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- PDB-8ihy: X-ray crystal structure of Q387E mutant of endo-1,4-beta glucanas... -

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Basic information

Entry
Database: PDB / ID: 8ihy
TitleX-ray crystal structure of Q387E 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: 1.6 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 13, 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)51,8865
Polymers51,7061
Non-polymers1794
Water7,584421
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 area14850 Å2
MethodPISA
Unit cell
Length a, b, c (Å)53.360, 71.639, 55.184
Angle α, β, γ (deg.)90.000, 114.037, 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

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Protein , 1 types, 1 molecules A

#1: Protein Endoglucanase


Mass: 51706.066 Da / Num. of mol.: 1 / Mutation: Q387E
Source method: isolated from a genetically manipulated source
Details: Residues 1-4 and 440-462 are derived from the expression vector.
Source: (gene. exp.) Eisenia fetida (common brandling worm) / Gene: EF-EG2 / Production host: Komagataella pastoris (fungus) / References: UniProt: I2FI81

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Non-polymers , 5 types, 425 molecules

#2: Chemical ChemComp-MG / MAGNESIUM ION


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


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-NA / SODIUM ION


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


Mass: 92.094 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C3H8O3
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 421 / 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: 1.86 Å3/Da / Density % sol: 33.98 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 18% 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-17A / Wavelength: 0.98 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: May 29, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 1.6→50 Å / Num. obs: 49767 / % possible obs: 99.4 % / Redundancy: 3.5 % / Biso Wilson estimate: 10.61 Å2 / CC1/2: 0.996 / Rrim(I) all: 0.112 / Net I/σ(I): 8.3
Reflection shellResolution: 1.6→1.63 Å / Mean I/σ(I) obs: 2.5 / Num. unique obs: 2443 / CC1/2: 0.84 / Rrim(I) all: 0.523

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Processing

Software
NameVersionClassification
PHENIX1.10.1_2155refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.6→40.29 Å / SU ML: 0.1295 / Cross valid method: FREE R-VALUE / σ(F): 1.336 / Phase error: 17.03
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.1767 2482 4.996 %
Rwork0.1471 47193 -
obs0.1486 49675 99.16 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 11.84 Å2
Refinement stepCycle: LAST / Resolution: 1.6→40.29 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3413 0 9 421 3843
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0049013655
X-RAY DIFFRACTIONf_angle_d0.79925005
X-RAY DIFFRACTIONf_chiral_restr0.0487493
X-RAY DIFFRACTIONf_plane_restr0.005242686
X-RAY DIFFRACTIONf_dihedral_angle_d12.742108
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.6-1.630.23081300.1812597X-RAY DIFFRACTION97.36
1.63-1.660.23161330.17032584X-RAY DIFFRACTION98.62
1.66-1.70.19911410.16822548X-RAY DIFFRACTION98.43
1.7-1.740.20791350.1722632X-RAY DIFFRACTION99.03
1.74-1.780.21581260.17182606X-RAY DIFFRACTION97.92
1.78-1.830.19881460.16162566X-RAY DIFFRACTION98.87
1.83-1.890.21151440.15592624X-RAY DIFFRACTION99.57
1.89-1.950.16791300.14682615X-RAY DIFFRACTION99.46
1.95-2.020.1861240.13722640X-RAY DIFFRACTION99.57
2.02-2.10.16771370.14352626X-RAY DIFFRACTION99.5
2.1-2.190.18081330.14092653X-RAY DIFFRACTION99.68
2.19-2.310.17671390.14242631X-RAY DIFFRACTION99.68
2.31-2.450.1841220.14922625X-RAY DIFFRACTION99.35
2.45-2.640.18111250.15232646X-RAY DIFFRACTION99
2.64-2.910.15291710.15012604X-RAY DIFFRACTION99.82
2.91-3.330.17431520.14592649X-RAY DIFFRACTION99.89
3.33-4.190.15711350.12572672X-RAY DIFFRACTION99.89
4.19-40.290.15641590.14072675X-RAY DIFFRACTION99.3

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