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- PDB-5c5l: Crystal structure of Staphylococcal nuclease variant Delta+PHS T6... -

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Basic information

Entry
Database: PDB / ID: 5c5l
TitleCrystal structure of Staphylococcal nuclease variant Delta+PHS T62E / L125E at cryogenic temperature
ComponentsThermonucleaseMicrococcal nuclease
KeywordsHYDROLASE / nuclease / hyperstable / pdTp / ionizable group / pH-sensitive switch
Function / homology
Function and homology information


endonuclease activity, active with either ribo- or deoxyribonucleic acids and producing 3'-phosphomonoesters / micrococcal nuclease / nucleic acid binding / extracellular region / membrane / metal ion binding
Similarity search - Function
Thermonuclease family signature 1. / OB fold (Dihydrolipoamide Acetyltransferase, E2P) - #90 / 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) ...Thermonuclease family signature 1. / OB fold (Dihydrolipoamide Acetyltransferase, E2P) - #90 / 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
PHOSPHATE ION / THYMIDINE-3',5'-DIPHOSPHATE / Thermonuclease
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.75 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 Staphylococcal nuclease variant Delta+PHS T62E / L125E at cryogenic temperature
Authors: Bell-Upp, P.C. / Schlessman, J.L. / Garcia-Moreno E., B.
History
DepositionJun 20, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 8, 2015Provider: repository / Type: Initial release
Revision 1.1Sep 6, 2017Group: Author supporting evidence / Derived calculations / Source and taxonomy
Category: entity_src_gen / pdbx_audit_support ...entity_src_gen / pdbx_audit_support / pdbx_struct_assembly / pdbx_struct_assembly_prop / 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_struct_assembly.oligomeric_details / _pdbx_struct_assembly_prop.type / _pdbx_struct_assembly_prop.value / _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
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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)16,7254
Polymers16,1871
Non-polymers5373
Water2,054114
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area990 Å2
ΔGint-19 kcal/mol
Surface area7230 Å2
MethodPISA
Unit cell
Length a, b, c (Å)30.709, 58.135, 34.796
Angle α, β, γ (deg.)90.000, 98.790, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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


Mass: 16187.430 Da / Num. of mol.: 1 / Fragment: UNP residues 83-231 / Mutation: G50F/V51N/T62E/P117G/H124L/L125E/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 / Thymidine diphosphate


Type: DNA linking / Mass: 402.188 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N2O11P2
#4: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 114 / 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: 1.9 Å3/Da / Density % sol: 35.13 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 6
Details: 35% MPD, 25 mM potassium phosphate, calcium chloride, pdTp
PH range: 6

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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 27, 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: 1.75→58.13 Å / Num. all: 12299 / Num. obs: 12299 / % possible obs: 100 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 8.11 % / Biso Wilson estimate: 20.1 Å2 / Rmerge(I) obs: 0.0359 / Net I/σ(I): 22.4 / Num. measured all: 103986
Reflection shellResolution: 1.75→1.77 Å / Redundancy: 5.68 % / Rmerge(I) obs: 0.1902 / % possible all: 100

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation1.75 Å34.39 Å
Translation1.75 Å34.39 Å

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Processing

Software
NameVersionClassification
SAINTdata scaling
APEX2013/1 for Windowsdata scaling
PHASER2.5.5phasing
REFMACrefinement
PDB_EXTRACT3.15data extraction
APEXdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 3BDC

3bdc


Resolution: 1.75→34.39 Å / Cor.coef. Fo:Fc: 0.963 / Cor.coef. Fo:Fc free: 0.935 / WRfactor Rfree: 0.1825 / WRfactor Rwork: 0.137 / FOM work R set: 0.8903 / SU B: 3.642 / SU ML: 0.067 / SU R Cruickshank DPI: 0.1076 / SU Rfree: 0.1093 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.108 / ESU R Free: 0.109 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.1907 587 4.8 %RANDOM
Rwork0.1436 ---
obs0.1459 11694 99.98 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 40.49 Å2 / Biso mean: 13.082 Å2 / Biso min: 5.69 Å2
Baniso -1Baniso -2Baniso -3
1--0.17 Å20 Å20.5 Å2
2---0.14 Å2-0 Å2
3---0.15 Å2
Refinement stepCycle: final / Resolution: 1.75→34.39 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1045 0 31 114 1190
Biso mean--13.06 19.8 -
Num. residues----130
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0180.0191120
X-RAY DIFFRACTIONr_bond_other_d0.0010.021104
X-RAY DIFFRACTIONr_angle_refined_deg1.9082.0091511
X-RAY DIFFRACTIONr_angle_other_deg0.90732560
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.035137
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.34125.29451
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.52315223
X-RAY DIFFRACTIONr_dihedral_angle_4_deg7.957155
X-RAY DIFFRACTIONr_chiral_restr0.1190.2159
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.021228
X-RAY DIFFRACTIONr_gen_planes_other0.0030.02237
X-RAY DIFFRACTIONr_mcbond_it0.7340.537527
X-RAY DIFFRACTIONr_mcbond_other0.6620.534526
X-RAY DIFFRACTIONr_mcangle_it1.1110.799659
LS refinement shellResolution: 1.75→1.795 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.183 31 -
Rwork0.154 886 -
all-917 -
obs--100 %
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)
10.7677-0.48410.01242.48290.21680.3730.01420.0738-0.0021-0.03290.028-0.00820.08390.0416-0.04220.03010.0114-0.01150.02850.00340.022539.5048-4.05136.0393
20.98110.14070.29870.69330.31872.1543-0.0053-0.1039-0.03160.1090.0456-0.02640.10880.0774-0.04040.04460.0171-0.01040.0539-0.00440.047136.05622.961948.049
30.6291-0.31070.23771.2583-0.07780.98520.00590.04270.0147-0.0477-0.01210.06640.06680.05750.00610.03880.0036-0.0180.03440.00360.04732.88412.016535.9733
40.8907-0.0761-0.56890.9516-0.43513.0080.0460.02080.0413-0.02820.05240.0297-0.168-0.0706-0.09830.04080.0044-0.00890.04290.00690.060830.718412.102134.9812
52.30490.13471.00151.22270.39337.2679-0.033-0.07980.11440.0815-0.01530.1482-0.2082-0.26880.04830.04220.01250.00820.02170.01270.058524.544113.474648.2612
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A7 - 35
2X-RAY DIFFRACTION2A36 - 70
3X-RAY DIFFRACTION3A71 - 107
4X-RAY DIFFRACTION4A108 - 124
5X-RAY DIFFRACTION5A125 - 142

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