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- PDB-5i6y: Crystal structure of Staphylococcal nuclease variant Delta+PHS A9... -

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Basic information

Entry
Database: PDB / ID: 5i6y
TitleCrystal structure of Staphylococcal nuclease variant Delta+PHS A90E 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: 1.9 Å
AuthorsSkerritt, L.A. / 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)GM061597 United States
CitationJournal: To be Published
Title: Crystal structure of Staphylococcal nuclease variant Delta+PHS A90E at cryogenic temperature
Authors: Skerritt, L.A. / Schlessman, J.L. / Garcia-Moreno E., B.
History
DepositionFeb 16, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 24, 2016Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2017Group: Author supporting evidence / Derived calculations / Category: pdbx_audit_support / 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 1.3Sep 27, 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
Water1,33374
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area830 Å2
ΔGint-15 kcal/mol
Surface area7110 Å2
MethodPISA
Unit cell
Length a, b, c (Å)31.178, 60.353, 38.425
Angle α, β, γ (deg.)90.000, 93.520, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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


Mass: 16201.500 Da / Num. of mol.: 1 / Fragment: UNP residues 80-228 / Mutation: G50F/V51N/A90E/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.: 74 / 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: 2.23 Å3/Da / Density % sol: 44.77 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 25% 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: 1.5418 Å
DetectorType: AGILENT ATLAS CCD / Detector: CCD / Date: Jan 20, 2016
RadiationMonochromator: mirrors / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.9→50 Å / Num. obs: 11262 / % possible obs: 99.9 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 10 % / Biso Wilson estimate: 30.77 Å2 / Rmerge(I) obs: 0.015 / Net I/σ(I): 61.95
Reflection shellResolution: 1.9→1.97 Å / Redundancy: 6.4 % / Rmerge(I) obs: 0.108 / Mean I/σ(I) obs: 8.01 / % possible all: 100

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation1.88 Å24.93 Å
Translation1.88 Å24.93 Å

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Processing

Software
NameVersionClassification
CrysalisPro1.171.36.32data scaling
PHASER2.5.7phasing
REFMAC5.8.0103refinement
PDB_EXTRACT3.2data extraction
CrysalisPro1.171.36.32data reduction
Coot0.7.1model building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 3BDC

3bdc


Resolution: 1.9→38.35 Å / Cor.coef. Fo:Fc: 0.961 / Cor.coef. Fo:Fc free: 0.943 / SU B: 6.574 / SU ML: 0.11 / SU R Cruickshank DPI: 0.1499 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.15 / ESU R Free: 0.144 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2215 604 5.4 %RANDOM
Rwork0.1719 ---
obs0.1744 10643 97.02 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 70.38 Å2 / Biso mean: 28.212 Å2 / Biso min: 13.26 Å2
Baniso -1Baniso -2Baniso -3
1--0.65 Å20 Å20.28 Å2
2---0.46 Å2-0 Å2
3---1.07 Å2
Refinement stepCycle: final / Resolution: 1.9→38.35 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1037 0 26 74 1137
Biso mean--20.59 28.43 -
Num. residues----129
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0180.0191147
X-RAY DIFFRACTIONr_bond_other_d0.0010.021156
X-RAY DIFFRACTIONr_angle_refined_deg1.7832.0051556
X-RAY DIFFRACTIONr_angle_other_deg0.83832687
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.6865147
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.2472550
X-RAY DIFFRACTIONr_dihedral_angle_3_deg17.7415236
X-RAY DIFFRACTIONr_dihedral_angle_4_deg10.289155
X-RAY DIFFRACTIONr_chiral_restr0.1130.2168
X-RAY DIFFRACTIONr_gen_planes_refined0.0130.021261
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02246
X-RAY DIFFRACTIONr_mcbond_it1.3911.571535
X-RAY DIFFRACTIONr_mcbond_other1.3651.568534
X-RAY DIFFRACTIONr_mcangle_it2.0262.345672
LS refinement shellResolution: 1.9→1.931 Å
RfactorNum. reflection% reflection
Rfree0.244 29 -
Rwork0.207 497 -
obs--60.74 %
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)
12.1873-0.4287-0.4564.37150.5891.6788-0.0685-0.0554-0.05030.03140.1152-0.10930.27250.1563-0.04680.04630.0179-0.00970.05570.01220.022145.03390.50614.9829
22.6424-0.0506-0.19343.04360.21442.0176-0.0514-0.00570.1650.05650.15020.3330.0991-0.1185-0.09870.0125-0.0166-0.0070.04470.0340.057436.97356.38625.7845
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A7 - 53
2X-RAY DIFFRACTION2A54 - 141

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