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- PDB-4hmj: Crystal structure of Staphylococcal nuclease variant Delta+PHS L3... -

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Basic information

Entry
Database: PDB / ID: 4hmj
TitleCrystal structure of Staphylococcal nuclease variant Delta+PHS L36D 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 / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.35 Å
AuthorsRobinson, A.C. / Schlessman, J.L. / Heroux, A. / Garcia-Moreno E., B.
CitationJournal: To be Published
Title: Crystal structure of Staphylococcal nuclease variant Delta+PHS L36D at cryogenic temperature
Authors: Robinson, A.C. / Schlessman, J.L. / Heroux, A. / Garcia-Moreno E., B.
History
DepositionOct 18, 2012Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 31, 2012Provider: 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)16,5883
Polymers16,1451
Non-polymers4422
Water2,108117
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)31.168, 60.289, 38.342
Angle α, β, γ (deg.)90.000, 93.300, 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: 16145.393 Da / Num. of mol.: 1 / Fragment: Nuclease A (UNP residues 83-231) / Mutation: L36D/G50F/V51N/P117G/H124L/S128A/Del44-49
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-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 / Thymidine diphosphate


Type: DNA linking / Mass: 402.188 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N2O11P2
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 117 / 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: 44.78 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 20% MPD, 25 mM potassium phosphate, with calcium chloride and pdTp added to protein in a 3:2:1 ratio, pH 8, 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: Jul 17, 2012
Details: Meridionally-bent fused silica mirror with palladium and uncoated stripes vertically-focusing at 6.6:1 demagnification
RadiationMonochromator: double crystal Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 1.35→50 Å / Num. all: 30941 / Num. obs: 30941 / % possible obs: 99.8 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 6.6 % / Biso Wilson estimate: 23.2 Å2 / Rmerge(I) obs: 0.094 / Χ2: 2.966 / Net I/σ(I): 46.7
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allΧ2Diffraction-ID% possible all
1.35-1.376.50.30715661.0091100
1.37-1.46.50.27515201.051100
1.4-1.436.30.23715281.113199.9
1.43-1.456.70.21515611.241100
1.45-1.496.60.19815501.21100
1.49-1.526.20.17315151.3891100
1.52-1.566.80.16315481.4451100
1.56-1.66.70.1515371.6091100
1.6-1.656.60.13315711.8321100
1.65-1.76.40.12315201.9621100
1.7-1.766.80.11615452.0681100
1.76-1.836.60.10915912.4791100
1.83-1.926.70.10415272.9111100
1.92-2.026.90.115433.4841100
2.02-2.146.60.09515434.2371100
2.14-2.316.80.0915604.5351100
2.31-2.546.60.08715614.561199.9
2.54-2.916.90.08815345.087199.7
2.91-3.666.50.08615416.598198.2
3.66-506.50.09415809.112198.4

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation2.5 Å32.32 Å
Translation2.5 Å32.32 Å

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Processing

Software
NameVersionClassificationNB
SCALEPACKdata scaling
PHASER2.2.1phasing
REFMACrefinement
PDB_EXTRACT3.11data extraction
CBASSdata collection
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 3BDC

3bdc


Resolution: 1.35→32.32 Å / Cor.coef. Fo:Fc: 0.967 / Cor.coef. Fo:Fc free: 0.959 / WRfactor Rfree: 0.2095 / WRfactor Rwork: 0.1911 / Occupancy max: 1 / Occupancy min: 0.4 / FOM work R set: 0.8892 / SU B: 1.441 / SU ML: 0.031 / SU R Cruickshank DPI: 0.0552 / SU Rfree: 0.0561 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.055 / ESU R Free: 0.056 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.1998 1552 5 %RANDOM
Rwork0.1797 ---
all0.1808 29368 --
obs0.1808 29368 99.3 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso max: 64.08 Å2 / Biso mean: 20.986 Å2 / Biso min: 10.36 Å2
Baniso -1Baniso -2Baniso -3
1--0.08 Å20 Å20.02 Å2
2---0.04 Å20 Å2
3---0.13 Å2
Refinement stepCycle: LAST / Resolution: 1.35→32.32 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1033 0 26 117 1176
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.020.0221130
X-RAY DIFFRACTIONr_angle_refined_deg1.96821533
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.4555144
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.71125.09851
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.01415224
X-RAY DIFFRACTIONr_dihedral_angle_4_deg8.209155
X-RAY DIFFRACTIONr_chiral_restr0.1340.2165
X-RAY DIFFRACTIONr_gen_planes_refined0.0160.021831
X-RAY DIFFRACTIONr_mcbond_it1.3051.5663
X-RAY DIFFRACTIONr_mcangle_it2.02921071
X-RAY DIFFRACTIONr_scbond_it3.2673467
X-RAY DIFFRACTIONr_scangle_it4.6694.5454
LS refinement shellResolution: 1.35→1.385 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.227 101 -
Rwork0.241 2009 -
all-2110 -
obs-2110 93.03 %
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)
11.3214-0.4045-0.54242.5710.65930.378-0.0925-0.0421-0.17070.12240.09350.00140.06650.0802-0.00090.11720.0166-0.00610.0980.01220.085813.192-4.2413.372
21.4408-3.4712-0.87728.61471.92530.42450.04570.0684-0.0726-0.0897-0.0662-0.1496-0.0092-0.03270.02050.09560.01740.00370.0905-0.01820.115717.809-3.7990.048
31.9829-0.3141-0.55942.6983-0.17552.5814-0.0704-0.33850.19910.37490.0766-0.19-0.1230.273-0.00620.09310.0052-0.02950.1231-0.020.045113.50510.85710.584
43.26110.8804-0.08895.97852.32692.7969-0.1911-0.4828-0.3160.53810.1070.19760.26550.06890.08410.17880.04430.03060.130.07490.01368.767-0.29514.826
52.3474-0.0446-0.21673.8279-0.64672.26910.06020.2789-0.1212-0.1711-0.0780.0895-0.11570.06170.01780.1156-0.0034-0.00980.1131-0.01390.04769.097-0.3-4.269
62.59130.36450.24532.97750.24150.1948-0.01690.1378-0.1277-0.00420.0415-0.03050.030.0117-0.02460.11120.0034-0.00460.10930.00430.058912.1131.8720.061
71.6545-0.2601-0.28124.42120.33562.76-0.0925-0.19370.02910.2550.05690.24530.0646-0.06360.03560.0850.01710.02110.0960.01010.05393.3987.669.554
83.3295-0.4852-0.28122.38520.3670.25740.01540.26380.0615-0.2649-0.03230.0903-0.0727-0.02520.01690.08830.0004-0.01760.09340.02010.06273.65811.654-1.661
97.7662-0.59473.26794.4422-0.35557.7541-0.2326-0.50310.33620.47290.21760.2596-0.3677-0.29090.0150.12910.05360.04010.1025-0.04710.071-0.69415.84714.052
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A7 - 27
2X-RAY DIFFRACTION2A28 - 35
3X-RAY DIFFRACTION3A36 - 53
4X-RAY DIFFRACTION4A54 - 71
5X-RAY DIFFRACTION5A72 - 80
6X-RAY DIFFRACTION6A81 - 97
7X-RAY DIFFRACTION7A98 - 110
8X-RAY DIFFRACTION8A111 - 128
9X-RAY DIFFRACTION9A129 - 141

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