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- PDB-7b2k: Structure of the M298F mutant of the Streptomyces coelicolor smal... -

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Basic information

Entry
Database: PDB / ID: 7b2k
TitleStructure of the M298F mutant of the Streptomyces coelicolor small laccase T1 copper axial ligand.
ComponentsPutative copper oxidase
KeywordsOXIDOREDUCTASE / small laccase / T1 copper / point mutation / M298F
Function / homology
Function and homology information


oxidoreductase activity / copper ion binding
Similarity search - Function
Multicopper oxidase, copper-binding site / Multicopper oxidases signature 2. / Multicopper oxidase / Multicopper oxidase, C-terminal / 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 / Copper oxidase
Similarity search - Component
Biological speciesStreptomyces coelicolor (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.2 Å
AuthorsZovo, K. / Majumdar, S. / Lukk, T.
Funding support Estonia, 1items
OrganizationGrant numberCountry
Estonian Research CouncilMOBTT60 Estonia
CitationJournal: Acs Omega / Year: 2022
Title: Substitution of the Methionine Axial Ligand of the T1 Copper for the Fungal-like Phenylalanine Ligand (M298F) Causes Local Structural Perturbations that Lead to Thermal Instability and Reduced ...Title: Substitution of the Methionine Axial Ligand of the T1 Copper for the Fungal-like Phenylalanine Ligand (M298F) Causes Local Structural Perturbations that Lead to Thermal Instability and Reduced Catalytic Efficiency of the Small Laccase from Streptomyces coelicolor A3(2).
Authors: Zovo, K. / Pupart, H. / Van Wieren, A. / Gillilan, R.E. / Huang, Q. / Majumdar, S. / Lukk, T.
History
DepositionNov 27, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 9, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 23, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Mar 9, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Putative copper oxidase
B: Putative copper oxidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)74,40410
Polymers73,8962
Non-polymers5088
Water3,819212
1
A: Putative copper oxidase
hetero molecules

A: Putative copper oxidase
hetero molecules

A: Putative copper oxidase
hetero molecules


  • defined by author&software
  • Evidence: gel filtration, The protein is an obligatory trimer as has been determined by crystallography as well as gel filtration chromatography. In this crystal form the monomers belong to separate ...Evidence: gel filtration, The protein is an obligatory trimer as has been determined by crystallography as well as gel filtration chromatography. In this crystal form the monomers belong to separate trimers, both completed by crystallographic symmetry.
  • trimer
  • 112 kDa, 3 polymers
  • Search similar-shape structures of this assembly by Omokage search (details)
Theoretical massNumber of molelcules
Total (without water)111,60715
Polymers110,8443
Non-polymers76312
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation8_544-z,x-1/2,-y-1/21
crystal symmetry operation11_545y+1/2,-z-1/2,-x1
Buried area12850 Å2
ΔGint-182 kcal/mol
Surface area29310 Å2
MethodPISA
2
B: Putative copper oxidase
hetero molecules

B: Putative copper oxidase
hetero molecules

B: Putative copper oxidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)111,60715
Polymers110,8443
Non-polymers76312
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation5_555z,x,y1
crystal symmetry operation9_555y,z,x1
Buried area12680 Å2
ΔGint-187 kcal/mol
Surface area29040 Å2
MethodPISA
Unit cell
Length a, b, c (Å)177.620, 177.620, 177.620
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number198
Space group name H-MP213

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Components

#1: Protein Putative copper oxidase / small laccase


Mass: 36947.984 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) (bacteria)
Strain: ATCC BAA-471 / A3(2) / M145 / Gene: SCO6712 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9XAL8
#2: Chemical
ChemComp-CU / COPPER (II) ION / Copper


Mass: 63.546 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Cu / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 212 / Source method: isolated from a natural source / Formula: H2O
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

Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: Protein concentration was 20 mg/mL in 20 mM Tris-HCl buffer (pH 7.5). Mother liquor was made up of 40% MPD, 200 mM NH4-OAc and 100 mM HEPES (pH 7.5). The protein was mixed in 2:1 ratio with ...Details: Protein concentration was 20 mg/mL in 20 mM Tris-HCl buffer (pH 7.5). Mother liquor was made up of 40% MPD, 200 mM NH4-OAc and 100 mM HEPES (pH 7.5). The protein was mixed in 2:1 ratio with protein to mother liquor.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: CHESS / Beamline: F1 / Wavelength: 0.9782 Å
DetectorType: ADSC QUANTUM 270 / Detector: CCD / Date: Apr 18, 2018
RadiationMonochromator: C111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9782 Å / Relative weight: 1
ReflectionResolution: 2.2→29.2 Å / Num. obs: 92609 / % possible obs: 98.1 % / Redundancy: 8.733 % / Biso Wilson estimate: 44.052 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.085 / Rrim(I) all: 0.09 / Χ2: 1.031 / Net I/σ(I): 16.5 / Num. measured all: 808727 / Scaling rejects: 89
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsCC1/2Rrim(I) all% possible all
2.2-2.264.3090.8051.6925774692959820.6520.90786.3
2.26-2.324.9850.6782.2131149675762490.7460.75292.5
2.32-2.395.7650.5492.9836588657763470.8520.60196.5
2.39-2.466.8480.4963.7543577640263630.8910.53699.4
2.46-2.548.810.4245.1454356617161700.9470.451100
2.54-2.6310.1660.3586.760912599359920.9670.377100
2.63-2.7310.1680.278.7558697577357730.9790.285100
2.73-2.8410.180.21310.8356572555855570.9870.224100
2.84-2.9710.1650.15714.1854730538453840.9920.165100
2.97-3.1110.1610.12517.7251648508450830.9950.131100
3.11-3.2810.1470.121.3949711490048990.9960.106100
3.28-3.4810.1020.0825.9746592461246120.9980.084100
3.48-3.7210.0510.06730.743733435343510.9980.071100
3.72-4.029.9950.0633.7440341403640360.9980.063100
4.02-4.49.9560.05237.4137206373737370.9990.055100
4.4-4.929.8990.04939.433725340734070.9990.052100
4.92-5.689.8750.04739.9229663300430040.9990.05100
5.68-6.969.8070.04839.0525018255125510.9990.051100
6.96-9.849.5890.04540.4219294201220120.9990.048100
9.84-29.28.5830.04439.559441115511000.9980.04795.2

