 / Source method: isolated from a natural 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
#3: 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.: 8 / Source method: isolated from a natural 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
#4: Sugar
ChemComp-MAN / alpha-D-mannopyranose / alpha-D-mannose / D-mannose / mannose


Type: D-saccharide, alpha linking / Mass: 180.156 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Formula: C6H12O6
IdentifierTypeProgram
DManpaCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
a-D-mannopyranoseCOMMON NAMEGMML 1.0
a-D-ManpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
ManSNFG CARBOHYDRATE SYMBOLGMML 1.0
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1060 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 50.21 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7 / Details: 0.1 M SPG Buffer pH 7.0, 27% (w/v) PEG 1500

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL44XU / Wavelength: 0.9 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Oct 15, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9 Å / Relative weight: 1
ReflectionResolution: 2.2→48.71 Å / Num. obs: 127314 / % possible obs: 99.37 % / Redundancy: 3.5 % / CC1/2: 0.999 / Rmerge(I) obs: 0.059 / Net I/σ(I): 14.54
Reflection shellResolution: 2.2→2.28 Å / Redundancy: 3.6 % / Rmerge(I) obs: 0.641 / Mean I/σ(I) obs: 2 / Num. unique obs: 12641 / CC1/2: 0.685 / % possible all: 99.58

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
PDB_EXTRACT3.25data extraction
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4GXP

4gxp


Resolution: 2.2→48.71 Å / SU ML: 0.23 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 19.73 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1862 6359 5 %
Rwork0.186 120787 -
obs0.186 127145 99.38 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 93.63 Å2 / Biso mean: 41.937 Å2 / Biso min: 22.41 Å2
Refinement stepCycle: final / Resolution: 2.2→48.71 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17166 0 391 1060 18617
Biso mean--64.01 43.38 -
Num. residues----2171
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.2-2.230.31882130.306540424255100
2.23-2.250.32672100.286639924202100
2.25-2.280.2772090.274939714180100
2.28-2.310.23852130.267540354248100
2.31-2.340.24952110.268340054216100
2.34-2.370.2432120.260640354247100
2.37-2.40.21292110.238740054216100
2.4-2.440.22092100.237939844194100
2.44-2.480.23732120.231440324244100
2.48-2.520.20782090.232439844193100
2.52-2.560.20912130.216840294242100
2.56-2.610.19242130.202740544267100
2.61-2.660.21472070.204839324139100
2.66-2.710.23052120.21654025423799
2.71-2.770.21462100.216139944204100
2.77-2.840.20892140.205740644278100
2.84-2.910.2142110.205440164227100
2.91-2.990.22222100.21533996420699
2.99-3.070.1952130.204640474260100
3.07-3.170.21682110.194240094220100
3.17-3.290.18962120.185640134225100
3.29-3.420.17262130.1824060427399
3.42-3.570.18732120.18394010422299
3.57-3.760.18512120.17824034424699
3.76-40.16342120.15764030424299
4-4.310.13922100.14393996420699
4.31-4.740.14222130.14234044425799
4.74-5.420.15052140.15034073428799
5.42-6.830.1582160.16694098431499
6.83-48.710.17632210.1724178439998

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