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- PDB-4wrd: Crystal structure of Staphylcoccal nulease variant Delta+PHS V66E... -

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Basic information

Entry
Database: PDB / ID: 4wrd
TitleCrystal structure of Staphylcoccal nulease variant Delta+PHS V66E L125E at cryogenic temperature
ComponentsThermonuclease
KeywordsHYDROLASE / nuclease / pdTp / pH-sensitive switch / internal ionizable groups
Function / homology
Function and homology information


micrococcal nuclease / : / nucleic acid binding / extracellular region / 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: 1.65 Å
AuthorsBell-Upp, P.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)GM073838 United States
CitationJournal: to be published
Title: Crystal structure of Staphylcoccal nulease variant Delta+PHS V66E L125E at cryogenic temperature
Authors: Bell-Upp, P.C. / Schlessman, J.L. / Garcia-Moreno E., B.
History
DepositionOct 23, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 8, 2015Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2017Group: Author supporting evidence / Derived calculations ...Author supporting evidence / Derived calculations / Other / Source and taxonomy
Category: entity_src_gen / pdbx_audit_support ...entity_src_gen / pdbx_audit_support / pdbx_database_status / pdbx_struct_oper_list
Item: _entity_src_gen.pdbx_alt_source_flag / _pdbx_audit_support.funding_organization ..._entity_src_gen.pdbx_alt_source_flag / _pdbx_audit_support.funding_organization / _pdbx_database_status.pdb_format_compatible / _pdbx_struct_oper_list.symmetry_operation
Revision 1.2Nov 22, 2017Group: Refinement description / Category: software / Item: _software.classification
Revision 1.3Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Sep 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 / refine_hist
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _refine_hist.number_atoms_total / _refine_hist.pdbx_number_atoms_nucleic_acid / _refine_hist.pdbx_number_atoms_protein

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Thermonuclease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,6323
Polymers16,1891
Non-polymers4422
Water1,69394
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area830 Å2
ΔGint-15 kcal/mol
Surface area6940 Å2
MethodPISA
Unit cell
Length a, b, c (Å)31.140, 60.315, 38.364
Angle α, β, γ (deg.)90.000, 94.050, 90.000
Int Tables number4
Space group name H-MP1211
Detailsbiological unit is a monomer.

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Components

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


Mass: 16189.404 Da / Num. of mol.: 1 / Fragment: UNP residues 80-228 / Mutation: G50F, V51N, V66E, P117G, H124L, L125E, S128A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Strain: COL / Gene: nuc, SACOL0860 / Plasmid: pET24a+ / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q5HHM4, 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.: 94 / 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: 2.22 Å3/Da / Density % sol: 44.59 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 6
Details: 20% MPD, 25mM 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: APEX II CCD / Detector: CCD / Date: Aug 4, 2014 / Details: multi-layer optics
RadiationMonochromator: Ge(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 1.65→38.27 Å / Num. all: 17155 / Num. obs: 17155 / % possible obs: 99.9 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 27.81 % / Biso Wilson estimate: 24.6 Å2 / Rmerge(I) obs: 0.01 / Net I/σ(I): 142.68 / Num. measured all: 477720
Reflection shellResolution: 1.65→1.67 Å / Redundancy: 8.71 % / Rmerge(I) obs: 0.1398 / Mean I/σ(I) obs: 6.63 / % possible all: 97.6

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation1.65 Å38.27 Å
Translation1.65 Å38.27 Å

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Processing

Software
NameVersionClassification
SAINTdata scaling
SAINT2013/3 for Windowsdata reduction
PHASER2.5.5phasing
REFMAC5.8.0049refinement
PDB_EXTRACT3.15data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3BDC

3bdc


Resolution: 1.65→38.27 Å / Cor.coef. Fo:Fc: 0.959 / Cor.coef. Fo:Fc free: 0.945 / WRfactor Rfree: 0.1773 / WRfactor Rwork: 0.1551 / FOM work R set: 0.8392 / SU B: 1.944 / SU ML: 0.067 / SU R Cruickshank DPI: 0.0993 / SU Rfree: 0.1005 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.099 / ESU R Free: 0.1 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.22 864 5.1 %RANDOM
Rwork0.1823 16239 --
obs0.1842 16239 99.68 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 79.73 Å2 / Biso mean: 20.713 Å2 / Biso min: 9.02 Å2
Baniso -1Baniso -2Baniso -3
1--1.02 Å20 Å20.53 Å2
2---1.06 Å2-0 Å2
3---1.98 Å2
Refinement stepCycle: final / Resolution: 1.65→38.27 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1036 0 26 94 1156
Biso mean--14.48 23.9 -
Num. residues----129
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0170.021136
X-RAY DIFFRACTIONr_bond_other_d0.0020.021123
X-RAY DIFFRACTIONr_angle_refined_deg1.622.0031541
X-RAY DIFFRACTIONr_angle_other_deg0.74132611
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.5875146
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.44925.38552
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.88915229
X-RAY DIFFRACTIONr_dihedral_angle_4_deg10.214155
X-RAY DIFFRACTIONr_chiral_restr0.1030.2165
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.021262
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02243
X-RAY DIFFRACTIONr_mcbond_it1.9871.784533
X-RAY DIFFRACTIONr_mcbond_other1.951.779532
X-RAY DIFFRACTIONr_mcangle_it2.7522.674669
LS refinement shellResolution: 1.65→1.69 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.268 66 -
Rwork0.19 1150 -
all-1216 -
obs--98.7 %

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