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- PDB-6eeg: Crystal structure of Staphylcoccal nuclease variant Delta+PHS T62... -

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Basic information

Entry
Database: PDB / ID: 6eeg
TitleCrystal structure of Staphylcoccal nuclease variant Delta+PHS T62E at cryogenic temperature
ComponentsThermonuclease
KeywordsHYDROLASE / nuclease / hyperstable / pdTp / ionizable group
Function / homology
Function and homology information


micrococcal nuclease / endonuclease activity, active with either ribo- or deoxyribonucleic acids and producing 3'-phosphomonoesters / nucleic acid binding / extracellular region / membrane / metal ion binding
Similarity search - Function
OB fold (Dihydrolipoamide Acetyltransferase, E2P) - #90 / Thermonuclease family signature 1. / Thermonuclease active site / Thermonuclease family signature 2. / Staphylococcal nuclease (SNase-like), OB-fold / Staphylococcal nuclease homologue / Thermonuclease domain profile. / Staphylococcal nuclease homologues / SNase-like, OB-fold superfamily / OB fold (Dihydrolipoamide Acetyltransferase, E2P) ...OB fold (Dihydrolipoamide Acetyltransferase, E2P) - #90 / Thermonuclease family signature 1. / Thermonuclease active site / Thermonuclease family signature 2. / Staphylococcal nuclease (SNase-like), OB-fold / Staphylococcal nuclease homologue / Thermonuclease domain profile. / Staphylococcal nuclease homologues / SNase-like, OB-fold superfamily / OB fold (Dihydrolipoamide Acetyltransferase, E2P) / Beta Barrel / Mainly Beta
Similarity search - Domain/homology
THYMIDINE-3',5'-DIPHOSPHATE / Thermonuclease
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.95 Å
AuthorsKougentakis, C.M. / Schlessman, J.L. / Garcia-Moreno E., B.
CitationJournal: To be Published
Title: Crystal structure of Staphylcoccal nuclease variant Delta+PHS T62E at cryogenic temperature
Authors: Kougentakis, C.M. / Schlessman, J.L. / Garcia-Moreno E., B.
History
DepositionAug 14, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 29, 2018Provider: repository / Type: Initial release
Revision 1.1Oct 11, 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: Thermonuclease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,6143
Polymers16,1711
Non-polymers4422
Water1,47782
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area830 Å2
ΔGint-15 kcal/mol
Surface area7010 Å2
MethodPISA
Unit cell
Length a, b, c (Å)31.037, 60.508, 38.184
Angle α, β, γ (deg.)90.000, 93.230, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Thermonuclease / TNase / Micrococcal nuclease / Staphylococcal nuclease


Mass: 16171.475 Da / Num. of mol.: 1 / Fragment: UNP residues 83-231 / Mutation: G50F/V51N/T62E/P117G/H124L/S128A/Del44-49
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: nuc / Plasmid: pET24a+ / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P00644, micrococcal nuclease
#2: Chemical ChemComp-THP / THYMIDINE-3',5'-DIPHOSPHATE


Type: DNA linking / Mass: 402.188 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N2O11P2
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 82 / Source method: isolated from a natural source / Formula: H2O

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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: 2.43 Å3/Da / Density % sol: 44.44 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 9
Details: 22% MPD, 25 mM potassium phosphate, calcium chloride, pdTp

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Data collection

DiffractionMean temperature: 110 K
Diffraction sourceSource: SEALED TUBE / Type: BRUKER IMUS MICROFOCUS / Wavelength: 1.54 Å
DetectorType: APEX II CCD / Detector: CCD / Date: Aug 12, 2016 / Details: mirrors
RadiationMonochromator: Ge(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 1.95→38.12 Å / Num. obs: 10364 / % possible obs: 100 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 14.79 % / Biso Wilson estimate: 28.22 Å2 / Rmerge(I) obs: 0.0257 / Net I/σ(I): 27.46
Reflection shellResolution: 1.95→1.98 Å / Redundancy: 10.8 % / Rmerge(I) obs: 0.1244 / Mean I/σ(I) obs: 7.27 / Num. unique obs: 456 / % possible all: 100

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation1.95 Å32.26 Å
Translation1.95 Å32.26 Å

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Processing

Software
NameVersionClassification
PHASER2.5.7phasing
REFMAC5.8.0124refinement
PDB_EXTRACT3.22data extraction
APEX 2data reduction
SHELXPREPdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 3BDC

3bdc


Resolution: 1.95→38.12 Å / Cor.coef. Fo:Fc: 0.956 / Cor.coef. Fo:Fc free: 0.924 / SU B: 3.248 / SU ML: 0.095 / SU R Cruickshank DPI: 0.1614 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.161 / ESU R Free: 0.149 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2186 520 5 %RANDOM
Rwork0.1723 ---
obs0.1746 9827 99.98 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 67.01 Å2 / Biso mean: 22.347 Å2 / Biso min: 9.57 Å2
Baniso -1Baniso -2Baniso -3
1-0 Å20 Å20.03 Å2
2---0.01 Å20 Å2
3---0.01 Å2
Refinement stepCycle: final / Resolution: 1.95→38.12 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1035 0 26 82 1143
Biso mean--17.1 24.72 -
Num. residues----129
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0190.0191097
X-RAY DIFFRACTIONr_bond_other_d0.0020.021084
X-RAY DIFFRACTIONr_angle_refined_deg1.9592.0041481
X-RAY DIFFRACTIONr_angle_other_deg1.02232508
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.7915134
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.56325.10249
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.63615214
X-RAY DIFFRACTIONr_dihedral_angle_4_deg12.221155
X-RAY DIFFRACTIONr_chiral_restr0.1250.2159
X-RAY DIFFRACTIONr_gen_planes_refined0.010.021206
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02235
LS refinement shellResolution: 1.95→2.001 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.256 47 -
Rwork0.176 707 -
all-754 -
obs--100 %

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