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- PDB-5kee: Crystal structure of Staphylococcal nuclease variant Delta+PHS L2... -

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Basic information

Entry
Database: PDB / ID: 5kee
TitleCrystal structure of Staphylococcal nuclease variant Delta+PHS L25K/I92F at cryogenic temperature
ComponentsThermonuclease
KeywordsHYDROLASE / Staphylococcal nuclease / hyperstable variant / pdtp
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 / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.5 Å
AuthorsSkerritt, L.A. / Caro, J.A. / Heroux, A. / Schlessman, J.L. / Garcia-Moreno E., B.
Funding support United States, 1items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)MCB-1517378 United States
CitationJournal: To be Published
Title: Crystal structure of Staphylococcal nuclease variant Delta+PHS L25K/I92F at cryogenic temperature
Authors: Skerritt, L.A. / Caro, J.A. / Heroux, A. / Schlessman, J.L. / Garcia-Moreno E., B.
History
DepositionJun 9, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 22, 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 1.2Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Sep 27, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr2_auth_seq_id

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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)16,6363
Polymers16,1941
Non-polymers4422
Water1,58588
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area830 Å2
ΔGint-16 kcal/mol
Surface area7100 Å2
MethodPISA
Unit cell
Length a, b, c (Å)32.283, 60.023, 74.175
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

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


Mass: 16193.504 Da / Num. of mol.: 1 / Fragment: UNP residues 83-231 / Mutation: L25K/G50F/V51N/I92F/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.: 88 / 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: 2.32 Å3/Da / Density % sol: 47 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 30% MPD, 25 mM potassium phosphate, calcium chloride, pdTp

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X25 / Wavelength: 1.1 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jul 25, 2013
RadiationMonochromator: double crystal Si(111) with cryogenically-cooled first crystal and sagittally-bent second crystal horizontally-focusing at 3.3:1 demagnification
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1 Å / Relative weight: 1
ReflectionResolution: 1.5→50 Å / Num. obs: 23798 / % possible obs: 100 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 11.9 % / Biso Wilson estimate: 24.05 Å2 / Rmerge(I) obs: 0.054 / Net I/av σ(I): 78.709 / Net I/σ(I): 20.6
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsDiffraction-ID% possible all
1.5-1.5310.50.156199.8
1.53-1.5511.80.147199.8
1.55-1.5812.20.13199.9
1.58-1.62120.1191100
1.62-1.6511.80.1091100
1.65-1.6912.40.0951100
1.69-1.73120.0881100
1.73-1.7811.90.0831100
1.78-1.8312.50.0771100
1.83-1.8911.90.071100
1.89-1.9612.10.0651100
1.96-2.0412.20.0611100
2.04-2.1311.80.0561100
2.13-2.2412.40.0551100
2.24-2.3811.60.0541100
2.38-2.5612.60.0511100
2.56-2.8211.90.051100
2.82-3.2311.90.049199.8
3.23-4.0711.50.0491100
4.07-5010.70.052199.9

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation1.5 Å46.66 Å
Translation1.5 Å46.66 Å

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Processing

Software
NameVersionClassification
REFMAC5.8.0131refinement
HKL-2000data scaling
PHASER2.5.7phasing
PDB_EXTRACT3.2data extraction
HKL-2000data reduction
Coot0.7.1model building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 3BDC

3bdc


Resolution: 1.5→46.66 Å / Cor.coef. Fo:Fc: 0.963 / Cor.coef. Fo:Fc free: 0.955 / SU B: 1.02 / SU ML: 0.04 / SU R Cruickshank DPI: 0.0728 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.073 / ESU R Free: 0.074 / Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflectionSelection details
Rfree0.2034 1174 4.9 %RANDOM
Rwork0.1761 ---
obs0.1775 22572 99.92 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 75.62 Å2 / Biso mean: 19.117 Å2 / Biso min: 8.47 Å2
Baniso -1Baniso -2Baniso -3
1--0.18 Å20 Å2-0 Å2
2---0.71 Å20 Å2
3---0.9 Å2
Refinement stepCycle: final / Resolution: 1.5→46.66 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1037 0 51 88 1176
Biso mean--15.96 26.94 -
Num. residues----129
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0160.021233
X-RAY DIFFRACTIONr_angle_refined_deg1.6742.0291688
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.2545164
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.36825.18554
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.57115257
X-RAY DIFFRACTIONr_dihedral_angle_4_deg12.815155
X-RAY DIFFRACTIONr_chiral_restr0.1240.2180
X-RAY DIFFRACTIONr_gen_planes_refined0.0160.021900
X-RAY DIFFRACTIONr_mcbond_it1.9711.548553
X-RAY DIFFRACTIONr_mcangle_it2.7712.331699
X-RAY DIFFRACTIONr_scbond_it3.6271.971680
LS refinement shellResolution: 1.5→1.54 Å
RfactorNum. reflection% reflection
Rfree0.193 85 -
Rwork0.171 1648 -
obs--99.2 %

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