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- PDB-3fbd: Crystal structure of the nuclease domain of COLE7(D493Q mutant) i... -

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Basic information

Entry
Database: PDB / ID: 3fbd
TitleCrystal structure of the nuclease domain of COLE7(D493Q mutant) in complex with an 18-BP duplex DNA
Components
  • 5'-D(*DGP*DGP*DAP*DAP*DTP*DTP*DCP*DGP*DAP*DTP*DCP*DGP*DAP*DAP*DTP*DTP*DCP*DC)-3'
  • Colicin-E7
KeywordsHYDROLASE/DNA / Computational redesign / Protein engineering / Protein-nucleic acid interactions / DNase / DNA hydrolysis. / Antibiotic / Antimicrobial / Bacteriocin / Endonuclease / Hydrolase / Metal-binding / Nuclease / Plasmid / Zinc / HYDROLASE-DNA COMPLEX
Function / homology
Function and homology information


extrachromosomal circular DNA / endonuclease activity / killing of cells of another organism / Hydrolases; Acting on ester bonds / defense response to bacterium / metal ion binding
Similarity search - Function
Colicin E7 immunity protein; Chain B, fragment: Endonuclease domain / Colicin/pyocin, DNase domain / Colicin/Pyocin-S2, DNase domain / Colicin/pyocin, DNase domain superfamily / Colicin, receptor domain / Coiled-coil receptor-binding R-domain of colicin E2 / Cloacin colicin family / Colicin-like bacteriocin tRNase domain / Pyosin/cloacin translocation domain / Pyosin/cloacin translocation domain superfamily ...Colicin E7 immunity protein; Chain B, fragment: Endonuclease domain / Colicin/pyocin, DNase domain / Colicin/Pyocin-S2, DNase domain / Colicin/pyocin, DNase domain superfamily / Colicin, receptor domain / Coiled-coil receptor-binding R-domain of colicin E2 / Cloacin colicin family / Colicin-like bacteriocin tRNase domain / Pyosin/cloacin translocation domain / Pyosin/cloacin translocation domain superfamily / HNH nucleases / His-Me finger superfamily / HNH nuclease / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / Colicin-E7
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.9 Å
AuthorsWang, Y.T. / Doudeva, L.G. / Yuan, H.S.
CitationJournal: To be Published
Title: Redesign of high-affinity nonspecific nucleases with altered sequence preference
Authors: Wang, Y.T. / Wright, J.D. / Doudeva, L.G. / Jhang, H.C. / Lim, C. / Yuan, H.S.
History
DepositionNov 19, 2008Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Nov 3, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Nov 10, 2021Group: Database references / Category: database_2 / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details
Revision 1.3Nov 1, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Colicin-E7
B: 5'-D(*DGP*DGP*DAP*DAP*DTP*DTP*DCP*DGP*DAP*DTP*DCP*DGP*DAP*DAP*DTP*DTP*DCP*DC)-3'
C: 5'-D(*DGP*DGP*DAP*DAP*DTP*DTP*DCP*DGP*DAP*DTP*DCP*DGP*DAP*DAP*DTP*DTP*DCP*DC)-3'
D: Colicin-E7
E: 5'-D(*DGP*DGP*DAP*DAP*DTP*DTP*DCP*DGP*DAP*DTP*DCP*DGP*DAP*DAP*DTP*DTP*DCP*DC)-3'
F: 5'-D(*DGP*DGP*DAP*DAP*DTP*DTP*DCP*DGP*DAP*DTP*DCP*DGP*DAP*DAP*DTP*DTP*DCP*DC)-3'


Theoretical massNumber of molelcules
Total (without water)52,3876
Polymers52,3876
Non-polymers00
Water1,27971
1
A: Colicin-E7
B: 5'-D(*DGP*DGP*DAP*DAP*DTP*DTP*DCP*DGP*DAP*DTP*DCP*DGP*DAP*DAP*DTP*DTP*DCP*DC)-3'
C: 5'-D(*DGP*DGP*DAP*DAP*DTP*DTP*DCP*DGP*DAP*DTP*DCP*DGP*DAP*DAP*DTP*DTP*DCP*DC)-3'


