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- PDB-4k2k: Crystal structure of Staphylococcal nuclease variant Delta+PHS L2... -

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Basic information

Entry
Database: PDB / ID: 4k2k
TitleCrystal structure of Staphylococcal nuclease variant Delta+PHS L25A/L36A/I92A at cryogenic temperature
ComponentsThermonuclease
KeywordsHYDROLASE / Staphylococcal nuclease / hyperstable variant / pdtp / cavity / pressure
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: 1.65 Å
AuthorsCaro, J.A. / Schlessman, J.L. / Heroux, A. / Garcia-Moreno E., B.
CitationJournal: To be Published
Title: Pressure effects on proteins
Authors: Caro, J.A. / Schlessman, J.L. / Garcia-Moreno E., B.
History
DepositionApr 9, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 24, 2013Provider: repository / Type: Initial release
Revision 1.1Sep 20, 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 / struct_ref_seq_dif / struct_site
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_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_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)16,4593
Polymers16,0171
Non-polymers4422
Water2,360131
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)31.155, 60.004, 38.879
Angle α, β, γ (deg.)90.000, 95.060, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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


Mass: 16017.223 Da / Num. of mol.: 1 / Fragment: Nuclease A (UNP residues 83-231)
Mutation: L25A,L36A,G50F,V51N,I92A,P117G,H124L,S128A,del(44-49)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: nuc / Plasmid: pET24a+ / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / 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.: 131 / 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: 2.26 Å3/Da / Density % sol: 45.57 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 9
Details: 20% MPD, 25 mM potassium phosphate, calcium chloride, pdTp, pH 9.0, VAPOR DIFFUSION, HANGING DROP, temperature 277K

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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: Sep 25, 2011
Details: Meridionally-bent fused silica mirror with palladium and uncoated stripes vertically-focusing at 6.6:1 demagnification
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: 1.65→50 Å / Num. all: 17188 / Num. obs: 17188 / % possible obs: 99.9 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 6.4 % / Biso Wilson estimate: 28.5 Å2 / Rmerge(I) obs: 0.042 / Χ2: 1.467 / Net I/σ(I): 16.6
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allΧ2Diffraction-ID% possible all
1.65-1.685.80.1358360.979199.4
1.68-1.715.80.1248441.072199.8
1.71-1.746.40.1218821.011100
1.74-1.786.50.1028461.0571100
1.78-1.826.40.0948541.1441100
1.82-1.8660.0878581.1961100
1.86-1.96.40.088461.2531100
1.9-1.966.40.0718701.3061100
1.96-2.016.20.0688551.4041100
2.01-2.086.70.0618631.4591100
2.08-2.156.60.0588481.5571100
2.15-2.246.20.0518551.6091100
2.24-2.346.50.0518581.6541100
2.34-2.466.30.0478641.6541100
2.46-2.626.60.0418731.5621100
2.62-2.826.50.0428621.7561100
2.82-3.116.40.0388631.891199.8
3.11-3.556.40.0378561.985199.9
3.55-4.486.40.0368661.885199.2
4.48-506.40.0388891.768199.2

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation2.5 Å32.54 Å
Translation2.5 Å32.54 Å

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Processing

Software
NameVersionClassificationNB
SCALEPACKdata scaling
PHASER2.3.0phasing
REFMACrefinement
PDB_EXTRACT3.11data extraction
CBASSdata collection
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 3BDC

3bdc


Resolution: 1.65→38.73 Å / Cor.coef. Fo:Fc: 0.966 / Cor.coef. Fo:Fc free: 0.953 / WRfactor Rfree: 0.242 / WRfactor Rwork: 0.1967 / Occupancy max: 1 / Occupancy min: 0.4 / FOM work R set: 0.8473 / SU B: 1.966 / SU ML: 0.067 / SU R Cruickshank DPI: 0.0978 / SU Rfree: 0.0999 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.098 / ESU R Free: 0.1 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2175 869 5.1 %RANDOM
Rwork0.1785 ---
obs0.1803 17170 99.72 %-
all-17170 --
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 78.09 Å2 / Biso mean: 25.1209 Å2 / Biso min: 12.13 Å2
Baniso -1Baniso -2Baniso -3
1--1.4 Å20 Å20.7 Å2
2---1.91 Å20 Å2
3---3.43 Å2
Refinement stepCycle: LAST / Resolution: 1.65→38.73 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1024 0 26 131 1181
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.020.021111
X-RAY DIFFRACTIONr_angle_refined_deg1.9821.9941506
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.2925140
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.42824.89849
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.06215214
X-RAY DIFFRACTIONr_dihedral_angle_4_deg11.792155
X-RAY DIFFRACTIONr_chiral_restr0.1410.2163
X-RAY DIFFRACTIONr_gen_planes_refined0.0140.021815
LS refinement shellResolution: 1.65→1.693 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.282 55 -
Rwork0.255 1148 -
all-1203 -
obs-1203 97.49 %

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