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- PDB-8p9u: Crystal Structure of Two-Domain Laccase mutant M199A/D268N from S... -

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Basic information

Entry
Database: PDB / ID: 8p9u
TitleCrystal Structure of Two-Domain Laccase mutant M199A/D268N from Streptomyces griseoflavus
Components(Two-domain laccase) x 2
KeywordsOXIDOREDUCTASE / Two-domain laccase / enzyme engineering / SgfSL / TNC / proton transport / laccase trinuclear cluster / oxygen reduction / structural biology
Function / homology
Function and homology information


hydroquinone:oxygen oxidoreductase activity / laccase / iron ion transport / copper ion binding / plasma membrane
Similarity search - Function
Multicopper oxidase, copper-binding site / Multicopper oxidases signature 2. / Multicopper oxidase, C-terminal / Multicopper oxidase / Multicopper oxidase / Multicopper oxidase, N-terminal / Multicopper oxidase / Twin arginine translocation (Tat) signal profile. / Twin-arginine translocation pathway, signal sequence / Cupredoxin
Similarity search - Domain/homology
COPPER (II) ION / OXYGEN MOLECULE / Two-domain laccase
Similarity search - Component
Biological speciesStreptomyces griseoflavus (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsKolyadenko, I.A. / Tishchenko, S.V. / Gabdulkhakov, A.G.
Funding support Russian Federation, 1items
OrganizationGrant numberCountry
Russian Science Foundation Russian Federation
CitationJournal: Int J Mol Sci / Year: 2023
Title: Structural Insight into the Amino Acid Environment of the Two-Domain Laccase's Trinuclear Copper Cluster.
Authors: Kolyadenko, I. / Tishchenko, S. / Gabdulkhakov, A.
History
DepositionJun 6, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 10, 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: Two-domain laccase
B: Two-domain laccase
C: Two-domain laccase
D: Two-domain laccase
E: Two-domain laccase
F: Two-domain laccase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)182,93433
Polymers181,4236
Non-polymers1,51127
Water13,655758
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)74.302, 94.026, 118.927
Angle α, β, γ (deg.)90.000, 91.100, 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 , 2 types, 6 molecules ABDEFC

#1: Protein
Two-domain laccase


Mass: 30227.605 Da / Num. of mol.: 5 / Mutation: M199A/D268N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces griseoflavus (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0M4FJ81, laccase
#2: Protein Two-domain laccase


Mass: 30284.660 Da / Num. of mol.: 1 / Mutation: M199A/D268N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces griseoflavus (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0M4FJ81

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Non-polymers , 4 types, 785 molecules

#3: Chemical
ChemComp-CU / COPPER (II) ION


Mass: 63.546 Da / Num. of mol.: 20 / Source method: obtained synthetically / Formula: Cu / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-OXY / OXYGEN MOLECULE


Mass: 31.999 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: O2 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 758 / 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: 2.31 Å3/Da / Density % sol: 46.73 %
Crystal growTemperature: 296 K / Method: vapor diffusion / pH: 5
Details: 15 % v/v PEG Smear High, 0.15 M Ammonium acetate, 0.1 M Sodium citrate condition #31 of BCS-1 from Molecular Dimensions, UK

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Data collection

DiffractionMean temperature: 120 K / Serial crystal experiment: N
Diffraction sourceSource: SEALED TUBE / Type: RIGAKU PhotonJet-S / Wavelength: 1.54 Å
DetectorType: RIGAKU HyPix-6000HE / Detector: PIXEL / Date: Mar 18, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 2→23.54 Å / Num. obs: 104468 / % possible obs: 94.6 % / Redundancy: 2.9 % / Biso Wilson estimate: 21.5 Å2 / CC1/2: 0.98 / Net I/σ(I): 8.01
Reflection shellResolution: 2→2.07 Å / Redundancy: 2.9 % / Mean I/σ(I) obs: 1.43 / Num. unique obs: 10454 / CC1/2: 0.54 / % possible all: 95.3

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
CrysalisProdata reduction
CrysalisProdata scaling
MOLREP11.6.03phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2→23.54 Å / SU ML: 0.2775 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 24.6378
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2418 5231 5.02 %
Rwork0.2011 99069 -
obs0.2031 104300 94.51 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 27.42 Å2
Refinement stepCycle: LAST / Resolution: 2→23.54 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12756 0 32 758 13546
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.007613474
X-RAY DIFFRACTIONf_angle_d0.894718351
X-RAY DIFFRACTIONf_chiral_restr0.0571877
X-RAY DIFFRACTIONf_plane_restr0.00782471
X-RAY DIFFRACTIONf_dihedral_angle_d15.64224824
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2-2.020.37391610.35493273X-RAY DIFFRACTION94.57
2.02-2.050.34092000.3063307X-RAY DIFFRACTION95.12
2.05-2.070.36211670.28723320X-RAY DIFFRACTION95.04
2.07-2.10.33041500.28313340X-RAY DIFFRACTION95.43
2.1-2.130.35851680.27633317X-RAY DIFFRACTION95.22
2.13-2.150.27241760.2643259X-RAY DIFFRACTION95.23
2.15-2.190.27851830.26533352X-RAY DIFFRACTION95.31
2.19-2.220.32061580.26653271X-RAY DIFFRACTION94.78
2.22-2.250.32491750.26493352X-RAY DIFFRACTION95.3
2.25-2.290.30751890.26313285X-RAY DIFFRACTION94.94
2.29-2.330.29281810.25553305X-RAY DIFFRACTION95.09
2.33-2.370.27912030.24633267X-RAY DIFFRACTION95.09
2.37-2.420.27861960.23613301X-RAY DIFFRACTION95.05
2.42-2.470.29471660.24383337X-RAY DIFFRACTION95.27
2.47-2.520.29441940.23273266X-RAY DIFFRACTION95.03
2.52-2.580.2671900.22253336X-RAY DIFFRACTION95.14
2.58-2.640.24681680.2223310X-RAY DIFFRACTION95.29
2.64-2.710.24961870.21793331X-RAY DIFFRACTION95.47
2.71-2.790.25881520.22023340X-RAY DIFFRACTION95.44
2.79-2.880.25811610.20363387X-RAY DIFFRACTION95.69
2.88-2.990.29751700.20923323X-RAY DIFFRACTION95.25
2.99-3.110.24631770.1873356X-RAY DIFFRACTION95.69
3.11-3.250.22071610.18663357X-RAY DIFFRACTION95.29
3.25-3.420.2211810.17353282X-RAY DIFFRACTION94.85
3.42-3.630.20361790.16583351X-RAY DIFFRACTION94.74
3.63-3.910.18261510.16213323X-RAY DIFFRACTION94.15
3.91-4.30.17062160.14393212X-RAY DIFFRACTION92.87
4.3-4.920.17561660.13553266X-RAY DIFFRACTION92.68
4.92-6.180.18581400.14283276X-RAY DIFFRACTION91.51
6.18-23.540.18141650.16823067X-RAY DIFFRACTION85.21

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