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- PDB-2xe0: Molecular basis of engineered meganuclease targeting of the endog... -

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Basic information

Entry
Database: PDB / ID: 2xe0
TitleMolecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus
Components
  • (24MER DNA) x 2
  • (I-CREI V2V3 VARIANT) x 2
KeywordsDNA BINDING PROTEIN/DNA / DNA BINDING PROTEIN-DNA COMPLEX / HOMING ENDONUCLEASES / DOUBLE-STRAND BREAK / HOMOLOGOUS RECOMBINATION / HUMAN RAG1 GENE / SEVERE COMBINED IMMUNODEFICIENCY (SCID) / DNA BINDING PROTEIN
Function / homologyHoming endonucleases / Endonuclease I-creI / Roll / Alpha Beta / ACETATE ION / S-1,2-PROPANEDIOL / DNA / DNA (> 10)
Function and homology information
Biological speciesCHLAMYDOMONAS REINHARDTII (plant)
HOMO SAPIENS (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.31 Å
AuthorsMunoz, I.G. / Prieto, J. / Subramanian, S. / Coloma, J. / Redondo, P. / Villate, M. / Merino, N. / Marenchino, M. / D'Abramo, M. / Gervasio, F.L. ...Munoz, I.G. / Prieto, J. / Subramanian, S. / Coloma, J. / Redondo, P. / Villate, M. / Merino, N. / Marenchino, M. / D'Abramo, M. / Gervasio, F.L. / Grizot, S. / Daboussi, F. / Smith, J. / Chion-Sotine, I. / Paques, F. / Duchateau, P. / Alibes, A. / Stricher, F. / Serrano, L. / Blanco, F.J. / Montoya, G.
CitationJournal: Nucleic Acids Res. / Year: 2011
Title: Molecular Basis of Engineered Meganuclease Targeting of the Endogenous Human Rag1 Locus
Authors: Munoz, I.G. / Prieto, J. / Subramanian, S. / Coloma, J. / Redondo, P. / Villate, M. / Merino, N. / Marenchino, M. / D'Abramo, M. / Gervasio, F.L. / Grizot, S. / Daboussi, F. / Smith, J. / ...Authors: Munoz, I.G. / Prieto, J. / Subramanian, S. / Coloma, J. / Redondo, P. / Villate, M. / Merino, N. / Marenchino, M. / D'Abramo, M. / Gervasio, F.L. / Grizot, S. / Daboussi, F. / Smith, J. / Chion-Sotine, I. / Paques, F. / Duchateau, P. / Alibes, A. / Stricher, F. / Serrano, L. / Blanco, F.J. / Montoya, G.
History
DepositionMay 10, 2010Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 29, 2010Provider: repository / Type: Initial release
Revision 1.1May 8, 2011Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Dec 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 / pdbx_struct_conn_angle / struct_conn / 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 / _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 / _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: I-CREI V2V3 VARIANT
B: I-CREI V2V3 VARIANT
C: 24MER DNA
D: 24MER DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,19312
Polymers48,8074
Non-polymers3858
Water1,13563
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12630 Å2
ΔGint-97.3 kcal/mol
Surface area16990 Å2
MethodPISA
2
C: 24MER DNA
D: 24MER DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)14,8125
Polymers14,7402
Non-polymers733
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2850 Å2
ΔGint-44.2 kcal/mol
Surface area9380 Å2
MethodPISA
3
A: I-CREI V2V3 VARIANT
B: I-CREI V2V3 VARIANT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,3807
Polymers34,0682
Non-polymers3125
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1880 Å2
ΔGint-9.1 kcal/mol
Surface area15520 Å2
MethodPISA
Unit cell
Length a, b, c (Å)177.857, 71.538, 45.005
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP21212

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein I-CREI V2V3 VARIANT


Mass: 17068.553 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) / Strain (production host): PET24D
#2: Protein I-CREI V2V3 VARIANT


