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- PDB-2oxv: Structure of the A138T promiscuous mutant of the EcoRI restrictio... -

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Basic information

Entry
Database: PDB / ID: 2oxv
TitleStructure of the A138T promiscuous mutant of the EcoRI restriction endonuclease bound to its cognate recognition site.
Components
  • DNA (5'-D(*TP*CP*GP*CP*GP*AP*AP*TP*TP*CP*GP*CP*G)-3')
  • Type II restriction enzyme EcoRI
Keywordshydrolase/DNA / EcoRI / type II restriction endonuclease / protein-DNA interactions / promiscuous mutant / relaxed specificity mutant / hydrolase-DNA COMPLEX
Function / homology
Function and homology information


type II site-specific deoxyribonuclease / type II site-specific deoxyribonuclease activity / DNA restriction-modification system / magnesium ion binding / DNA binding
Similarity search - Function
Restriction endonuclease, type II, EcoRI / Restriction endonuclease, type II, EcoRI, Proteobacteria / Restriction endonuclease EcoRI / ECO RI Endonuclease; Chain A / Eco RI Endonuclease, subunit A / Restriction endonuclease, type II, EcoRI/MunI / Restriction endonuclease type II-like / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / Type II restriction enzyme EcoRI
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.95 Å
AuthorsSapienza, P.J. / Rosenberg, J.M. / Jen-Jacobson, L.
CitationJournal: Structure / Year: 2007
Title: Structural and thermodynamic basis for enhanced DNA binding by a promiscuous mutant EcoRI endonuclease.
Authors: Sapienza, P.J. / Rosenberg, J.M. / Jen-Jacobson, L.
History
DepositionFeb 21, 2007Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 23, 2007Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Oct 18, 2017Group: Refinement description / Category: software
Item: _software.classification / _software.contact_author ..._software.classification / _software.contact_author / _software.contact_author_email / _software.date / _software.language / _software.location / _software.name / _software.type / _software.version
Revision 1.3Aug 30, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: DNA (5'-D(*TP*CP*GP*CP*GP*AP*AP*TP*TP*CP*GP*CP*G)-3')
A: Type II restriction enzyme EcoRI


Theoretical massNumber of molelcules
Total (without water)35,0992
Polymers35,0992
Non-polymers00
Water2,450136
1
C: DNA (5'-D(*TP*CP*GP*CP*GP*AP*AP*TP*TP*CP*GP*CP*G)-3')
A: Type II restriction enzyme EcoRI

C: DNA (5'-D(*TP*CP*GP*CP*GP*AP*AP*TP*TP*CP*GP*CP*G)-3')
A: Type II restriction enzyme EcoRI


Theoretical massNumber of molelcules
Total (without water)70,1984
Polymers70,1984
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_555y,x,-z1
Unit cell
Length a, b, c (Å)116.473, 116.473, 48.476
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number150
Space group name H-MP321
DetailsThe biological assembly consists of an enzyme dimer bound to a DNA double helix.

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Components

#1: DNA chain DNA (5'-D(*TP*CP*GP*CP*GP*AP*AP*TP*TP*CP*GP*CP*G)-3')


Mass: 3967.585 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: ABI3900 DNA synthesizer
#2: Protein Type II restriction enzyme EcoRI / Endonuclease EcoRI / R.EcoRI


Mass: 31131.406 Da / Num. of mol.: 1 / Mutation: A138T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: ecoRIR / Plasmid: pET24a / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: P00642, type II site-specific deoxyribonuclease
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 136 / 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: 2.69 Å3/Da / Density % sol: 54.3 %
Crystal growTemperature: 277.15 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: cryoprotectant solution is 40mM bis-tris Propane, 16% v/v PEG 400, 15% v/v glycerol, pH 6.5, VAPOR DIFFUSION, SITTING DROP, temperature 277.15K
Components of the solutions
IDNameCrystal-IDSol-ID
1PEG 40011
2glycerol11
3PEG 40012
4glycerol12
5bis-tris Propane13

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X6A / Wavelength: 0.979 Å
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: Nov 18, 2003
Details: Si(111) channel cut monochromator. Toroidal focusing mirror
RadiationMonochromator: Si(111) channel cut momochromator / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 1.95→99 Å / Num. all: 27884 / Num. obs: 27856 / % possible obs: 99.9 % / Observed criterion σ(F): 2 / Observed criterion σ(I): 2 / Redundancy: 17 % / Rmerge(I) obs: 0.077 / Χ2: 0.994 / Net I/σ(I): 25.1
Reflection shellResolution: 1.95→2.01 Å / Redundancy: 10.2 % / Rmerge(I) obs: 0.436 / Mean I/σ(I) obs: 4.2 / Num. unique all: 2283 / Χ2: 0.99 / % possible all: 100

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
CNSrefinement
PDB_EXTRACT2data extraction
ADSCQuantumdata collection
HKL-2000data reduction
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1CKQ

1ckq


Resolution: 1.95→12 Å / Cross valid method: THROUGHOUT / σ(F): 2 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.244 2523 9.1 %random
Rwork0.202 ---
all-27690 --
obs-25266 91.2 %-
Solvent computationBsol: 61.189 Å2
Displacement parametersBiso mean: 40.131 Å2
Baniso -1Baniso -2Baniso -3
1--3.683 Å2-4.593 Å20 Å2
2---3.683 Å20 Å2
3---7.366 Å2
Refine analyzeLuzzati coordinate error obs: 0.254 Å
Refinement stepCycle: LAST / Resolution: 1.95→12 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2053 263 0 136 2452
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.009
X-RAY DIFFRACTIONc_angle_d1.324
X-RAY DIFFRACTIONc_mcbond_it2.5511.5
X-RAY DIFFRACTIONc_scbond_it3.5362
X-RAY DIFFRACTIONc_mcangle_it3.4422
X-RAY DIFFRACTIONc_scangle_it4.6472.5
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1protein_rep.paramprotein.top
X-RAY DIFFRACTION2dna-rna_rep.paramdna-rna.top
X-RAY DIFFRACTION3water_rep.paramwater.top

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