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- PDB-5kyl: Crystal structure of Staphylococcal nuclease variant Delta+PHS T6... -

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Basic information

Entry
Database: PDB / ID: 5kyl
TitleCrystal structure of Staphylococcal nuclease variant Delta+PHS T62K/V66E pH 7 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: 1.85 Å
AuthorsKougentakis, C.M. / Sternke, M.C. / Schlessman, J.L. / Garcia-Moreno E., B.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM-061579 United States
CitationJournal: To be Published
Title: Crystal structure of Staphylococcal nuclease variant Delta+PHS T62K/V66E pH 7 at cryogenic temperature
Authors: Kougentakis, C.M. / Sternke, M.C. / Schlessman, J.L. / Garcia-Moreno E., B.
History
DepositionJul 21, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 3, 2016Provider: repository / Type: Initial release
Revision 1.1Apr 4, 2018Group: Author supporting evidence / Data collection / Derived calculations
Category: pdbx_audit_support / pdbx_prerelease_seq / pdbx_struct_oper_list
Item: _pdbx_audit_support.funding_organization / _pdbx_struct_oper_list.symmetry_operation
Revision 1.2Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Oct 4, 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)16,6443
Polymers16,2021
Non-polymers4422
Water1,67593
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area840 Å2
ΔGint-14 kcal/mol
Surface area6910 Å2
MethodPISA
Unit cell
Length a, b, c (Å)31.062, 60.498, 38.300
Angle α, β, γ (deg.)90.000, 94.280, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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


Mass: 16201.523 Da / Num. of mol.: 1 / Fragment: UNP residues 83-231 / Mutation: G50F/V51N/T62K/V66E/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-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#3: Chemical ChemComp-THP / THYMIDINE-3',5'-DIPHOSPHATE


Type: DNA linking / Mass: 402.188 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N2O11P2
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 93 / 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: 2.21 Å3/Da / Density % sol: 44.47 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 7
Details: 30% MPD, 25 mM potassium phosphate, calcium chloride, pdTp

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Data collection

DiffractionMean temperature: 110 K
Diffraction sourceSource: SEALED TUBE / Type: OTHER / Wavelength: 1.54 Å
DetectorType: AGILENT ATLAS CCD / Detector: CCD / Date: Aug 13, 2015 / Details: mirrors
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 1.85→50 Å / Num. obs: 12150 / % possible obs: 100 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 10.1 % / Biso Wilson estimate: 24.41 Å2 / Rmerge(I) obs: 0.02 / Net I/σ(I): 50.97
Reflection shellResolution: 1.85→1.92 Å / Redundancy: 6.7 % / Rmerge(I) obs: 0.105 / Mean I/σ(I) obs: 8.03 / % possible all: 100

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation1.85 Å23.09 Å
Translation1.85 Å23.09 Å

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Processing

Software
NameVersionClassification
PHASER2.5.7phasing
REFMAC5.8.0124refinement
PDB_EXTRACT3.2data extraction
CrysalisPro1.171.36.32data reduction
CrysalisPro1.171.36.32data scaling
CrysalisPro1.171.36.32data collection
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 3BDC

3bdc


Resolution: 1.85→38.19 Å / Cor.coef. Fo:Fc: 0.952 / Cor.coef. Fo:Fc free: 0.929 / SU B: 3.176 / SU ML: 0.096 / SU R Cruickshank DPI: 0.1435 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.143 / ESU R Free: 0.135 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.22 665 5.5 %RANDOM
Rwork0.1774 ---
obs0.1797 11469 99.93 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 69.21 Å2 / Biso mean: 19.315 Å2 / Biso min: 6.98 Å2
Baniso -1Baniso -2Baniso -3
1--0.02 Å20 Å20.01 Å2
2--0.02 Å20 Å2
3---0 Å2
Refinement stepCycle: final / Resolution: 1.85→38.19 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1037 0 26 95 1158
Biso mean--12.81 21.9 -
Num. residues----129
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0180.0191150
X-RAY DIFFRACTIONr_bond_other_d0.0020.021143
X-RAY DIFFRACTIONr_angle_refined_deg1.8542.0021560
X-RAY DIFFRACTIONr_angle_other_deg0.95132658
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.0625148
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.8625.3754
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.4915234
X-RAY DIFFRACTIONr_dihedral_angle_4_deg10.594155
X-RAY DIFFRACTIONr_chiral_restr0.1130.2165
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.021281
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02250
X-RAY DIFFRACTIONr_mcbond_it1.5681.663535
X-RAY DIFFRACTIONr_mcbond_other1.5461.658534
X-RAY DIFFRACTIONr_mcangle_it2.3182.486672
LS refinement shellResolution: 1.85→1.898 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.282 49 -
Rwork0.218 841 -
all-890 -
obs--100 %

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