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- PDB-1b97: ANALYSIS OF A MUTATIONAL HOT-SPOT IN THE ECORV RESTRICTION ENDONU... -

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Basic information

Entry
Database: PDB / ID: 1b97
TitleANALYSIS OF A MUTATIONAL HOT-SPOT IN THE ECORV RESTRICTION ENDONUCLEASE: A CATALYTIC ROLE FOR A MAIN CHAIN CARBONYL GROUP
Components
  • DNA (5'-D(*AP*AP*AP*GP*AP*TP*AP*TP*CP*TP*T)-3')
  • RESTRICTION ENDONUCLEASE ECORV
KeywordsHYDROLASE/DNA / ENDONUCLEASE / RESTRICTION / ECORV / 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)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.9 Å
AuthorsThomas, M.P. / Halford, S.E. / Brady, R.L.
CitationJournal: Nucleic Acids Res. / Year: 1999
Title: Structural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group.
Authors: Thomas, M.P. / Brady, R.L. / Halford, S.E. / Sessions, R.B. / Baldwin, G.S.
History
DepositionFeb 19, 1999Deposition site: PDBE / Processing site: RCSB
Revision 1.0Feb 26, 1999Provider: repository / Type: Initial release
Revision 1.1Apr 26, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Nov 3, 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 1.4Aug 9, 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

Downloads & links

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Assembly

Deposited unit
C: DNA (5'-D(*AP*AP*AP*GP*AP*TP*AP*TP*CP*TP*T)-3')
D: DNA (5'-D(*AP*AP*AP*GP*AP*TP*AP*TP*CP*TP*T)-3')
A: RESTRICTION ENDONUCLEASE ECORV
B: RESTRICTION ENDONUCLEASE ECORV


Theoretical massNumber of molelcules
Total (without water)63,8014
Polymers63,8014
Non-polymers00
Water6,449358
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)48.651, 49.224, 64.039
Angle α, β, γ (deg.)96.65, 108.91, 107.53
Int Tables number1
Cell settingtriclinic
Space group name H-MP1

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Components

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


Mass: 3356.235 Da / Num. of mol.: 2 / Source method: obtained synthetically
#2: Protein RESTRICTION ENDONUCLEASE ECORV / E.C.3.1.21.4


Mass: 28544.186 Da / Num. of mol.: 2 / Mutation: Q69L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Cellular location: CYTOPLASM / Gene: ECORVR / Plasmid: PBSKRV / Production host: Escherichia coli (E. coli)
References: UniProt: P04390, type II site-specific deoxyribonuclease
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 358 / 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: 2.11 Å3/Da / Density % sol: 41.62 %
Crystal growMethod: vapor diffusion, sitting drop / pH: 7 / Details: pH 7.0, VAPOR DIFFUSION, SITTING DROP
Components of the solutions
IDNameCrystal-IDSol-ID
1PHOSPHATE BUFFER11
2NACL11
3EDTA11
4DTT11
5NAN311
6SODIUM CACODYLATE11
7PEG 400011
8PEG 400012
Crystal grow
*PLUS
Temperature: 18 ℃ / pH: 7.5
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDDetailsChemical formula
115 mg/mlprotein1drop0.260mM dimer
210 mMphosphate1droppH7.5
3250 mM1dropNaCl
41 mMEDTA1drop
50.1 mMdithiothreitol1drop
60.1 %(w/v)sodium azide1drop
73 mg/mlDNA1drop0.460mM
810 mMcacodylic1droppH6.0
90.8-1.4 %(w/v)PEG40001drop

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SRS / Beamline: PX7.2 / Wavelength: 1.5418
DetectorDate: Oct 15, 1996
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.9→15 Å / Num. obs: 17144 / % possible obs: 92.7 % / Redundancy: 1.8 % / Biso Wilson estimate: 27.5 Å2 / Rmerge(I) obs: 0.052 / Net I/σ(I): 15.1
Reflection shellResolution: 1.9→1.99 Å / Redundancy: 1.7 % / Rmerge(I) obs: 0.207 / Mean I/σ(I) obs: 3.6 / % possible all: 87.5
Reflection
*PLUS
Num. obs: 38240 / Num. measured all: 113353
Reflection shell
*PLUS
% possible obs: 87.5 %

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Processing

Software
NameClassification
AMoREphasing
REFMACrefinement
DENZOdata reduction
SCALEPACKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1RVA

1rva


Resolution: 1.9→15 Å / Cross valid method: THROUGHOUT / σ(F): 0
RfactorNum. reflection% reflectionSelection details
Rfree0.279 1714 10 %RANDOM
Rwork0.219 ---
all-17144 --
obs-17144 92.7 %-
Refinement stepCycle: LAST / Resolution: 1.9→15 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4088 446 0 358 4892
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONp_bond_d0.0130.02
X-RAY DIFFRACTIONp_angle_d2.430.028
X-RAY DIFFRACTIONp_angle_deg
X-RAY DIFFRACTIONp_planar_d
X-RAY DIFFRACTIONp_hb_or_metal_coord
X-RAY DIFFRACTIONp_mcbond_it
X-RAY DIFFRACTIONp_mcangle_it
X-RAY DIFFRACTIONp_scbond_it
X-RAY DIFFRACTIONp_scangle_it
X-RAY DIFFRACTIONp_plane_restr
X-RAY DIFFRACTIONp_chiral_restr
X-RAY DIFFRACTIONp_singtor_nbd
X-RAY DIFFRACTIONp_multtor_nbd
X-RAY DIFFRACTIONp_xhyhbond_nbd
X-RAY DIFFRACTIONp_xyhbond_nbd
X-RAY DIFFRACTIONp_planar_tor
X-RAY DIFFRACTIONp_staggered_tor
X-RAY DIFFRACTIONp_orthonormal_tor
X-RAY DIFFRACTIONp_transverse_tor
X-RAY DIFFRACTIONp_special_tor
Software
*PLUS
Name: REFMAC / Classification: refinement
Refinement
*PLUS
Lowest resolution: 15 Å / σ(F): 0 / % reflection Rfree: 10 % / Rfactor obs: 0.219
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Type: p_bond_d / Dev ideal target: 0.02

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