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- PDB-5t8d: Engineered variant of I-OnuI meganuclease targeting the HIV integ... -

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Basic information

Entry
Database: PDB / ID: 5t8d
TitleEngineered variant of I-OnuI meganuclease targeting the HIV integrase gene; harbors 47 point mutations relative to wild-type I-OnuI
Components
  • (DNA (26-MER)) x 2
  • I-OnuI_e-vHIVInt_v2
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: 2.15 Å
AuthorsStoddard, B.L. / Werther, R. / Lambert, A.R.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM105691 United States
bluebird bio, inc. (Corporate funding) United States
CitationJournal: Protein Eng.Des.Sel. / Year: 2017
Title: Tuning DNA binding affinity and cleavage specificity of an engineered gene-targeting nuclease via surface display, flow cytometry and cellular analyses.
Authors: Niyonzima, N. / Lambert, A.R. / Werther, R. / De Silva Feelixge, H. / Roychoudhury, P. / Greninger, A.L. / Stone, D. / Stoddard, B.L. / Jerome, K.R.
History
DepositionSep 7, 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 1.3Mar 18, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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
Revision 1.4Oct 4, 2023Group: 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.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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: I-OnuI_e-vHIVInt_v2
B: DNA (26-MER)
C: DNA (26-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,5216
Polymers50,4013
Non-polymers1203
Water4,666259
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9230 Å2
ΔGint-95 kcal/mol
Surface area18320 Å2
MethodPISA
Unit cell
Length a, b, c (Å)39.701, 75.007, 165.158
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-vHIVInt_v2


Mass: 34426.754 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: 8036.203 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (26-MER)


Mass: 7938.127 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.: 3 / Source method: obtained synthetically / Formula: Ca
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 259 / 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: 2.44 Å3/Da / Density % sol: 49.58 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7
Details: 0.2 M Ammonium sulfate, 0.1 M HEPES pH 7.0, 22.5% w/v Polyethylene glycol 3,350

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Data collection

DiffractionMean temperature: 108 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Mar 3, 2016
RadiationMonochromator: Double crystal, Si(111) liquid N2 cooled / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.15→50 Å / Num. obs: 27792 / % possible obs: 99.9 % / Redundancy: 12.6 % / Biso Wilson estimate: 34.29 Å2 / Rmerge(I) obs: 0.093 / Rpim(I) all: 0.027 / Rrim(I) all: 0.097 / Χ2: 0.87 / Net I/av σ(I): 29.278 / Net I/σ(I): 6.7 / Num. measured all: 351202
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.15-2.23110.61827070.9040.1930.6490.75399.2
2.23-2.3212.30.53127130.9540.1570.5550.76199.9
2.32-2.4213.40.48127330.9660.1360.50.768100
2.42-2.5513.10.3427320.9830.0970.3540.788100
2.55-2.7112.60.25127490.9870.0730.2610.808100
2.71-2.92130.17627460.9950.0510.1830.847100
2.92-3.2113.30.11227740.9960.0320.1170.895100
3.21-3.6812.50.06428050.9980.0190.0671.06100
3.68-4.6313.20.05228230.9990.0150.0540.979100
4.63-50120.04730100.9990.0140.0491.008100

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation2.15 Å44.38 Å
Translation2.15 Å44.38 Å

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Processing

Software
NameVersionClassification
PHENIX1.10.1_2155refinement
HKL-2000data scaling
PHASER2.6.0phasing
PDB_EXTRACT3.2data extraction
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3QQY

3qqy


Resolution: 2.15→44.381 Å / SU ML: 0.22 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 25.33 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2281 1384 5 %
Rwork0.1824 26310 -
obs0.1847 27694 99.77 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 93.65 Å2 / Biso mean: 37.7676 Å2 / Biso min: 19.51 Å2
Refinement stepCycle: final / Resolution: 2.15→44.381 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2335 1066 3 259 3663
Biso mean--32.53 39.31 -
Num. residues----344
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0073576
X-RAY DIFFRACTIONf_angle_d0.9965051
X-RAY DIFFRACTIONf_chiral_restr0.058565
X-RAY DIFFRACTIONf_plane_restr0.006459
X-RAY DIFFRACTIONf_dihedral_angle_d21.2491935
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 10

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.1497-2.22650.31021360.23642572270899
2.2265-2.31570.28021350.21925752710100
2.3157-2.4210.27861360.223625912727100
2.421-2.54870.24791370.219425932730100
2.5487-2.70830.30441370.223426092746100
2.7083-2.91740.30541360.220326022738100
2.9174-3.21090.29881390.21726312770100
3.2109-3.67540.2281390.17422631277099
3.6754-4.62980.16491400.140526742814100
4.6298-44.39080.17451490.152328322981100

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