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- PDB-5thg: Engineered variant of I-OnuI meganuclease targeting the HIV CCR5 ... -

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Basic information

Entry
Database: PDB / ID: 5thg
TitleEngineered variant of I-OnuI meganuclease targeting the HIV CCR5 gene; harbors 43 point mutations relative to wild-type I-OnuI
Components
  • (DNA (29-MER)) x 2
  • I-OnuI_e-hCCR5
KeywordsHYDROLASE/DNA / Meganuclease / Engineered protein / DNA complex / 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 / Resolution: 3.106 Å
AuthorsHallinan, J.P. / Stoddard, B.L.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH R01 GM105691 United States
Bill & Melinda Gates FoundationTarget Malaria Program United States
bluebird bio, inc.Corporate Funding United States
CitationJournal: to be published
Title: The structural basis of altered gene specificity resulting from meganuclease and MegaTAL engineering
Authors: Werther, R. / Hallinan, J.P. / Lambert, A. / Haven, K. / Jarjor, J. / Stoddard, B.L.
History
DepositionSep 29, 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 6, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / 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: I-OnuI_e-hCCR5
B: DNA (29-MER)
C: DNA (29-MER)
D: I-OnuI_e-hCCR5
E: DNA (29-MER)
F: DNA (29-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)106,29512
Polymers105,9506
Non-polymers3456
Water21612
1
A: I-OnuI_e-hCCR5
B: DNA (29-MER)
C: DNA (29-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,1476
Polymers52,9753
Non-polymers1723
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8440 Å2
ΔGint-57 kcal/mol
Surface area19740 Å2
MethodPISA
2
D: I-OnuI_e-hCCR5
E: DNA (29-MER)
F: DNA (29-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,1476
Polymers52,9753
Non-polymers1723
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8270 Å2
ΔGint-55 kcal/mol
Surface area19650 Å2
MethodPISA
Unit cell
Length a, b, c (Å)75.959, 112.351, 122.879
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 1 types, 2 molecules AD

#1: Protein I-OnuI_e-hCCR5


Mass: 35144.617 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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DNA chain , 2 types, 4 molecules BECF

#2: DNA chain DNA (29-MER)


Mass: 8781.647 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (29-MER)


Mass: 9048.833 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 18 molecules

#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
#5: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 12 / 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: 2.53 Å3/Da / Density % sol: 51.37 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 6.6
Details: 28% PEG 8000, 100mM HEPES pH 6.6, 5mM Calcium Chloride

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.54 Å
DetectorType: RIGAKU SATURN 944+ / Detector: CCD / Date: Dec 12, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 3.1→47.77 Å / Num. obs: 19313 / % possible obs: 100 % / Redundancy: 7.2 % / CC1/2: 0.924 / Rmerge(I) obs: 0.157 / Net I/σ(I): 12.7
Reflection shellResolution: 3.1→3.21 Å / Redundancy: 6.8 % / Rmerge(I) obs: 0.694 / Mean I/σ(I) obs: 2.2 / CC1/2: 0.924 / % possible all: 99.9

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Processing

Software
NameVersionClassification
PHENIX(1.10_2155)refinement
HKL-2000data reduction
HKL-2000data scaling
PHENIX(1.10_2155)phasing
RefinementResolution: 3.106→47.769 Å / SU ML: 0.4 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 34.28
RfactorNum. reflection% reflection
Rfree0.2788 1930 9.99 %
Rwork0.2206 --
obs0.2264 19313 98.65 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 3.106→47.769 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4410 2318 16 12 6756
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0067102
X-RAY DIFFRACTIONf_angle_d0.6910100
X-RAY DIFFRACTIONf_dihedral_angle_d22.4463759
X-RAY DIFFRACTIONf_chiral_restr0.0431155
X-RAY DIFFRACTIONf_plane_restr0.004887
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.1063-3.1840.42051250.31941136X-RAY DIFFRACTION93
3.184-3.27010.39541360.27841221X-RAY DIFFRACTION98
3.2701-3.36630.36881320.26931193X-RAY DIFFRACTION97
3.3663-3.47490.29391370.24111231X-RAY DIFFRACTION99
3.4749-3.5990.30721380.25491241X-RAY DIFFRACTION100
3.599-3.74310.27621370.23261228X-RAY DIFFRACTION100
3.7431-3.91340.29371390.23181241X-RAY DIFFRACTION100
3.9134-4.11960.28111390.2191254X-RAY DIFFRACTION100
4.1196-4.37750.30241400.19921262X-RAY DIFFRACTION100
4.3775-4.71520.22741380.19941250X-RAY DIFFRACTION100
4.7152-5.18920.24371400.19811259X-RAY DIFFRACTION100
5.1892-5.93890.32341410.20491275X-RAY DIFFRACTION100
5.9389-7.47780.23171430.2291286X-RAY DIFFRACTION100
7.4778-47.77490.2311450.18641306X-RAY DIFFRACTION96

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