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- PDB-8ihw: X-ray crystal structure of D43R mutant of endo-1,4-beta glucanase... -

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Basic information

Entry
Database: PDB / ID: 8ihw
TitleX-ray crystal structure of D43R mutant of endo-1,4-beta glucanase from Eisenia fetida
ComponentsEndoglucanase
KeywordsHYDROLASE / earthworm / cellulase / cold-adapted enzyme
Function / homology
Function and homology information


cellulase / cellulase activity / cellulose catabolic process / metal ion binding
Similarity search - Function
Glycosyl hydrolases family 9, Asp/Glu active sites / Glycosyl hydrolases family 9 (GH9) active site signature 3. / Glycoside hydrolase family 9 / Glycosyl hydrolase family 9 / Six-hairpin glycosidase-like superfamily / Six-hairpin glycosidase superfamily
Similarity search - Domain/homology
Biological speciesEisenia fetida (common brandling worm)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.7 Å
AuthorsKuroki, C. / Hirano, Y. / Nakazawa, M. / Sakamoto, T. / Tamada, T. / Ueda, M.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)19H03090 Japan
CitationJournal: Curr Res Biotechnol / Year: 2023
Title: A single mutation Asp43Arg was increased 2.5-fold the catalytic activity and maintained the stability of cold-adapted endo-1,4-beta glucanase (Ef-EG2) from Eisenia fetida.
Authors: Kuroki, C. / Hirano, Y. / Nakazawa, M. / Sakamoto, T. / Tamada, T. / Ueda, M.
History
DepositionFeb 24, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 6, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2024Group: Database references / Structure summary
Category: citation / pdbx_entry_details / pdbx_modification_feature
Item: _citation.country / _citation.journal_id_ISSN / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Endoglucanase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,9044
Polymers51,7471
Non-polymers1563
Water8,863492
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area90 Å2
ΔGint-13 kcal/mol
Surface area14930 Å2
MethodPISA
Unit cell
Length a, b, c (Å)53.292, 71.682, 55.359
Angle α, β, γ (deg.)90.000, 114.233, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein Endoglucanase


Mass: 51747.188 Da / Num. of mol.: 1 / Mutation: D43R
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Eisenia fetida (common brandling worm) / Gene: EF-EG2 / Production host: Komagataella pastoris (fungus) / References: UniProt: I2FI81
#2: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 492 / Source method: isolated from a natural source / Formula: H2O
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: 1.86 Å3/Da / Density % sol: 33.99 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 16% PEG3350, 0.2 M magnesium chloride, 50 mM sodium acetate pH 5.0

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Photon Factory / Beamline: BL-5A / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Oct 25, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.7→50 Å / Num. obs: 41828 / % possible obs: 99.9 % / Redundancy: 6.8 % / Biso Wilson estimate: 9.645 Å2 / CC1/2: 0.996 / Rrim(I) all: 0.134 / Net I/σ(I): 11.6
Reflection shellResolution: 1.7→1.73 Å / Mean I/σ(I) obs: 3.6 / Num. unique obs: 2194 / CC1/2: 0.921 / Rrim(I) all: 0.499 / % possible all: 99.2

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Processing

Software
NameVersionClassification
PHENIX1.10.1_2155refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.7→48.6 Å / SU ML: 0.1392 / Cross valid method: FREE R-VALUE / σ(F): 1.348 / Phase error: 16.85
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.1758 2092 5 %
Rwork0.1439 39707 -
obs0.1455 41799 99.93 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 6.964 Å2
Refinement stepCycle: LAST / Resolution: 1.7→48.6 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3425 0 8 492 3925
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.004123603
X-RAY DIFFRACTIONf_angle_d0.75394922
X-RAY DIFFRACTIONf_chiral_restr0.0471488
X-RAY DIFFRACTIONf_plane_restr0.004664667
X-RAY DIFFRACTIONf_dihedral_angle_d10.212073
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.7-1.740.20741390.168532626X-RAY DIFFRACTION99.35
1.74-1.780.22421300.157922671X-RAY DIFFRACTION99.96
1.78-1.830.20351450.158692599X-RAY DIFFRACTION100
1.83-1.890.21981440.156182629X-RAY DIFFRACTION100
1.89-1.950.18251270.150582636X-RAY DIFFRACTION100
1.95-2.020.19791300.144892655X-RAY DIFFRACTION99.96
2.02-2.10.19121360.147942641X-RAY DIFFRACTION100
2.1-2.190.15521310.144522648X-RAY DIFFRACTION99.96
2.19-2.310.1871420.139912654X-RAY DIFFRACTION99.96
2.31-2.450.18891250.152972647X-RAY DIFFRACTION100
2.45-2.640.18041240.149982672X-RAY DIFFRACTION99.96
2.64-2.910.17131730.152352612X-RAY DIFFRACTION100
2.91-3.330.17471510.144432651X-RAY DIFFRACTION99.93
3.33-4.190.15571360.122892671X-RAY DIFFRACTION100
4.19-48.60.13981590.130332695X-RAY DIFFRACTION99.79

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