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- PDB-6qm2: NlaIV restriction endonuclease -

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Basic information

Entry
Database: PDB / ID: 6qm2
TitleNlaIV restriction endonuclease
ComponentsType-2 restriction enzyme NlaIV
KeywordsHYDROLASE / RESTRICTION ENDONUCLEASE / NLAIV / BLUNT END CUTTER
Function / homologyType-2 restriction enzyme NlaIV / NgoBV restriction endonuclease / type II site-specific deoxyribonuclease / type II site-specific deoxyribonuclease activity / DNA restriction-modification system / Type-2 restriction enzyme NlaIV
Function and homology information
Biological speciesNeisseria lactamica (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2.8 Å
AuthorsCzapinska, H. / Siwek, W. / Szczepanowski, R.H. / Bujnicki, J.M. / Bochtler, M. / Skowronek, K.
Citation
Journal: J.Mol.Biol. / Year: 2019
Title: Crystal Structure and Directed Evolution of Specificity of NlaIV Restriction Endonuclease.
Authors: Czapinska, H. / Siwek, W. / Szczepanowski, R.H. / Bujnicki, J.M. / Bochtler, M. / Skowronek, K.J.
#1: Journal: Protein Eng. Des. Sel. / Year: 2005
Title: A theoretical model of restriction endonuclease NlaIV in complex with DNA, predicted by fold recognition and validated by site-directed mutagenesis and circular dichroism spectroscopy.
Authors: Chmiel, A.A. / Radlinska, M. / Pawlak, S.D. / Krowarsch, D. / Bujnicki, J.M. / Skowronek, K.J.
Validation Report
SummaryFull reportAbout validation report
History
DepositionFeb 1, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 1, 2019Provider: repository / Type: Initial release
Revision 1.1Jun 5, 2019Group: Data collection / Database references / Category: citation / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Type-2 restriction enzyme NlaIV
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,2163
Polymers29,1541
Non-polymers622
Water2,216123
1
A: Type-2 restriction enzyme NlaIV
hetero molecules

A: Type-2 restriction enzyme NlaIV
hetero molecules


Theoretical massNumber of molelcules
Total (without water)58,4316
Polymers58,3072
Non-polymers1244
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation9_555-x,-x+y,-z+1/31
Buried area3490 Å2
ΔGint-44 kcal/mol
Surface area24770 Å2
MethodPISA
Unit cell
γ
α
β
Length a, b, c (Å)110.380, 110.380, 209.006
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number180
Space group name H-MP6222

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Components

#1: Protein/peptide Type-2 restriction enzyme NlaIV / R.NlaIV / Endonuclease NlaIV / Type II restriction enzyme NlaIV


Mass: 29153.611 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria lactamica (bacteria) / Strain: NRCC 2118 / Gene: nlaIVR / Plasmid: PET28a / Production host: Escherichia coli (E. coli) / Strain (production host): ER2566
References: UniProt: P50183, type II site-specific deoxyribonuclease
#2: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na / Sodium
#3: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: K / Potassium
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 123 / 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

Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop / pH: 5
Details: 10 mg/ml of the enzyme in 15 % glycerol, 50 mM NaOH/HEPES pH 7,5, 50 mM KCl, 10 mM DTT and 5 mM CaCl2 was mixed in 1:1 ratio with double stranded DNA composed of the 5'-ATGGTACCTGC-3' and 5'-CAGGTACCATG-3' strands. The protein-DNA solutions were in turn mixed in 1:1 ratio with the precipitant solution containing 2 M NaCl and 100 mM citric acid, pH 5.
PH range: 5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: MPG/DESY, HAMBURG / Beamline: BW6 / Wavelength: 1.05 Å
DetectorType: MARRESEARCH / Detector: CCD / Date: Dec 17, 2008 / Details: BENT MIRROR
RadiationMonochromator: TRIANGULAR MONOCHROMATOR / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.05 Å / Relative weight: 1
ReflectionResolution: 2.8→30 Å / Num. obs: 18997 / % possible obs: 98.5 % / Redundancy: 7.5 % / Rmerge(I) obs: 0.043 / Rsym value: 0.04 / Net I/σ(I): 28.92
Reflection shellResolution: 2.8→2.97 Å / Redundancy: 7.7 % / Rmerge(I) obs: 1.052 / Mean I/σ(I) obs: 1.72 / Rsym value: 0.986 / % possible all: 98.6

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Processing

Software
NameVersionClassification
REFMAC5.8.0189refinement
XDSdata reduction
XDSdata scaling
STARANISOdata scaling
autoSHARPphasing
SHELXDEphasing
SOLOMONphasing
DMphasing
PARROTphasing
ARP/wARPmodel building
BUCCANEERmodel building
RefinementMethod to determine structure: MAD / Resolution: 2.8→29.72 Å / Cor.coef. Fo:Fc: 0.966 / Cor.coef. Fo:Fc free: 0.956 / SU B: 11.197 / SU ML: 0.189 / Cross valid method: THROUGHOUT / ESU R: 0.267 / ESU R Free: 0.221
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. The ION IDENTITY HAS BEEN ASSIGNED TENTATIVELY.
RfactorNum. reflection% reflectionSelection details
Rfree0.22103 912 4.8 %RANDOM
Rwork0.19095 ---
Obs0.19245 18083 98.46 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 115.889 Å2
Baniso -1Baniso -2Baniso -3
1-1.68 Å20.84 Å20 Å2
2--1.68 Å2-0 Å2
3----5.44 Å2
Refinement stepCycle: LAST / Resolution: 2.8→29.72 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2056 0 2 123 2181
Refine LS restraints

Refinement-ID: X-RAY DIFFRACTION

TypeDev idealDev ideal targetNumber
r_bond_refined_d0.0070.0192142
r_bond_other_d0.0010.021994
r_angle_refined_deg1.0831.9362893
r_angle_other_deg0.84934637
r_dihedral_angle_1_deg5.2895253
r_dihedral_angle_2_deg33.83424.352108
r_dihedral_angle_3_deg13.63915405
r_dihedral_angle_4_deg16.2891512
r_chiral_restr0.0620.2299
r_gen_planes_refined0.0030.022365
r_gen_planes_other0.0010.02455
r_nbd_refined
r_nbd_other
r_nbtor_refined
r_nbtor_other
r_xyhbond_nbd_refined
r_xyhbond_nbd_other
r_metal_ion_refined
r_metal_ion_other
r_symmetry_vdw_refined
r_symmetry_vdw_other
r_symmetry_hbond_refined
r_symmetry_hbond_other
r_symmetry_metal_ion_refined
r_symmetry_metal_ion_other
r_mcbond_it3.82511.6481003
r_mcbond_other3.81411.6431002
r_mcangle_it6.42717.4821259
r_mcangle_other6.42717.4891260
r_scbond_it3.76511.8941139
r_scbond_other3.76511.8931139
r_scangle_it
r_scangle_other6.54217.6321634
r_long_range_B_refined9.963127.8012402
r_long_range_B_other9.927127.7342390
r_rigid_bond_restr
r_sphericity_free
r_sphericity_bonded
LS refinement shellResolution: 2.8→2.872 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.456 54 -
Rwork0.392 1305 -
Obs--98.48 %

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