Mass: 16999.404 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) / Strain (production host): PET24D

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DNA chain , 2 types, 2 molecules CD

#3: DNA chain 24MER DNA / 5'-D(*TP*TP*GP*TP*TP*CP*TP*CP*AP*GP*GP*TP *AP*CP*CP*TP*CP*AP*GP*CP*CP*AP*GP*A)-3'


Mass: 7320.728 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) HOMO SAPIENS (human)
#4: DNA chain 24MER DNA / 5'-D(*TP*CP*TP*GP*GP*CP*TP*GP*AP*GP*GP*TP*AP*CP CP*TP*GP*AP*GP*AP*AP*CP*AP*A)-3'


Mass: 7418.804 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) HOMO SAPIENS (human)

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Non-polymers , 4 types, 71 molecules

#5: Chemical
ChemComp-ACT / ACETATE ION / Acetate


Mass: 59.044 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H3O2
#6: Chemical ChemComp-PGO / S-1,2-PROPANEDIOL / Propanediol


Mass: 76.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O2
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 63 / Source method: isolated from a natural source / Formula: H2O

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Details

Sequence detailsAUTHOR MENTIONED MUTATIONS TO THE CRYSTALLIZED SEQUENCE AT THE FOLLOWING POSITIONS V2 ...AUTHOR MENTIONED MUTATIONS TO THE CRYSTALLIZED SEQUENCE AT THE FOLLOWING POSITIONS V2 N30R,Y33N,Q44A,R68Y,R70S, I77R V3 K28N,Y33S,Q38R,S40R,Q44Y,R70S,D75Q,I77V

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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: 2.95 Å3/Da / Density % sol: 57.91 % / Description: NONE

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Type: SLS / Wavelength: 0.98
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 2.31→35.76 Å / Num. obs: 24763 / % possible obs: 74.4 % / Observed criterion σ(I): 2 / Redundancy: 2.7 % / Biso Wilson estimate: 49.04 Å2 / Rmerge(I) obs: 0.44 / Net I/σ(I): 1.1
Reflection shellResolution: 2.4→35.76 Å / Redundancy: 5.3 % / Rmerge(I) obs: 0.06 / Mean I/σ(I) obs: 12.1 / % possible all: 94.9

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Processing

Software
NameVersionClassification
PHENIX(PHENIX.REFINE)refinement
XDSdata reduction
XSCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1G9Z

1g9z


Resolution: 2.31→35.769 Å / SU ML: 0.32 / σ(F): 1.34 / Phase error: 27.01 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2557 1239 5 %
Rwork0.2131 --
obs0.2152 24762 95.07 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 48.687 Å2 / ksol: 0.35 e/Å3
Displacement parameters
Baniso -1Baniso -2Baniso -3
1-0 Å20 Å20 Å2
2--0 Å20 Å2
3---0 Å2
Refinement stepCycle: LAST / Resolution: 2.31→35.769 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2405 980 24 63 3472
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0083563
X-RAY DIFFRACTIONf_angle_d1.3095025
X-RAY DIFFRACTIONf_dihedral_angle_d22.9341352
X-RAY DIFFRACTIONf_chiral_restr0.068579
X-RAY DIFFRACTIONf_plane_restr0.004472
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.3101-2.40260.31991030.29532036X-RAY DIFFRACTION75
2.4026-2.51190.31321170.2682392X-RAY DIFFRACTION89
2.5119-2.64430.35691280.25342625X-RAY DIFFRACTION96
2.6443-2.80990.3131280.24332689X-RAY DIFFRACTION98
2.8099-3.02670.26231510.22352691X-RAY DIFFRACTION99
3.0267-3.33110.24551520.21032688X-RAY DIFFRACTION99
3.3311-3.81270.27211540.19512741X-RAY DIFFRACTION99
3.8127-4.80180.22071620.16892765X-RAY DIFFRACTION100
4.8018-35.77330.22751440.20552896X-RAY DIFFRACTION98

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