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- PDB-4du9: Crystal structure of Staphylococcal nuclease variant Delta+PHS I9... -

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Basic information

Entry
Database: PDB / ID: 4du9
TitleCrystal structure of Staphylococcal nuclease variant Delta+PHS I92A/V74A at cryogenic temperature
ComponentsThermonuclease
KeywordsHYDROLASE / Staphylococcal nuclease / hyperstable variant / pdtp / cavity / pressure
Function / homology
Function and homology information


micrococcal nuclease / : / nucleic acid binding / extracellular region / metal ion binding / membrane
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: 1.63 Å
AuthorsCaro, J.A. / Clark, I. / Schlessman, J.L. / Heroux, A. / Garcia-Moreno E., B.
CitationJournal: To be Published
Title: Pressure effects in proteins
Authors: Caro, J.A. / Schlessman, J.L. / Garcia-Moreno E., B.
History
DepositionFeb 21, 2012Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 7, 2012Provider: repository / Type: Initial release
Revision 1.1Sep 30, 2015Group: Data collection
Revision 1.2Sep 13, 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)16,5163
Polymers16,0731
Non-polymers4422
Water1,71195
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)31.050, 60.517, 38.601
Angle α, β, γ (deg.)90.000, 93.850, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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


Mass: 16073.328 Da / Num. of mol.: 1 / Fragment: Nuclease A (UNP residues 83-231) / Mutation: 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.: 95 / 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: 2.25 Å3/Da / Density % sol: 45.36 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 6
Details: 20% MPD, 25 mM potassium phosphate, calcium chloride, pdTp, pH 6.0, 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
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1 Å / Relative weight: 1
ReflectionResolution: 1.63→50 Å / Num. all: 17867 / Num. obs: 17867 / % possible obs: 99.7 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 12.1 % / Biso Wilson estimate: 33.2 Å2 / Rmerge(I) obs: 0.051 / Χ2: 1.636 / Net I/σ(I): 16.8
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique allΧ2% possible all
1.63-1.669.80.28511.38821.07897.4
1.66-1.6910.30.2698591.06298.6
1.69-1.7210.70.2539061.012100
1.72-1.7611.60.2218971.054100
1.76-1.79120.1938771.142100
1.79-1.8411.80.1679191.123100
1.84-1.8813.10.1568471.19100
1.88-1.9312.90.1329321.286100
1.93-1.9912.60.1118661.386100
1.99-2.0512.10.0968861.464100
2.05-2.1312.90.0868891.567100
2.13-2.2112.30.0689041.558100
2.21-2.3113.20.0688911.676100
2.31-2.43130.0598961.708100
2.43-2.5912.50.0529061.88100
2.59-2.79130.058832.047100
2.79-3.0712.80.0459012.255100
3.07-3.5112.20.0439032.513100
3.51-4.4211.30.0439042.68299
4.42-5011.70.0459192.72699.1

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation2.5 Å38.51 Å
Translation2.5 Å38.51 Å

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Processing

Software
NameVersionClassificationNB
SCALEPACKdata scaling
PHASER2.3.0phasing
REFMACrefinement
PDB_EXTRACT3.1data extraction
CBASSdata collection
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 3BDC

3bdc


Resolution: 1.63→38.51 Å / Cor.coef. Fo:Fc: 0.97 / Cor.coef. Fo:Fc free: 0.959 / WRfactor Rfree: 0.2315 / WRfactor Rwork: 0.1967 / Occupancy max: 1 / Occupancy min: 0.3 / FOM work R set: 0.8614 / SU B: 3.137 / SU ML: 0.063 / SU R Cruickshank DPI: 0.0911 / SU Rfree: 0.0909 / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / ESU R: 0.091 / ESU R Free: 0.091 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.208 914 5.1 %RANDOM
Rwork0.1773 ---
all0.1789 17849 --
obs0.1789 17849 99.4 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 82.41 Å2 / Biso mean: 34.6367 Å2 / Biso min: 18.1 Å2
Baniso -1Baniso -2Baniso -3
1--2.18 Å20 Å20.73 Å2
2---1.88 Å20 Å2
3---4.16 Å2
Refinement stepCycle: LAST / Resolution: 1.63→38.51 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1028 0 26 95 1149
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0150.021102
X-RAY DIFFRACTIONr_angle_refined_deg1.86521490
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.495137
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.46625.250
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.49315213
X-RAY DIFFRACTIONr_dihedral_angle_4_deg11.429155
X-RAY DIFFRACTIONr_chiral_restr0.1380.2160
X-RAY DIFFRACTIONr_gen_planes_refined0.0150.021809
LS refinement shellResolution: 1.63→1.669 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.241 58 -
Rwork0.24 1136 -
all-1194 -
obs-1194 93.5 %
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)
13.8512-1.0367-0.40263.54741.04770.8011-0.1207-0.0099-0.57520.04360.07350.01480.08580.03270.04720.10680.02050.00650.05570.00650.178414.4435-3.83422.5361
25.86220.0031-0.22593.9946-0.25091.8392-0.1358-0.88150.1230.56290.08470.15080.14730.19030.05110.15890.04070.0150.1986-0.0070.050411.10495.088913.3414
35.3685-0.1554-0.208410.8782-5.23918.477-0.07140.8455-0.6473-0.3921-0.03480.26150.0643-0.16470.10630.1049-0.0165-0.02090.1709-0.06970.16037.9357-1.2184-3.4973
45.9929-0.7267-0.65393.26930.27141.3111-0.0850.24270.2046-0.04290.08390.18260.0655-0.05970.00110.0871-0.0125-0.01860.08650.01690.13488.72596.50462.0404
58.1844-2.3918-2.7363.73852.33435.4803-0.02830.34420.4943-0.084-0.00870.5356-0.0914-0.14210.0370.0313-0.0068-0.02490.080.04170.2885-0.561211.68111.5759
613.17712.00694.61886.16041.88927.8456-0.2547-1.27591.43710.68580.2010.5429-0.2068-0.47220.05370.17870.08170.06770.2233-0.17260.343-0.429516.220814.9125
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A7 - 35
2X-RAY DIFFRACTION2A36 - 70
3X-RAY DIFFRACTION3A71 - 80
4X-RAY DIFFRACTION4A81 - 119
5X-RAY DIFFRACTION5A120 - 130
6X-RAY DIFFRACTION6A131 - 141

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