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Basic information

Entry
Database: PDB / ID: 3owv
TitleStructural insights into catalytic and substrate binding mechanisms of the strategic EndA nuclease from Streptococcus pneumoniae
ComponentsDNA-entry nuclease
KeywordsHYDROLASE / sequence nonspecific endonuclease
Function / homology
Function and homology information


Hydrolases; Acting on ester bonds; Endoribonucleases that are active with either ribo- or deoxyribonucleic acids and produce 5'-phosphomonoesters / establishment of competence for transformation / endonuclease activity / nucleic acid binding / membrane => GO:0016020 / metal ion binding / plasma membrane
Similarity search - Function
DNA/RNA non-specific endonuclease, active site / DNA/RNA non-specific endonucleases active site. / Extracellular Endonuclease; Chain A / Extracellular Endonuclease, subunit A / DNA/RNA non-specific endonuclease / DNA/RNA non-specific endonuclease / DNA/RNA non-specific endonuclease / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Biological speciesStreptococcus pneumoniae (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.75 Å
AuthorsMoon, A.F. / Midon, M. / Meiss, G. / Pingoud, A.M. / London, R.E. / Pedersen, L.C.
CitationJournal: Nucleic Acids Res. / Year: 2011
Title: Structural insights into catalytic and substrate binding mechanisms of the strategic EndA nuclease from Streptococcus pneumoniae.
Authors: Moon, A.F. / Midon, M. / Meiss, G. / Pingoud, A. / London, R.E. / Pedersen, L.C.
History
DepositionSep 20, 2010Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 22, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Nov 8, 2017Group: Refinement description / Category: software / Item: _software.name
Revision 1.3Feb 21, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / 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_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
Revision 1.4Apr 3, 2024Group: Refinement description / Category: pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA-entry nuclease
B: DNA-entry nuclease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,7859
Polymers53,5592
Non-polymers2267
Water8,917495
1
A: DNA-entry nuclease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,8754
Polymers26,7801
Non-polymers953
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: DNA-entry nuclease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,9105
Polymers26,7801
Non-polymers1314
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)67.963, 75.931, 90.784
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein DNA-entry nuclease / Competence-specific nuclease


Mass: 26779.693 Da / Num. of mol.: 2 / Fragment: UNP residues 31 to 274 / Mutation: H160G
Source method: isolated from a genetically manipulated source
Details: N-terminal His6x-GST fusion with a TEV protease cleavage site immediately preceding the EndA sequence
Source: (gene. exp.) Streptococcus pneumoniae (bacteria) / Strain: TIGR4 / Gene: endA, SP_1964 / Plasmid: pET30M / Production host: Escherichia coli (E. coli)
References: UniProt: P0A3S3, Hydrolases; Acting on ester bonds; Endoribonucleases that are active with either ribo- or deoxyribonucleic acids and produce 5'-phosphomonoesters
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#3: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Cl
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 495 / 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: 2.19 Å3/Da / Density % sol: 43.72 %
Crystal growTemperature: 297 K / Method: vapor diffusion, sitting drop / pH: 7
Details: Crystallization solution contained 0.1M Tris pH 7, 17% PEG8000, and 0.2M MgCl2. The crystal was transferred to a hanging drop containing 0.1M Tris pH 7, 75mM MaCl, 0.2M MgCl2, and 20% ...Details: Crystallization solution contained 0.1M Tris pH 7, 17% PEG8000, and 0.2M MgCl2. The crystal was transferred to a hanging drop containing 0.1M Tris pH 7, 75mM MaCl, 0.2M MgCl2, and 20% PEG800, which was then placed over a vapor diffusion chamber containing a sitting drop bridge with 25% glutaraldehyde. The crystal was cross-linked for 8 minutes at room temperature, then transferred to a soaking solution containing 0.1M Tris pH 7, 75mM NaCl, 0.2M MgCl2,20% PEG8000, 11.6mM trimethyl lead acetate, and 9.63mM triethyl lead acetate for 43 hrs at room temperature. The soaked crystal was then transferred to a cryoprotectant (0.1M Tris pH 7, 75mM NaCl, 0.2M MgCl2, 20% PEG8000, 15% ethylene glycol, 11.9mM trimethyl lead acetate, and 13.75mM triethyl lead acetate) in four steps, then flash frozen in liquid nitrogen. , VAPOR DIFFUSION, SITTING DROP, temperature 297K

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Data collection

DiffractionMean temperature: 93 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-ID / Wavelength: 0.94 Å
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Dec 10, 2009
RadiationMonochromator: double crystal monochrometer Si-220 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.94 Å / Relative weight: 1
ReflectionResolution: 1.75→50 Å / Num. all: 46402 / Num. obs: 46402 / % possible obs: 96.8 % / Observed criterion σ(F): -3 / Observed criterion σ(I): -3 / Redundancy: 13.6 % / Biso Wilson estimate: 17.8 Å2 / Rsym value: 0.097 / Net I/σ(I): 26.5
Reflection shellResolution: 1.75→1.78 Å / Redundancy: 7.2 % / Mean I/σ(I) obs: 3.56 / Rsym value: 0.371 / % possible all: 68.7

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Processing

Software
NameVersionClassification
MAR345data collection
AutoBuildmodel building
CNS1.2refinement
HKL-2000data reduction
HKL-2000data scaling
AutoBuildphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: incomplete manually built model of EndA from a low resolution incompletely refined data set from an MBP-EndA fusion protein

Resolution: 1.75→37.97 Å / Rfactor Rfree error: 0.005 / Data cutoff high absF: 91449.58 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber / Details: BULK SOLVENT MODEL USED
RfactorNum. reflection% reflectionSelection details
Rfree0.217 1962 4.3 %RANDOM
Rwork0.195 ---
obs0.195 45460 94.7 %-
all-45460 --
Solvent computationSolvent model: FLAT MODEL / Bsol: 50.0725 Å2 / ksol: 0.35 e/Å3
Displacement parametersBiso mean: 23.3 Å2
Baniso -1Baniso -2Baniso -3
1--5.55 Å20 Å2-0 Å2
2---2.71 Å2-0 Å2
3---8.27 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.23 Å0.19 Å
Luzzati d res low-5 Å
Luzzati sigma a0.18 Å0.15 Å
Refinement stepCycle: LAST / Resolution: 1.75→37.97 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3337 0 7 495 3839
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.004
X-RAY DIFFRACTIONc_bond_d_na
X-RAY DIFFRACTIONc_bond_d_prot
X-RAY DIFFRACTIONc_angle_d
X-RAY DIFFRACTIONc_angle_d_na
X-RAY DIFFRACTIONc_angle_d_prot
X-RAY DIFFRACTIONc_angle_deg1.2
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d25
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d0.68
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it1.211.5
X-RAY DIFFRACTIONc_mcangle_it1.832
X-RAY DIFFRACTIONc_scbond_it3.832
X-RAY DIFFRACTIONc_scangle_it4.682.5
Refine LS restraints NCSNCS model details: NONE
LS refinement shellResolution: 1.75→1.86 Å / Rfactor Rfree error: 0.017 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.28 256 4.4 %
Rwork0.248 5593 -
obs--74.1 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1protein_rep.paramprotein.top
X-RAY DIFFRACTION2ion.paramprotein.link
X-RAY DIFFRACTION3water_rep.paramwater.top
X-RAY DIFFRACTION4ion.top

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