Theoretical massNumber of molelcules
Total (without water)26,1933
Polymers26,1933
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3570 Å2
ΔGint-24 kcal/mol
Surface area12370 Å2
MethodPISA
2
D: Colicin-E7
E: 5'-D(*DGP*DGP*DAP*DAP*DTP*DTP*DCP*DGP*DAP*DTP*DCP*DGP*DAP*DAP*DTP*DTP*DCP*DC)-3'
F: 5'-D(*DGP*DGP*DAP*DAP*DTP*DTP*DCP*DGP*DAP*DTP*DCP*DGP*DAP*DAP*DTP*DTP*DCP*DC)-3'


Theoretical massNumber of molelcules
Total (without water)26,1933
Polymers26,1933
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3590 Å2
ΔGint-23 kcal/mol
Surface area12320 Å2
MethodPISA
Unit cell
Length a, b, c (Å)60.587, 49.423, 92.400
Angle α, β, γ (deg.)90.00, 102.64, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Colicin-E7


Mass: 15162.222 Da / Num. of mol.: 2 / Fragment: NUCLEASE DOMAIN / Mutation: D493Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: W3110 / Gene: cea, colE7 / Plasmid: PQE70 / Production host: Escherichia coli (E. coli) / Strain (production host): M15
References: UniProt: Q47112, Hydrolases; Acting on ester bonds
#2: DNA chain
5'-D(*DGP*DGP*DAP*DAP*DTP*DTP*DCP*DGP*DAP*DTP*DCP*DGP*DAP*DAP*DTP*DTP*DCP*DC)-3'


Mass: 5515.591 Da / Num. of mol.: 4 / Source method: obtained synthetically
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 71 / 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: 2.58 Å3/Da / Density % sol: 52.27 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 15% PEG 3350, 16.25% MPD, 0.15M ammonium acetate, 0.025M sodium acetate, pH 8, VAPOR DIFFUSION, HANGING DROP, temperature 298K
Components of the solutions
IDNameCrystal-IDSol-ID
1PEG 335011
2MPD11
3ammonium acetate11
4sodium acetate11
5PEG 335012
6MPD12
7ammonium acetate12
8sodium acetate12

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Data collection

Diffraction sourceSource: SYNCHROTRON / Site: NSRRC / Beamline: BL13C1 / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: May 25, 2007
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.9→50 Å / Num. all: 12069 / Num. obs: 11321

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Processing

Software
NameVersionClassification
HKL-2000data collection
MOLREPphasing
CNS1.2refinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2IVH

2ivh


Resolution: 2.9→27.2 Å / Rfactor Rfree error: 0.008 / Data cutoff high absF: 184463.11 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Details: BULK SOLVENT MODEL USED
RfactorNum. reflection% reflectionSelection details
Rfree0.264 1166 10.3 %RANDOM
Rwork0.201 ---
obs0.201 11321 93.9 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 18.558 Å2 / ksol: 0.3 e/Å3
Displacement parametersBiso mean: 50.3 Å2
Baniso -1Baniso -2Baniso -3
1-25.28 Å20 Å28.86 Å2
2---10.98 Å20 Å2
3----14.3 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.46 Å0.33 Å
Luzzati d res low-5 Å
Luzzati sigma a0.54 Å0.38 Å
Refinement stepCycle: LAST / Resolution: 2.9→27.2 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2128 1464 0 71 3663
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.011
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_deg1.4
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d21.3
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d1.48
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it1.751.5
X-RAY DIFFRACTIONc_mcangle_it2.952
X-RAY DIFFRACTIONc_scbond_it2.952
X-RAY DIFFRACTIONc_scangle_it4.782.5
LS refinement shellResolution: 2.9→3.08 Å / Rfactor Rfree error: 0.035 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.411 142 9.4 %
Rwork0.317 1366 -
obs--76.5 %
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
X-RAY DIFFRACTION4ion.paramion.top

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