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- PDB-5deh: Crystal structure of Staphylococcal nuclease variant Delta+PHS N1... -

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Basic information

Entry
Database: PDB / ID: 5deh
TitleCrystal structure of Staphylococcal nuclease variant Delta+PHS N100D 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: 1.7 Å
AuthorsSkerritt, L.A. / Bell-Upp, P.C. / Siegler, M.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 N100D at cryogenic temperature
Authors: Skerritt, L.A. / Bell-Upp, P.C. / Siegler, M.A. / Schlessman, J.L. / Garcia-Moreno E., B.
History
DepositionAug 25, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 2, 2015Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2017Group: Author supporting evidence / Derived calculations / Refinement description
Category: pdbx_audit_support / pdbx_struct_oper_list / software
Item: _pdbx_audit_support.funding_organization / _pdbx_struct_oper_list.symmetry_operation / _software.name
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)16,5873
Polymers16,1441
Non-polymers4422
Water1,946108
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area840 Å2
ΔGint-14 kcal/mol
Surface area6960 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: 16144.447 Da / Num. of mol.: 1 / Fragment: UNP residues 83-231 / Mutation: G50F/V51N/N100D/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.: 108 / 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: 2.45 Å3/Da / Density % sol: 44.97 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 20% 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: Jul 20, 2015
RadiationMonochromator: mirrors / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.7→50 Å / Num. all: 15566 / Num. obs: 15566 / % possible obs: 100 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 10.2 % / Biso Wilson estimate: 26.65 Å2 / Rmerge(I) obs: 0.015 / Net I/σ(I): 58.5
Reflection shellResolution: 1.7→1.76 Å / Redundancy: 7 % / Rmerge(I) obs: 0.119 / Mean I/σ(I) obs: 6.56 / % possible all: 100

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation1.7 Å24.93 Å
Translation1.7 Å24.93 Å

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Processing

Software
NameVersionClassification
REFMAC5.8.0103refinement
CrysalisPro1.171.36.32data scaling
PHASER2.5.6phasing
PDB_EXTRACT3.15data extraction
CrysalisPro1.171.36.32data reduction
Coot0.7.1model building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 3BDC

3bdc


Resolution: 1.7→38.35 Å / Cor.coef. Fo:Fc: 0.964 / Cor.coef. Fo:Fc free: 0.953 / WRfactor Rfree: 0.19 / WRfactor Rwork: 0.1646 / FOM work R set: 0.8569 / SU B: 3.988 / SU ML: 0.073 / SU R Cruickshank DPI: 0.1028 / SU Rfree: 0.0995 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.103 / ESU R Free: 0.1 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.1995 747 4.8 %RANDOM
Rwork0.1687 ---
obs0.1701 14802 99.25 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 66.93 Å2 / Biso mean: 23.039 Å2 / Biso min: 10.96 Å2
Baniso -1Baniso -2Baniso -3
1--0.93 Å20 Å20.43 Å2
2---1.12 Å2-0 Å2
3---1.98 Å2
Refinement stepCycle: final / Resolution: 1.7→38.35 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1033 0 26 108 1167
Biso mean--16.53 26.92 -
Num. residues----129
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0180.0191145
X-RAY DIFFRACTIONr_bond_other_d0.0010.021154
X-RAY DIFFRACTIONr_angle_refined_deg1.8762.0051553
X-RAY DIFFRACTIONr_angle_other_deg0.84932683
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.3635147
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.7222550
X-RAY DIFFRACTIONr_dihedral_angle_3_deg17.26515235
X-RAY DIFFRACTIONr_dihedral_angle_4_deg10.799155
X-RAY DIFFRACTIONr_chiral_restr0.1210.2167
X-RAY DIFFRACTIONr_gen_planes_refined0.0140.021261
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02246
X-RAY DIFFRACTIONr_mcbond_it1.1921.26535
X-RAY DIFFRACTIONr_mcbond_other1.1331.256534
X-RAY DIFFRACTIONr_mcangle_it1.711.886672
LS refinement shellResolution: 1.702→1.746 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.237 50 -
Rwork0.214 1013 -
all-1063 -
obs--90.62 %
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)
12.0168-0.6966-0.25623.32830.50970.232-0.03560.0096-0.13270.05740.0775-0.10770.08630.0661-0.04190.05470.021-0.01930.0517-0.00390.038213.5843-1.62172.8955
23.70552.12931.26239.94253.05823.7755-0.067-0.4725-0.05450.52810.0638-0.04030.08310.14340.00320.12120.02780.00420.14640.03450.013210.56112.490415.4972
32.2195-0.4716-0.05652.49840.4060.7207-0.00640.12150.0617-0.10510.01520.15590.0091-0.0157-0.00880.0316-0.0074-0.01490.03810.01990.02136.90186.02090.894
412.4728-0.23053.12196.2253-0.28145.2783-0.1971-0.45430.59660.52860.13980.549-0.4174-0.27150.05730.09690.04420.05240.082-0.0350.1063-0.994616.130814.9283
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A7 - 43
2X-RAY DIFFRACTION2A50 - 71
3X-RAY DIFFRACTION3A72 - 130
4X-RAY DIFFRACTION4A131 - 141

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