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- PDB-4rkl: Crystal structure of Staphylococcal nuclease variant Delta+PHS V2... -

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Basic information

Entry
Database: PDB / ID: 4rkl
TitleCrystal structure of Staphylococcal nuclease variant Delta+PHS V23T/V66T 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 / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.66 Å
AuthorsCaro, J.A. / Mercedes, R. / Schlessman, J.L. / Garcia-Moreno E., B.
CitationJournal: to be published
Title: Cavities in proteins
Authors: Caro, J.A. / Schlessman, J.L. / Garcia-Moreno E., B.
History
DepositionOct 13, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 26, 2014Provider: repository / Type: Initial release
Revision 1.1Jun 24, 2015Group: Other
Revision 1.2Mar 7, 2018Group: Data collection / Category: diffrn_source / Item: _diffrn_source.source
Revision 1.3Sep 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)16,5903
Polymers16,1471
Non-polymers4422
Water2,054114
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)31.153, 60.402, 38.472
Angle α, β, γ (deg.)90.000, 94.480, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Thermonuclease


Mass: 16147.410 Da / Num. of mol.: 1 / Fragment: UNP residues 83-232 / Mutation: V23T, V60T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Plasmid: pET24a+ / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P00644, micrococcal nuclease
#2: Chemical ChemComp-THP / THYMIDINE-3',5'-DIPHOSPHATE


Type: DNA linking / Mass: 402.188 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N2O11P2
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#4: Water ChemComp-HOH / 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: 2.23 Å3/Da / Density % sol: 44.96 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 20% MPD, 25 mM Potassium Phosphate, Calcium Chloride, pdTp, pH 8.0, vapor diffusion, hanging drop, temperature 277K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: BRUKER AXS MICROSTAR / Wavelength: 1.54 Å
DetectorType: APEX II CCD / Detector: CCD / Date: Jan 25, 2013 / Details: Multilayer
RadiationMonochromator: GE111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 1.66→50 Å / Num. all: 16877 / Num. obs: 16877 / % possible obs: 100 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 20.39 % / Biso Wilson estimate: 25.4 Å2 / Rmerge(I) obs: 0.015 / Net I/σ(I): 6.56
Reflection shellResolution: 1.66→1.76 Å / Redundancy: 5.83 % / Rmerge(I) obs: 0.1175 / Mean I/σ(I) obs: 6.56 / Num. unique all: 2701 / % possible all: 100

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
XPREP2013/1 for Windowsdata reduction
PHASERphasing
REFMAC5.6.0117refinement
PDB_EXTRACT3.15data extraction
APEXdata collection
APEXdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3BDC

3bdc


Resolution: 1.66→38.35 Å / Cor.coef. Fo:Fc: 0.959 / Cor.coef. Fo:Fc free: 0.93 / WRfactor Rfree: 0.2307 / WRfactor Rwork: 0.1858 / FOM work R set: 0.809 / SU B: 3.709 / SU ML: 0.073 / SU R Cruickshank DPI: 0.1037 / SU Rfree: 0.1078 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.104 / ESU R Free: 0.108 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2322 848 5 %RANDOM
Rwork0.1867 ---
all0.189 15965 --
obs0.189 15965 99.71 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 60.15 Å2 / Biso mean: 19.225 Å2 / Biso min: 8.64 Å2
Baniso -1Baniso -2Baniso -3
1--0.83 Å20 Å20.49 Å2
2---1.21 Å20 Å2
3---2.12 Å2
Refinement stepCycle: LAST / Resolution: 1.66→38.35 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1033 0 26 114 1173
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0190.021113
X-RAY DIFFRACTIONr_angle_refined_deg1.9752.0051506
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.5825139
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.78825.10249
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.4615220
X-RAY DIFFRACTIONr_dihedral_angle_4_deg7.15155
X-RAY DIFFRACTIONr_chiral_restr0.1580.2164
X-RAY DIFFRACTIONr_gen_planes_refined0.0140.021809
LS refinement shellResolution: 1.66→1.703 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.288 59 -
Rwork0.223 1144 -
all-1203 -
obs-1203 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)
11.1771-0.5645-0.0653.35830.65950.5085-0.0315-0.0366-0.09180.11560.0523-0.02960.07990.0619-0.02080.08590.0104-0.00170.08520.00920.077113.8668-0.86853.2939
24.07632.76371.03619.07352.7642.7153-0.0809-0.5839-0.05650.5236-0.01950.0770.1430.02150.10030.18340.04080.01020.15610.030.009610.51582.440415.3692
32.2093-0.4440.06182.5240.69280.2779-0.05510.0607-0.10810.04660.03750.11810.051-0.03220.01760.096-0.0005-0.00580.09480.01360.08928.42242.67681.8625
44.6248-0.6055-0.81472.87710.31771.4252-0.00060.16310.1834-0.1483-0.00420.2363-0.0644-0.0250.00480.1025-0.0032-0.02150.09370.0130.14162.91712.44680.388
519.1737-0.5542.15044.54571.88389.9509-0.2581-0.78410.82260.59450.02570.5878-0.3219-0.25910.23240.19580.04120.08270.1116-0.06330.13640.080716.207215.612
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A7 - 43
2X-RAY DIFFRACTION2A50 - 71
3X-RAY DIFFRACTION3A72 - 109
4X-RAY DIFFRACTION4A110 - 131
5X-RAY DIFFRACTION5A132 - 141

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