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- PDB-6ek1: Crystal structure of Type IIP restriction endonuclease PfoI -

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Basic information

Entry
Database: PDB / ID: 6ek1
TitleCrystal structure of Type IIP restriction endonuclease PfoI
Componentsrestriction endonuclease PfoI
KeywordsHYDROLASE / Restriction endonuclease / PD-(D/E)xK nuclease
Function / homologytype II site-specific deoxyribonuclease / type II site-specific deoxyribonuclease activity / Restriction endonuclease PfoI
Function and homology information
Biological speciesPseudomonas fluorescens (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2.601 Å
AuthorsTamulaitiene, G. / Manakova, E. / Jovaisaite, V. / Grazulis, S. / Siksnys, V.
Funding supportLithuania, 1items
OrganizationGrant numberCountry
Research Council of LithuaniaMIP-41/2013Lithuania
CitationJournal: Nucleic Acids Res. / Year: 2019
Title: Unique mechanism of target recognition by PfoI restriction endonuclease of the CCGG-family.
Authors: Tamulaitiene, G. / Manakova, E. / Jovaisaite, V. / Tamulaitis, G. / Grazulis, S. / Bochtler, M. / Siksnys, V.
History
DepositionSep 25, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 10, 2018Provider: repository / Type: Initial release
Revision 1.1Apr 24, 2019Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / entity / pdbx_database_proc
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _entity.formula_weight

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: restriction endonuclease PfoI
hetero molecules


Theoretical massNumber of molelcules
Total (without water)35,2572
Polymers35,1951
Non-polymers621
Water1086
1
A: restriction endonuclease PfoI
hetero molecules

A: restriction endonuclease PfoI
hetero molecules


Theoretical massNumber of molelcules
Total (without water)70,5134
Polymers70,3892
Non-polymers1242
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation7_465y-1,x+1,-z1
Buried area4210 Å2
ΔGint-15 kcal/mol
Surface area25610 Å2
MethodPISA
Unit cell
γ
α
β
Length a, b, c (Å)48.067, 48.067, 266.693
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number96
Space group name H-MP43212

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Components

#1: Protein restriction endonuclease PfoI


Mass: 35194.527 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas fluorescens (bacteria) / Strain: biovar 126 / Plasmid: pBAD24 / Production host: Escherichia coli BH10B (bacteria)
References: UniProt: A0A452CST7*PLUS, type II site-specific deoxyribonuclease
#2: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 6 / 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: 43.84 % / Description: Bipyramidal crystals
Crystal growTemperature: 292 K / Method: vapor diffusion
Details: 6.8 mg/ml of PfoI in complex with DNA oligoduplex was mixed with reservoir solution 1:1, microseeding was used. NaHepes 0.1M, magnesium formate 0.2M, 18-20% PEG3350
PH range: 7-7.5

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RUH3R / Wavelength: 1.54 Å
DetectorType: RIGAKU RAXIS IV++ / Detector: IMAGE PLATE / Date: May 9, 2009 / Details: OSMIC CONFOCAL MIRRORS
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 2.6→45.22 Å / Num. obs: 17795 / % possible obs: 98.6 % / Redundancy: 5.3 % / Biso Wilson estimate: 49.262 Å2 / Rmerge(I) obs: 0.112 / Rpim(I) all: 0.074 / Rsym value: 0.112 / Net I/σ(I): 5.7
Reflection shellResolution: 2.6→2.74 Å / Redundancy: 6.2 % / Rmerge(I) obs: 0.44 / Mean I/σ(I) obs: 3.4 / Num. unique obs: 1470 / Rsym value: 0.44 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX1.8.3_1479refinement
MOSFLM6.2.6data reduction
SCALA3.2.19data scaling
MOLREP9.2.10phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.601→45.219 Å / SU ML: 0.34 / Cross valid method: FREE R-VALUE / σ(F): 1.2 / Phase error: 27.41
RfactorNum. reflection% reflectionSelection details
Rfree0.274 1710 9.61 %Random selection
Rwork0.2181 ---
obs0.2233 17795 96.94 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.601→45.219 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2395 0 4 6 2405
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0022457
X-RAY DIFFRACTIONf_angle_d0.5743318
X-RAY DIFFRACTIONf_dihedral_angle_d12.425898
X-RAY DIFFRACTIONf_chiral_restr0.022374
X-RAY DIFFRACTIONf_plane_restr0.002426
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.6011-2.67760.4141420.29311411X-RAY DIFFRACTION99
2.6776-2.7640.31831640.28131331X-RAY DIFFRACTION99
2.764-2.86280.33531650.26571356X-RAY DIFFRACTION99
2.8628-2.97740.33951380.24521406X-RAY DIFFRACTION100
2.9774-3.11290.27211240.24341351X-RAY DIFFRACTION99
3.1129-3.27690.28741440.25121379X-RAY DIFFRACTION99
3.2769-3.48220.29851430.23921351X-RAY DIFFRACTION97
3.4822-3.75090.28851470.22671308X-RAY DIFFRACTION95
3.7509-4.12810.2421460.21331258X-RAY DIFFRACTION92
4.1281-4.72490.21291490.1721292X-RAY DIFFRACTION94
4.7249-5.95080.25041200.18881310X-RAY DIFFRACTION93
5.9508-45.22570.24881280.17691332X-RAY DIFFRACTION96

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