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- PDB-5t2n: Engineered variant of I-OnuI meganuclease targeting the Anopheles... -

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Basic information

Entry
Database: PDB / ID: 5t2n
TitleEngineered variant of I-OnuI meganuclease targeting the Anopheles AGAP007280 gene; harbors 38 point mutations relative to wild-type I-OnuI
Components
  • (DNA (26-MER)) x 2
  • I-OnuI_e-ag007820
KeywordsHydrolase/DNA / Meganuclease / Engineered protein / DNA complex / Homing Endonuclease / Hydrolase-DNA complex
Function / homologyHoming endonucleases / Endonuclease I-creI / Roll / Alpha Beta / DNA / DNA (> 10)
Function and homology information
Biological speciessynthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.079 Å
AuthorsStoddard, B.L. / Werther, R.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM105691 United States
Bill & Melinda Gates Foundation United States
bluebird bio, inc. (Corporate Funding) United States
CitationJournal: Nucleic Acids Res. / Year: 2017
Title: Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity.
Authors: Werther, R. / Hallinan, J.P. / Lambert, A.R. / Havens, K. / Pogson, M. / Jarjour, J. / Galizi, R. / Windbichler, N. / Crisanti, A. / Nolan, T. / Stoddard, B.L.
History
DepositionAug 23, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 3, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 27, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Jun 23, 2021Group: Database references / Derived calculations
Category: citation / citation_author ...citation / citation_author / pdbx_struct_conn_angle / struct_conn
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _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.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_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
Revision 1.4Oct 4, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: I-OnuI_e-ag007820
B: DNA (26-MER)
C: DNA (26-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,3317
Polymers50,1713
Non-polymers1604
Water1,06359
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9410 Å2
ΔGint-114 kcal/mol
Surface area17710 Å2
MethodPISA
Unit cell
Length a, b, c (Å)40.834, 67.421, 166.883
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein I-OnuI_e-ag007820


Mass: 34192.773 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)RIL
#2: DNA chain DNA (26-MER)


Mass: 7802.002 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (26-MER)


Mass: 8176.281 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 59 / 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: 2.29 Å3/Da / Density % sol: 46.27 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 0.05M ammonium sulfate, 0.05M BIS-TRIS pH 6.5, 35% pentaerythritol ethoxylate (15/4 EO/OH)

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Data collection

DiffractionMean temperature: 108 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 1.084 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: May 26, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.084 Å / Relative weight: 1
ReflectionResolution: 2.08→50 Å / Num. obs: 28554 / % possible obs: 99.6 % / Redundancy: 11.9 % / Biso Wilson estimate: 49.74 Å2 / Rmerge(I) obs: 0.094 / Rpim(I) all: 0.028 / Rrim(I) all: 0.098 / Χ2: 0.824 / Net I/av σ(I): 19.082 / Net I/σ(I): 10.1 / Num. measured all: 339574
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.08-2.158.90.79327820.8830.2660.8390.45799
2.15-2.2411.20.65827980.9660.1990.6880.482100
2.24-2.34120.50828100.9730.1510.530.517100
2.34-2.4711.90.35528380.980.1070.3720.585100
2.47-2.6212.80.23128360.9930.0670.2410.762100
2.62-2.8212.80.17528390.9940.0510.1831.109100
2.82-3.1112.30.12628500.9960.0370.1311.1100
3.11-3.5612.80.10428290.9940.0310.1081.09798.3
3.56-4.4812.40.08628960.9960.0250.091.0599.1
4.48-5011.80.07230760.9950.0220.0760.87499.4

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation2.08 Å42.91 Å
Translation2.08 Å42.91 Å

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Processing

Software
NameVersionClassification
PHENIX1.9_1692refinement
HKL-2000data scaling
PHASER2.5.6phasing
PDB_EXTRACT3.2data extraction
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3QQY

3qqy


Resolution: 2.079→42.908 Å / SU ML: 0.29 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 30.66 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2578 1997 7.01 %
Rwork0.2199 26476 -
obs0.2226 28473 99.39 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 111.61 Å2 / Biso mean: 56.1879 Å2 / Biso min: 31.27 Å2
Refinement stepCycle: final / Resolution: 2.079→42.908 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2269 1063 4 59 3395
Biso mean--51.2 48.17 -
Num. residues----343
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0033505
X-RAY DIFFRACTIONf_angle_d0.6974961
X-RAY DIFFRACTIONf_chiral_restr0.029561
X-RAY DIFFRACTIONf_plane_restr0.003446
X-RAY DIFFRACTIONf_dihedral_angle_d22.4461344
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.0785-2.13050.34181380.29731813195197
2.1305-2.18810.35311400.280518521992100
2.1881-2.25250.2971400.284918902030100
2.2525-2.32520.3531400.279518491989100
2.3252-2.40830.32181430.28318932036100
2.4083-2.50470.3651380.292118401978100
2.5047-2.61870.30511430.269818852028100
2.6187-2.75670.35271410.277718872028100
2.7567-2.92940.32371450.271419152060100
2.9294-3.15550.31761440.253418972041100
3.1555-3.47290.27651390.23371849198898
3.4729-3.97510.26361440.20941919206399
3.9751-5.0070.21431470.18791933208099
5.007-42.91720.19131550.17412054220999

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