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- PDB-2ewf: Crystal structure of the site-specific DNA nickase N.BspD6I -

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Entry
Database: PDB / ID: 2ewf
TitleCrystal structure of the site-specific DNA nickase N.BspD6I
ComponentsNicking endonuclease N.BspD6I
KeywordsHYDROLASE / helix-turn-helix / beta-alpha-barrel
Function / homologyAlwI restriction endonuclease / Restriction endonuclease, type II, AlwI / endonuclease activity / Heterodimeric restriction endonuclease R.BspD6I large subunit / Nicking endonuclease N.BspD6I
Function and homology information
Biological speciesBacillus sp. (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 1.84 Å
AuthorsKachalova, G.S. / Bartunik, H.D. / Artyukh, R.I. / Rogulin, E.A. / Perevyazova, T.A. / Zheleznaya, L.A. / Matvienko, N.I.
Citation
Journal: J.Mol.Biol. / Year: 2008
Title: Structural analysis of the heterodimeric type IIS restriction endonuclease R.BspD6I acting as a complex between a monomeric site-specific nickase and a catalytic subunit.
Authors: Kachalova, G.S. / Rogulin, E.A. / Yunusova, A.K. / Artyukh, R.I. / Perevyazova, T.A. / Matvienko, N.I. / Zheleznaya, L.A. / Bartunik, H.D.
#1: Journal: Acta Crystallogr.,Sect.F / Year: 2005
Title: Crystallization and preliminary crystallographic analysis of the site-specific DNA nickase Nb.BspD6I
Authors: Kachalova, G.S. / Rogulin, E.A. / Artyukh, R.I. / Perevyazova, T.A. / Zheleznaya, L.A. / Matvienko, N.I. / Bartunik, H.D.
#2: Journal: Biochemistry Mosc. / Year: 2003
Title: Cloning and sequencing of the gene of site-specific nickase N.BspD6I
Authors: Perevyazova, T.A. / Rogulin, E.A. / Zheleznaya, L.A. / Matvienko, N.I.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Nov 3, 2005 / Release: Nov 21, 2006
RevisionDateData content typeGroupProviderType
1.0Nov 21, 2006Structure modelrepositoryInitial release
1.1May 1, 2008Structure modelVersion format compliance
1.2Jul 13, 2011Structure modelSource and taxonomy / Version format compliance

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Nicking endonuclease N.BspD6I
hetero molecules


Theoretical massNumber of molelcules
Total (without water)72,74114
Polymers71,7021
Non-polymers1,03913
Water11,295627
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
γ
α
β
Length a, b, c (Å)59.553, 92.562, 76.349
Angle α, β, γ (deg.)90.00, 111.88, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein/peptide Nicking endonuclease N.BspD6I


Mass: 71702.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus sp. (bacteria) / Strain: D6 / Plasmid: pET28b / Production host: Escherichia coli (E. coli) / Strain (production host): TOP10F / References: UniProt: Q8GCA3, UniProt: A3FEV7*PLUS
#2: Chemical
ChemComp-BR / BROMIDE ION


Mass: 79.904 Da / Num. of mol.: 13 / Source method: obtained synthetically / Formula: Br / Bromide
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 627 / Source method: isolated from a natural source / Formula: H2O / Water

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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: 2.72 Å3/Da / Density % sol: 54.82 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 7.2
Details: 0.04 M KH2PO4, 16% (w/v) PEG 8000, 20% (v/v) glycerol, pH 7.2, VAPOR DIFFUSION, SITTING DROP, temperature 298.0K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: MPG/DESY, HAMBURG / Beamline: BW6 / Wavelength: 0.9198, 0.9200, 1.05
DetectorType: MARRESEARCH / Detector: CCD / Date: Jul 25, 2005
RadiationMonochromator: graphite / Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
10.91981
20.921
31.051
ReflectionResolution: 1.84→20 Å / Num. obs: 67523 / % possible obs: 99.9 % / Observed criterion σ(F): 2 / Observed criterion σ(I): 2 / Rmerge(I) obs: 0.08 / Net I/σ(I): 12.6
Reflection shellResolution: 1.84→1.87 Å / % possible all: 100

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Processing

Software
NameClassification
SHELXmodel building
SHELXL-97refinement
DENZOdata reduction
SCALEPACKdata scaling
SHELXSphasing
RefinementMethod to determine structure: MAD / Resolution: 1.84→10 Å / Num. parameters: 22116 / Num. restraintsaints: 20045 / Cross valid method: FREE R / σ(F): 0 / Stereochemistry target values: ENGH AND HUBER / Details: ANISOTROPIC REFINEMENT
RfactorNum. reflection% reflectionSelection details
Rfree0.236 3330 -RANDOM
Rwork0.1829 ---
All0.2003 66117 --
Obs0.2 62804 99.8 %-
Refine analyzeNum. disordered residues: 7 / Occupancy sum hydrogen: 0 / Occupancy sum non hydrogen: 5358.79
FreeObs
Luzzati coordinate error0.1 Å0.08 Å
Luzzati d res low-8 Å
Luzzati sigma a0.23 Å0.2 Å
Refinement stepCycle: LAST / Resolution: 1.84→10 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4861 0 13 627 5501
Refine LS restraints
Refinement-IDTypeDev ideal
X-RAY DIFFRACTIONs_bond_d0.007
X-RAY DIFFRACTIONs_angle_d0.022
X-RAY DIFFRACTIONs_similar_dist0
X-RAY DIFFRACTIONs_from_restr_planes0.024
X-RAY DIFFRACTIONs_zero_chiral_vol0.034
X-RAY DIFFRACTIONs_non_zero_chiral_vol0.04
X-RAY DIFFRACTIONs_anti_bump_dis_restr0.008
X-RAY DIFFRACTIONs_rigid_bond_adp_cmpnt0
X-RAY DIFFRACTIONs_similar_adp_cmpnt0.087
X-RAY DIFFRACTIONs_approx_iso_adps0.12
LS refinement shellResolution: 1.84→1.87 Å / Redundancy reflection obs: 3131
RfactorNum. reflection% reflection
Rfree0.29 150 -
Rwork0.23 --
Obs--99.8 %

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