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- PDB-4aad: Crystal structure of the mutant D75N I-CreI in complex with its w... -

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Basic information

Entry
Database: PDB / ID: 4aad
TitleCrystal structure of the mutant D75N I-CreI in complex with its wild- type target in absence of metal ions at the active site (The four central bases, 2NN region, are composed by GTAC from 5' to 3')
Components
  • 24MER DNA
  • DNA ENDONUCLEASE I-CREI
KeywordsHYDROLASE/DNA / HYDROLASE-DNA COMPLEX / GENE TARGETING / PROTEIN-DNA INTERACTION / HOMING ENDONUCLEASES
Function / homology
Function and homology information


intron homing / chloroplast / endonuclease activity / Hydrolases; Acting on ester bonds / identical protein binding / metal ion binding
Similarity search - Function
LAGLIDADG endonuclease / Homing endonucleases / Endonuclease I-creI / Homing endonuclease, LAGLIDADG / Homing endonuclease / Roll / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA endonuclease I-CreI
Similarity search - Component
Biological speciesCHLAMYDOMONAS REINHARDTII (plant)
SYNTHETIC CONSTRUCT (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.1 Å
AuthorsMolina, R. / Redondo, P. / Stella, S. / Marenchino, M. / D'Abramo, M. / Gervasio, F.L. / Epinat, J.C. / Valton, J. / Grizot, S. / Duchateau, P. ...Molina, R. / Redondo, P. / Stella, S. / Marenchino, M. / D'Abramo, M. / Gervasio, F.L. / Epinat, J.C. / Valton, J. / Grizot, S. / Duchateau, P. / Prieto, J. / Montoya, G.
CitationJournal: Nucleic Acids Res. / Year: 2012
Title: Non-Specific Protein-DNA Interactions Control I-Crei Target Binding and Cleavage.
Authors: Molina, R. / Redondo, P. / Stella, S. / Marenchino, M. / D'Abramo, M. / Gervasio, F.L. / Charles Epinat, J. / Valton, J. / Grizot, S. / Duchateau, P. / Prieto, J. / Montoya, G.
History
DepositionDec 1, 2011Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 2, 2012Provider: repository / Type: Initial release
Revision 1.1Aug 22, 2012Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other
Revision 1.2Dec 20, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / pdbx_database_status / pdbx_initial_refinement_model / struct_site
Item: _citation.page_last / _database_2.pdbx_DOI ..._citation.page_last / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA ENDONUCLEASE I-CREI
B: DNA ENDONUCLEASE I-CREI
E: 24MER DNA
F: 24MER DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,50512
Polymers49,7684
Non-polymers7378
Water46826
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12790 Å2
ΔGint-61.4 kcal/mol
Surface area17770 Å2
MethodPISA
Unit cell
Length a, b, c (Å)49.406, 84.668, 159.959
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP22121

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Components

#1: Protein DNA ENDONUCLEASE I-CREI / I-CREI / 23S RRNA INTRON PROTEIN


Mass: 17515.137 Da / Num. of mol.: 2 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) CHLAMYDOMONAS REINHARDTII (plant) / Production host: ESCHERICHIA COLI (E. coli)
References: UniProt: P05725, Hydrolases; Acting on ester bonds
#2: DNA chain 24MER DNA


Mass: 7368.778 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)
#3: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C3H8O3
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 26 / Source method: isolated from a natural source / Formula: H2O
Compound detailsENGINEERED RESIDUE IN CHAIN A, ASP 75 TO ASN ENGINEERED RESIDUE IN CHAIN B, ASP 75 TO ASN

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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: 3.36 Å3/Da / Density % sol: 67 % / Description: NONE
Crystal growpH: 8.5
Details: 20% PEG300, 0.1M TRIS PH 8.5, 5% PEG8000, 10% GLYCEROL

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Type: SLS / Wavelength: 1
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.1→47.44 Å / Num. obs: 12755 / % possible obs: 99.9 % / Observed criterion σ(I): 2 / Redundancy: 4.3 % / Biso Wilson estimate: 86.59 Å2 / Rmerge(I) obs: 0.05 / Net I/σ(I): 7.9
Reflection shellResolution: 3.1→3.27 Å / Redundancy: 4.3 % / Rmerge(I) obs: 0.31 / Mean I/σ(I) obs: 2.5 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX(PHENIX.REFINE)refinement
XDSdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 2XE0

2xe0


Resolution: 3.1→47.206 Å / SU ML: 0.67 / σ(F): 1.34 / Phase error: 24.3 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2378 1276 10.01 %
Rwork0.2097 --
obs0.2126 12750 99.79 %
Solvent computationShrinkage radii: 0.83 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 74.935 Å2 / ksol: 0.327 e/Å3
Displacement parameters
Baniso -1Baniso -2Baniso -3
1--4.0402 Å20 Å20 Å2
2--1.6569 Å20 Å2
3---2.3833 Å2
Refinement stepCycle: LAST / Resolution: 3.1→47.206 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2464 978 48 26 3516
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0043646
X-RAY DIFFRACTIONf_angle_d1.0365117
X-RAY DIFFRACTIONf_dihedral_angle_d25.0791437
X-RAY DIFFRACTIONf_chiral_restr0.063579
X-RAY DIFFRACTIONf_plane_restr0.003474
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.1-3.22410.36631370.31391229X-RAY DIFFRACTION100
3.2241-3.37080.30121390.2431246X-RAY DIFFRACTION100
3.3708-3.54850.27461390.23891263X-RAY DIFFRACTION99
3.5485-3.77070.25161410.22411255X-RAY DIFFRACTION100
3.7707-4.06170.28591380.21111253X-RAY DIFFRACTION100
4.0617-4.47010.25071430.19331281X-RAY DIFFRACTION100
4.4701-5.11630.2231390.18121259X-RAY DIFFRACTION100
5.1163-6.44340.2561460.21551307X-RAY DIFFRACTION100
6.4434-47.21110.18111540.19741381X-RAY DIFFRACTION100
Refinement TLS params.Method: refined / Origin x: -15.4485 Å / Origin y: 14.6971 Å / Origin z: -23.2561 Å
111213212223313233
T0.5022 Å20.1753 Å2-0.0307 Å2-0.7532 Å2-0.0596 Å2--0.6807 Å2
L2.1285 °21.3196 °21.4396 °2-7.5353 °25.5985 °2--7.0188 °2
S-0.081 Å °-0.0384 Å °-0.4563 Å °0.3164 Å °1.0031 Å °-0.7189 Å °0.5155 Å °1.2483 Å °-0.8328 Å °
Refinement TLS groupSelection details: ALL

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