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- PDB-5f8a: Crystal structure of the ternary EcoRV-DNA-Lu complex with unclea... -

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Basic information

Entry
Database: PDB / ID: 5f8a
TitleCrystal structure of the ternary EcoRV-DNA-Lu complex with uncleaved DNA substrate. Lanthanide binding to EcoRV-DNA complex inhibits cleavage.
Components
  • DNA (5'-D(*AP*AP*AP*GP*AP*TP*AP*TP*CP*TP*TP*T)-3')
  • Type-2 restriction enzyme EcoRV
KeywordsHYDROLASE/DNA / Hydrolase / Protein-DNA complex / Lutetium / HYDROLASE-DNA complex
Function / homology
Function and homology information


type II site-specific deoxyribonuclease / type II site-specific deoxyribonuclease activity / DNA restriction-modification system / DNA binding / metal ion binding
Similarity search - Function
Restriction endonuclease, type II, EcoRV / Restriction endonuclease, type II, EcoRV, Proteobacteria / Restriction endonuclease EcoRV / DNA mismatch repair MutH/Restriction endonuclease, type II / DNA mismatch repair MutH/Type II restriction endonuclease superfamily / ECO RV Endonuclease; Chain A / Restriction endonuclease type II-like / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
: / DNA / DNA (> 10) / Type II restriction enzyme EcoRV
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
synthetic construct (others)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.76 Å
AuthorsSangani, S.S. / Kehr, A.D. / Sinha, K. / Rule, G.S. / Jen-Jacobson, L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)2R01GM029207 United States
CitationJournal: Biochemistry / Year: 2016
Title: Metal Ion Binding at the Catalytic Site Induces Widely Distributed Changes in a Sequence Specific Protein-DNA Complex.
Authors: Sinha, K. / Sangani, S.S. / Kehr, A.D. / Rule, G.S. / Jen-Jacobson, L.
History
DepositionDec 9, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 9, 2016Provider: repository / Type: Initial release
Revision 1.1Dec 21, 2016Group: Database references
Revision 1.2Sep 27, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Sep 27, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: DNA (5'-D(*AP*AP*AP*GP*AP*TP*AP*TP*CP*TP*TP*T)-3')
D: DNA (5'-D(*AP*AP*AP*GP*AP*TP*AP*TP*CP*TP*TP*T)-3')
A: Type-2 restriction enzyme EcoRV
B: Type-2 restriction enzyme EcoRV
hetero molecules


Theoretical massNumber of molelcules
Total (without water)64,95910
Polymers64,4394
Non-polymers5206
Water5,423301
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9970 Å2
ΔGint-48 kcal/mol
Surface area22310 Å2
MethodPISA
Unit cell
Length a, b, c (Å)46.430, 52.860, 65.310
Angle α, β, γ (deg.)70.61, 73.24, 81.81
Int Tables number1
Space group name H-MP1

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Components

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DNA chain / Protein , 2 types, 4 molecules CDAB

#1: DNA chain DNA (5'-D(*AP*AP*AP*GP*AP*TP*AP*TP*CP*TP*TP*T)-3')


Mass: 3660.428 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: Protein Type-2 restriction enzyme EcoRV / R.EcoRV / Endonuclease EcoRV / Type II restriction enzyme EcoRV


Mass: 28559.158 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: ecoRVR / Production host: Escherichia coli (E. coli)
References: UniProt: P04390, type II site-specific deoxyribonuclease

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Non-polymers , 4 types, 307 molecules

#3: Chemical ChemComp-LU / LUTETIUM (III) ION / LU / Lutetium


Mass: 174.967 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Lu
#4: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#5: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6O2
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 301 / 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: 2.24 Å3/Da / Density % sol: 45.18 %
Crystal growTemperature: 285 K / Method: vapor diffusion, sitting drop / pH: 7.4
Details: 4.5 mg/mL (monomer), protein(dimer):DNA - 1:1, Enzyme:Well - 3uL:3uL, Well - 500uL of 100 mM HEPES pH 7.4, 8% Ethylene glycol, 4% Polyethylene glycol 8000, 10% glycerol.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU FR-E DW / Wavelength: 1.54178 Å
DetectorType: RIGAKU RAXIS HTC / Detector: IMAGE PLATE / Date: Nov 18, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54178 Å / Relative weight: 1
ReflectionResolution: 1.76→36.673 Å / Num. obs: 51697 / % possible obs: 93 % / Redundancy: 3.9 % / Biso Wilson estimate: 21.698 Å2 / Rmerge(I) obs: 0.051 / Rsym value: 0.059 / Net I/σ(I): 14.7
Reflection shellResolution: 1.76→1.85 Å / Redundancy: 3.2 % / Rmerge(I) obs: 0.196 / Mean I/σ(I) obs: 4.4 / % possible all: 88.9

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Processing

Software
NameVersionClassification
REFMAC5.8.0103refinement
iMOSFLM7.2.1data reduction
SCALACCP4interface 6.5.019data scaling
PHASERCCP4interface 6.5.019phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1B95

1b95


Resolution: 1.76→36.673 Å / Cor.coef. Fo:Fc: 0.969 / Cor.coef. Fo:Fc free: 0.955 / SU B: 2.494 / SU ML: 0.078 / Cross valid method: THROUGHOUT / ESU R: 0.127 / ESU R Free: 0.12 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.19914 5000 9.7 %RANDOM
Rwork0.16026 ---
obs0.16402 46685 92.94 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 27.868 Å2
Baniso -1Baniso -2Baniso -3
1-0.96 Å2-0.78 Å20.85 Å2
2--1.36 Å2-0.66 Å2
3----1.83 Å2
Refinement stepCycle: LAST / Resolution: 1.76→36.673 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4046 486 12 301 4845
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0170.0184732
X-RAY DIFFRACTIONr_bond_other_d0.0070.024326
X-RAY DIFFRACTIONr_angle_refined_deg1.5741.8486507
X-RAY DIFFRACTIONr_angle_other_deg1.05639845
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.5985496
X-RAY DIFFRACTIONr_dihedral_angle_2_deg38.75224.299214
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.43415743
X-RAY DIFFRACTIONr_dihedral_angle_4_deg20.1221522
X-RAY DIFFRACTIONr_chiral_restr0.1030.2668
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.025018
X-RAY DIFFRACTIONr_gen_planes_other0.0020.021116
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it2.6722.4961963
X-RAY DIFFRACTIONr_mcbond_other2.6662.4951962
X-RAY DIFFRACTIONr_mcangle_it3.523.7292454
X-RAY DIFFRACTIONr_mcangle_other3.5213.7292455
X-RAY DIFFRACTIONr_scbond_it3.8062.8652769
X-RAY DIFFRACTIONr_scbond_other3.8062.8652770
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other5.3934.1514050
X-RAY DIFFRACTIONr_long_range_B_refined6.46821.7225898
X-RAY DIFFRACTIONr_long_range_B_other6.46821.7185896
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.759→1.805 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.315 366 -
Rwork0.262 3227 -
obs--88.06 %

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