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
XDSVERSION Jan 26, 2018data reduction
XSCALEVERSION Jan 26, 2018data scaling
PHASER2.8.2phasing
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3CG8

3cg8


Resolution: 2.2→29.2 Å / SU ML: 0.22 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 20.28 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1851 4627 5 %
Rwork0.1709 87931 -
obs0.1716 92558 98.11 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 157.95 Å2 / Biso mean: 41.7343 Å2 / Biso min: 24.56 Å2
Refinement stepCycle: final / Resolution: 2.2→29.2 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4359 0 8 212 4579
Biso mean--64.81 42.92 -
Num. residues----562
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 (%)
2.2-2.230.30411310.27492485261683
2.23-2.250.26691370.24562608274588
2.25-2.280.31061410.25032675281691
2.28-2.310.27011460.23552782292893
2.31-2.340.25661480.23052806295495
2.34-2.370.27181520.22922880303297
2.37-2.40.24411540.21422923307799
2.4-2.440.23491540.21042939309399
2.44-2.480.26191560.20829643120100
2.48-2.520.22551550.199629353090100
2.52-2.560.20751570.197329803137100
2.56-2.610.20891570.19929863143100
2.61-2.660.21451550.19829463101100
2.66-2.710.23481560.195429703126100
2.71-2.770.21991580.193830093167100
2.77-2.840.21051570.199529673124100
2.84-2.910.19671560.185129643120100
2.91-2.990.21571570.182929873144100
2.99-3.070.20171560.184529723128100
3.07-3.170.18341570.178329733130100
3.17-3.290.22021570.176929853142100
3.29-3.420.18551580.167429993157100
3.42-3.570.17571580.159930003158100
3.57-3.760.15641560.151829733129100
3.76-3.990.14911580.153230043162100
4-4.30.1411590.13830183177100
4.31-4.730.15521590.127730173176100
4.74-5.410.16691590.142130253184100
5.42-6.810.14661610.16530593220100
6.81-29.20.15351620.16933100326298
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.11270.12-0.42440.77240.24460.9368-0.0002-0.08640.12580.12110.0537-0.0094-0.1745-0.0917-0.06720.41040.1203-0.03470.3218-0.00850.306922.1011-37.1629-14.1213
23.36230.0776-3.20340.7672-0.00014.3294-0.1523-0.3002-0.23780.2214-0.0544-0.134-0.13660.09450.14380.40650.0616-0.09850.3274-0.03670.35334.0252-39.8235-11.2547
31.3631-0.0752-0.26930.8276-0.18030.6626-0.04730.00980.04480.06920.0106-0.0276-0.0505-0.11870.06030.37360.062-0.03650.3305-0.03620.295226.4389-44.1825-19.867
42.21631.0406-0.04692.6436-1.38852.16050.14350.1757-0.10540.1279-0.1312-0.073-0.1247-0.2996-0.05990.37030.0942-0.02870.4196-0.06470.317810.7185-50.5837-23.7276
50.6751-0.3448-0.17180.63950.11121.0136-0.03080.1139-0.0050.07040.02160.0058-0.1417-0.1579-0.00390.31260.0511-0.02190.3345-0.03340.257918.243-51.0269-34.0282
63.9529-2.74033.9163.55140.14489.6442-0.0502-0.1627-0.04540.2083-0.3431.95930.2944-1.89860.29160.46720.0121-0.01160.79-0.14170.7523-1.484-44.8985-40.8144
70.6620.0983-0.01361.43670.68030.97760.03750.12490.0437-0.1113-0.0415-0.04620.08490.1419-0.00350.30780.08890.02050.39680.04670.29524.4834-18.4706-30.3076
80.81540.0188-0.26890.48680.02670.9545-0.017-0.0044-0.0470.1276-0.0120.00110.17690.0880.01870.33120.06630.02380.29760.02260.2507-4.6596-27.7669-11.9507
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 38 through 112 )A38 - 112
2X-RAY DIFFRACTION2chain 'A' and (resid 113 through 143 )A113 - 143
3X-RAY DIFFRACTION3chain 'A' and (resid 144 through 188 )A144 - 188
4X-RAY DIFFRACTION4chain 'A' and (resid 189 through 210 )A189 - 210
5X-RAY DIFFRACTION5chain 'A' and (resid 211 through 305 )A211 - 305
6X-RAY DIFFRACTION6chain 'A' and (resid 306 through 319 )A306 - 319
7X-RAY DIFFRACTION7chain 'B' and (resid 37 through 173 )B37 - 173
8X-RAY DIFFRACTION8chain 'B' and (resid 174 through 316 )B174 - 316

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