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- PDB-4qf4: Crystal structure of Staphylococcal nuclease variant V23M at cryo... -

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Basic information

Entry
Database: PDB / ID: 4qf4
TitleCrystal structure of Staphylococcal nuclease variant V23M at cryogenic temperature
ComponentsThermonuclease
KeywordsHYDROLASE / Staphylococcal nuclease / pdtp / cavity / pressure
Function / homology
Function and homology information


3' overhang single-stranded DNA endodeoxyribonuclease activity / 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
PHOSPHATE ION / THYMIDINE-3',5'-DIPHOSPHATE / Thermonuclease
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.8 Å
AuthorsCaro, J.A. / 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
DepositionMay 19, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 4, 2014Provider: repository / Type: Initial release
Revision 1.1Nov 22, 2017Group: Refinement description / Category: software
Item: _software.classification / _software.contact_author ..._software.classification / _software.contact_author / _software.contact_author_email / _software.date / _software.language / _software.location / _software.name / _software.type / _software.version
Revision 1.2Sep 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_asym_id / _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.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_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_asym_id / _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_asym_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
B: Thermonuclease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,8258
Polymers33,7512
Non-polymers1,0746
Water2,036113
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Thermonuclease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)17,4134
Polymers16,8751
Non-polymers5373
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
3
A: Thermonuclease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)17,4134
Polymers16,8751
Non-polymers5373
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)67.530, 67.530, 62.817
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number76
Space group name H-MP41

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Components

#1: Protein Thermonuclease / TNase / Micrococcal nuclease / Staphylococcal nuclease / Nuclease B / Nuclease A


Mass: 16875.395 Da / Num. of mol.: 2 / Mutation: V23M
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-THP / THYMIDINE-3',5'-DIPHOSPHATE


Type: DNA linking / Mass: 402.188 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N2O11P2
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#4: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 113 / 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: 2.12 Å3/Da / Density % sol: 42.03 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 6
Details: 32% 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: 0.979 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Sep 25, 2013
RadiationMonochromator: Monochromator: Double silicon(111) crystal monochromator with cryogenically- cooled first crystal and sagittally- bent second crystal horizontally- focusing at 3.3:1 demagnification. ...Monochromator: Monochromator: Double silicon(111) crystal monochromator with cryogenically- cooled first crystal and sagittally- bent second crystal horizontally- focusing at 3.3:1 demagnification. Mirror: Meridionally- bent fused silica mirror with palladium and uncoated stripes vertically- focusing at 6.6:1 demagnification
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 1.8→50 Å / Num. all: 26669 / Num. obs: 26669 / % possible obs: 99.7 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 12.1 % / Biso Wilson estimate: 36.6 Å2 / Rmerge(I) obs: 0.073 / Χ2: 2.301 / Net I/σ(I): 9.6
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique allΧ2% possible all
1.8-1.8312.70.316913390.914100
1.83-1.8612.40.313070.974100
1.86-1.911.90.27113371.117100
1.9-1.9412.20.23313251.308100
1.94-1.9812.70.19713341.425100
1.98-2.0312.40.17113351.47100
2.03-2.0811.80.15713091.703100
2.08-2.1312.70.1413531.937100
2.13-2.212.70.13713402.146100
2.2-2.2712.50.12613342.284100
2.27-2.3511.80.11413352.315100
2.35-2.4412.70.10413122.519100
2.44-2.5512.30.09613242.651100
2.55-2.6912.10.0913262.913100
2.69-2.8612.60.08113483100
2.86-3.0811.70.07313393.251100
3.08-3.3911.60.06613393.44599.7
3.39-3.8810.70.06113283.74198.4
3.88-4.8810.50.05713253.8297.3
4.88-5011.50.0613803.69699.5

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation2.5 Å38.01 Å
Translation2.5 Å38.01 Å

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
PHASER2.3.0phasing
REFMAC5.6.0117refinement
PDB_EXTRACT3.14data extraction
CBASSdata collection
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1SNC

1snc


Resolution: 1.8→38 Å / Cor.coef. Fo:Fc: 0.969 / Cor.coef. Fo:Fc free: 0.961 / WRfactor Rfree: 0.2647 / WRfactor Rwork: 0.2275 / FOM work R set: 0.7032 / SU B: 6.911 / SU ML: 0.11 / SU R Cruickshank DPI: 0.148 / SU Rfree: 0.1346 / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / ESU R: 0.148 / ESU R Free: 0.135 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2395 1329 5.1 %RANDOM
Rwork0.2082 ---
all0.2098 26206 --
obs0.2098 26206 99.73 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 81.42 Å2 / Biso mean: 35.298 Å2 / Biso min: 17.24 Å2
Baniso -1Baniso -2Baniso -3
1-1.09 Å20 Å20 Å2
2--1.09 Å20 Å2
3----2.18 Å2
Refinement stepCycle: LAST / Resolution: 1.8→38 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2122 0 62 113 2297
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0180.022270
X-RAY DIFFRACTIONr_angle_refined_deg1.9052.0093067
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.455276
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.62924.68194
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.74315449
X-RAY DIFFRACTIONr_dihedral_angle_4_deg14.3061510
X-RAY DIFFRACTIONr_chiral_restr0.1280.2331
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.0211628
LS refinement shellResolution: 1.8→1.847 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.355 93 -
Rwork0.312 1781 -
all-1874 -
obs-1874 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)
14.33780.3269-1.53773.9849-0.52175.1247-0.15020.2526-0.39890.2240.382-0.01350.2978-0.6098-0.23180.0569-0.0428-0.00610.2299-0.01710.07879.38174.285215.8893
22.22950.4157-1.46972.1247-2.34874.8136-0.04230.19920.0540.10880.39440.1157-0.3218-0.7033-0.35210.09430.02510.01110.2519-0.00970.0719.740513.057113.2154
31.55310.2638-1.16694.308-3.27617.75010.14310.1650.10760.50850.0486-0.2558-1.29220.0209-0.19170.2433-0.02570.0360.1082-0.02320.059717.617822.16929.5111
44.15030.0040.79954.07211.03374.4064-0.16080.28650.3640.17770.3887-0.0071-0.23380.5002-0.22790.0568-0.04730.00550.18170.03140.075524.037429.1366-16.062
52.24450.28541.29622.20452.02844.3664-0.04160.2341-0.03590.02740.3769-0.12690.20310.5879-0.33530.0745-0.0051-0.01440.23740.01330.0824.31420.88-18.1192
61.67460.46191.74694.40954.10277.37480.17760.2084-0.14540.46330.04770.27291.2102-0.0416-0.22540.2576-0.0474-0.06050.14350.02740.098116.437311.3048-21.9061
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A7 - 32
2X-RAY DIFFRACTION2A33 - 96
3X-RAY DIFFRACTION3A97 - 141
4X-RAY DIFFRACTION4B7 - 30
5X-RAY DIFFRACTION5B31 - 96
6X-RAY DIFFRACTION6B97 - 141

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