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- PDB-5cv5: Crystal structure of Staphylococcal nuclease variant Delta+PHS K6... -

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Basic information

Entry
Database: PDB / ID: 5cv5
TitleCrystal structure of Staphylococcal nuclease variant Delta+PHS K64G/V66K/E67G at cryogenic temperature
ComponentsThermonucleaseMicrococcal nuclease
KeywordsHYDROLASE / nuclease / hyperstable / pdTp / ionizable group
Function / homology
Function and homology information


endonuclease activity, active with either ribo- or deoxyribonucleic acids and producing 3'-phosphomonoesters / micrococcal nuclease / nucleic acid binding / extracellular region / membrane / metal ion binding
Similarity search - Function
Thermonuclease family signature 1. / OB fold (Dihydrolipoamide Acetyltransferase, E2P) - #90 / 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) ...Thermonuclease family signature 1. / OB fold (Dihydrolipoamide Acetyltransferase, E2P) - #90 / 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: 1.8 Å
AuthorsOrtega Quintanilla, G. / Robinson, A.C. / Schlessman, J.L. / Garcia-Moreno E., B.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH-GM 061597 United States
CitationJournal: To be Published
Title: Crystal structure of Staphylococcal nuclease variant Delta+PHS K64G/V66K/E67G at cryogenic temperature
Authors: Ortega Quintanilla, G. / Robinson, A.C. / Schlessman, J.L. / Garcia-Moreno E., B.
History
DepositionJul 25, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 12, 2015Provider: 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 1.2Dec 25, 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_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.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.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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Thermonuclease
B: Thermonuclease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,9436
Polymers32,0592
Non-polymers8854
Water2,522140
1
A: Thermonuclease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,4723
Polymers16,0291
Non-polymers4422
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Thermonuclease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,4723
Polymers16,0291
Non-polymers4422
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)30.831, 60.549, 77.901
Angle α, β, γ (deg.)90.000, 93.150, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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


Mass: 16029.320 Da / Num. of mol.: 2
Mutation: G50F/V51N/K64G/V66K/E67G/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.: 2 / Source method: obtained synthetically / Formula: Ca
#3: Chemical ChemComp-THP / THYMIDINE-3',5'-DIPHOSPHATE / Thymidine diphosphate


Type: DNA linking / Mass: 402.188 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N2O11P2
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 140 / 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: 45.69 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 6
Details: 25% MPD, 25 mM potassium phosphate, calcium chloride, pdTp

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Data collection

DiffractionMean temperature: 110 K
Diffraction sourceSource: SEALED TUBE / Type: OTHER / Wavelength: 1.5418 Å
DetectorType: APEX II CCD / Detector: CCD / Date: Jul 18, 2014 / Details: multi-layer optics
RadiationMonochromator: Ge(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.8→50 Å / Num. all: 26668 / Num. obs: 26668 / % possible obs: 100 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 18.84 % / Biso Wilson estimate: 25.2 Å2 / Rmerge(I) obs: 0.0261 / Net I/σ(I): 29.29 / Num. measured all: 510698
Reflection shell

Diffraction-ID: 1 / Rejects: 0

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. possibleNum. unique obsNet I/σ(I) obs% possible all
1.8-1.847.330.23364.24129112914.24100
1.84-1.8812.640.38155415546100
1.88-1.9214.350.323146314637.31100
1.92-1.9614.740.298135113517.98100
1.96-2.0115.330.25150415049.07100
2.01-2.0615.910.211418141811.09100
2.06-2.1216.140.2221457145712.18100
2.12-2.1917.040.1661547154715.05100
2.19-2.2717.190.1641518151816.79100
2.27-2.3518.190.1351334133419.51100
2.35-2.4519.120.1161431143122.04100
2.45-2.5620.050.0981341134126.92100
2.56-2.720.90.0871371137131.39100
2.7-2.8722.190.0771344134435.86100
2.87-3.0923.50.0621349134945.97100
3.09-3.4125.040.0531370137057.83100
3.41-3.9126.190.051341134172.23100
3.91-4.9626.940.0391346134683.23100
4.96-60.5527.30.0371339133885.1299.9

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation1.8 Å32.72 Å
Translation1.8 Å32.72 Å

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Processing

Software
NameVersionClassification
APEX2008/2 for Windowsdata scaling
PHASER2.5.7phasing
REFMAC5.8.0124refinement
PDB_EXTRACT3.15data extraction
APEXdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 3BDC

3bdc


Resolution: 1.8→50 Å / Cor.coef. Fo:Fc: 0.949 / Cor.coef. Fo:Fc free: 0.932 / WRfactor Rfree: 0.2199 / WRfactor Rwork: 0.1776 / FOM work R set: 0.8518 / SU B: 4.92 / SU ML: 0.086 / SU R Cruickshank DPI: 0.1249 / SU Rfree: 0.1225 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.125 / ESU R Free: 0.122 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2253 1319 5 %RANDOM
Rwork0.1849 ---
obs0.1869 25309 99.91 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 60.71 Å2 / Biso mean: 21.809 Å2 / Biso min: 7.58 Å2
Baniso -1Baniso -2Baniso -3
1-0.09 Å20 Å2-0.07 Å2
2--0.04 Å20 Å2
3----0.12 Å2
Refinement stepCycle: final / Resolution: 1.8→50 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2024 0 52 140 2216
Biso mean--18.55 23.66 -
Num. residues----254
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0170.0192130
X-RAY DIFFRACTIONr_bond_other_d0.0060.022100
X-RAY DIFFRACTIONr_angle_refined_deg1.7922.0062871
X-RAY DIFFRACTIONr_angle_other_deg0.83834855
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.9845259
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.01724.72591
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.30115409
X-RAY DIFFRACTIONr_dihedral_angle_4_deg11.7071510
X-RAY DIFFRACTIONr_chiral_restr0.1150.2308
X-RAY DIFFRACTIONr_gen_planes_refined0.0140.022329
X-RAY DIFFRACTIONr_gen_planes_other0.0030.02457
X-RAY DIFFRACTIONr_mcbond_it1.3981.1441021
X-RAY DIFFRACTIONr_mcbond_other1.3831.1431020
X-RAY DIFFRACTIONr_mcangle_it1.9921.7031273
LS refinement shellResolution: 1.8→1.847 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.276 105 -
Rwork0.216 1838 -
all-1943 -
obs--99.95 %
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)
11.7226-0.4539-0.03941.32710.32081.0377-0.00690.0069-0.00540.02190.01510.03120.0398-0.0483-0.00820.0477-0.00610.00880.020.01490.04839.50093.70585.6388
21.99650.02520.2291.88810.51131.1612-0.0302-0.3843-0.09220.00490.06730.1252-0.05410.0022-0.0370.00360.00150.00290.09990.03550.025752.80812.124233.7171
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A7 - 141
2X-RAY DIFFRACTION2B7 - 141

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