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- PDB-6fb0: Crystal Structure of a Tailored I-CreI Homing Endonuclease Protei... -

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Basic information

Entry
Database: PDB / ID: 6fb0
TitleCrystal Structure of a Tailored I-CreI Homing Endonuclease Protein (3115 variant) in complex with its target DNA (Haemoglobin beta subunit gene) in the presence of Calcium
Components
  • (DNA endonuclease I- ...) x 2
  • DNA (5'-D(*TP*CP*AP*GP*AP*CP*TP*TP*CP*TP*CP*CP*AP*CP*AP*GP*GP*AP*GP*TP*CP*AP*GP*A)-3')
  • DNA (5'-D(*TP*CP*TP*GP*AP*CP*TP*CP*CP*TP*GP*TP*GP*GP*AP*GP*AP*AP*GP*TP*CP*TP*GP*A)-3')
KeywordsDNA BINDING PROTEIN / Chlamydomonas reinhardtii
Function / homology
Function and homology information


intron homing / chloroplast / endonuclease activity / Hydrolases; Acting on ester bonds / metal ion binding / identical protein binding
Similarity search - Function
LAGLIDADG endonuclease / Homing endonucleases / Endonuclease I-creI / Homing endonuclease, LAGLIDADG / Homing endonuclease / Roll / Alpha Beta
Similarity search - Domain/homology
ACETATE ION / S-1,2-PROPANEDIOL / DNA / DNA (> 10) / DNA endonuclease I-CreI
Similarity search - Component
Biological speciesChlamydomonas reinhardtii (plant)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.15 Å
AuthorsMolina, R. / Prieto, J.
CitationJournal: Sci Rep / Year: 2018
Title: Understanding the indirect DNA read-out specificity of I-CreI Meganuclease.
Authors: Prieto, J. / Redondo, P. / Lopez-Mendez, B. / D'Abramo, M. / Merino, N. / Blanco, F.J. / Duchateau, P. / Montoya, G. / Molina, R.
History
DepositionDec 18, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 18, 2018Provider: repository / Type: Initial release
Revision 1.1Jan 17, 2024Group: 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_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_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 / _struct_conn.ptnr2_label_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
D: DNA (5'-D(*TP*CP*AP*GP*AP*CP*TP*TP*CP*TP*CP*CP*AP*CP*AP*GP*GP*AP*GP*TP*CP*AP*GP*A)-3')
F: DNA (5'-D(*TP*CP*TP*GP*AP*CP*TP*CP*CP*TP*GP*TP*GP*GP*AP*GP*AP*AP*GP*TP*CP*TP*GP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,45915
Polymers49,8514
Non-polymers60811
Water2,954164
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12660 Å2
ΔGint-108 kcal/mol
Surface area17640 Å2
MethodPISA
Unit cell
Length a, b, c (Å)45.437, 67.673, 83.525
Angle α, β, γ (deg.)90.00, 96.75, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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DNA endonuclease I- ... , 2 types, 2 molecules AB

#1: Protein DNA endonuclease I-CreI / 23S rRNA intron protein


Mass: 17528.154 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlamydomonas reinhardtii (plant) / Production host: Escherichia coli (E. coli)
References: UniProt: P05725*PLUS, Hydrolases; Acting on ester bonds
#2: Protein DNA endonuclease I-CreI / 23S rRNA intron protein


Mass: 17583.205 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlamydomonas reinhardtii (plant) / Production host: Escherichia coli (E. coli)
References: UniProt: P05725*PLUS, Hydrolases; Acting on ester bonds

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DNA chain , 2 types, 2 molecules DF

#3: DNA chain DNA (5'-D(*TP*CP*AP*GP*AP*CP*TP*TP*CP*TP*CP*CP*AP*CP*AP*GP*GP*AP*GP*TP*CP*AP*GP*A)-3')


Mass: 7338.757 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (5'-D(*TP*CP*TP*GP*AP*CP*TP*CP*CP*TP*GP*TP*GP*GP*AP*GP*AP*AP*GP*TP*CP*TP*GP*A)-3')


Mass: 7400.777 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 175 molecules

#5: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
#6: Chemical
ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C2H3O2
#7: Chemical ChemComp-PGO / S-1,2-PROPANEDIOL


Mass: 76.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O2
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 164 / 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: 2.56 Å3/Da / Density % sol: 51.92 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop
Details: 0.1 M Calcium Acetate 0.1M Sodium Acetate pH=4.6 35% 1,2-propanediol

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06SA / Wavelength: 0.87 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Mar 10, 2011
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.87 Å / Relative weight: 1
ReflectionResolution: 2.15→37.81 Å / Num. obs: 26697 / % possible obs: 100 % / Redundancy: 4.2 % / Rmerge(I) obs: 0.07 / Net I/σ(I): 10.5
Reflection shellResolution: 2.15→2.27 Å / Rmerge(I) obs: 0.46

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Processing

Software
NameVersionClassification
PHENIX1.6_289refinement
XDSdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1G9Y

1g9y


Resolution: 2.15→33.837 Å / SU ML: 0.3 / Cross valid method: FREE R-VALUE / σ(F): 0 / Phase error: 23.68 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2294 1937 7.26 %
Rwork0.1761 --
obs0.1799 26697 97.09 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 51.779 Å2 / ksol: 0.322 e/Å3
Displacement parameters
Baniso -1Baniso -2Baniso -3
1--4.4795 Å20 Å2-4.1299 Å2
2--4.0666 Å2-0 Å2
3---0.4129 Å2
Refinement stepCycle: LAST / Resolution: 2.15→33.837 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2477 978 34 167 3656
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0133677
X-RAY DIFFRACTIONf_angle_d1.1545196
X-RAY DIFFRACTIONf_dihedral_angle_d22.2121420
X-RAY DIFFRACTIONf_chiral_restr0.058586
X-RAY DIFFRACTIONf_plane_restr0.004481
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.15-2.22690.31421740.22442299X-RAY DIFFRACTION91
2.2269-2.3160.2921880.21362373X-RAY DIFFRACTION93
2.316-2.42140.28371950.20622421X-RAY DIFFRACTION96
2.4214-2.5490.27871880.19452463X-RAY DIFFRACTION96
2.549-2.70860.25561930.1982477X-RAY DIFFRACTION97
2.7086-2.91770.28851930.20192500X-RAY DIFFRACTION99
2.9177-3.21110.24992000.1932529X-RAY DIFFRACTION99
3.2111-3.67520.22972010.16142533X-RAY DIFFRACTION99
3.6752-4.62850.17342010.14112569X-RAY DIFFRACTION100
4.6285-33.84070.18542040.15532596X-RAY DIFFRACTION99
Refinement TLS params.Method: refined / Origin x: 11.6627 Å / Origin y: 16.0044 Å / Origin z: 20.2017 Å
111213212223313233
T0.116 Å2-0.0107 Å20.0322 Å2-0.1489 Å20.0306 Å2--0.1604 Å2
L1.861 °2-0.6164 °20.8418 °2-0.7284 °2-0.6189 °2--1.4634 °2
S-0.0004 Å °0.021 Å °0.1416 Å °0.0541 Å °-0.1789 Å °0.0304 Å °-0.0057 Å °0.0543 Å °0.1506 Å °
Refinement TLS groupSelection details: all

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