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- PDB-6uwj: Intermediate engineered variant of I-OnuI meganuclease with impro... -

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Basic information

Entry
Database: PDB / ID: 6uwj
TitleIntermediate engineered variant of I-OnuI meganuclease with improved thermostability and partially altered specificity
Components
  • (DNA (27-MER)) x 2
  • I-OnuI-e-Therm-hChr11v2
KeywordsDNA BINDING PROTEIN/DNA / Meganuclease / Homing Endonuclease / DNA BINDING PROTEIN / DNA BINDING PROTEIN-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: 1.852 Å
AuthorsWerther, R. / Stoddard, B.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM105691 United States
CitationJournal: Structure / Year: 2020
Title: Optimization of Protein Thermostability and Exploitation of Recognition Behavior to Engineer Altered Protein-DNA Recognition.
Authors: Lambert, A.R. / Hallinan, J.P. / Werther, R. / Glow, D. / Stoddard, B.L.
History
DepositionNov 5, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 18, 2019Provider: repository / Type: Initial release
Revision 1.1May 13, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jul 22, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Oct 11, 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_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr2_symmetry / _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_conn.ptnr2_symmetry

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: I-OnuI-e-Therm-hChr11v2
B: DNA (27-MER)
C: DNA (27-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,8997
Polymers50,7383
Non-polymers1604
Water2,504139
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9240 Å2
ΔGint-104 kcal/mol
Surface area17230 Å2
MethodPISA
Unit cell
Length a, b, c (Å)38.288, 62.586, 157.405
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-Therm-hChr11v2


Mass: 34144.758 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)
#2: DNA chain DNA (27-MER)


Mass: 8234.293 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (27-MER)


Mass: 8359.414 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.: 139 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 1.86 Å3/Da / Density % sol: 33.82 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 100mM sodium acetate pH 6.0, 200mM calcium acetate, 35% PEG 400

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Data collection

DiffractionMean temperature: 108 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.1 / Wavelength: 0.977 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Apr 25, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.977 Å / Relative weight: 1
ReflectionResolution: 1.85→50 Å / Num. obs: 33122 / % possible obs: 99.7 % / Redundancy: 11.3 % / Rmerge(I) obs: 0.035 / Rpim(I) all: 0.011 / Rrim(I) all: 0.037 / Χ2: 0.998 / Net I/σ(I): 20.2 / Num. measured all: 374757
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.85-1.927.30.16231660.9880.0630.1750.86996.7
1.92-1.999.50.13532540.9950.0460.1430.874100
1.99-2.0811.50.10232640.9970.0310.1070.929100
2.08-2.1912.10.0832580.9990.0240.0840.997100
2.19-2.3312.10.06832940.9990.020.0711.147100
2.33-2.5112.30.05532900.9990.0160.0571.093100
2.51-2.7612.10.04632970.9990.0140.0481.016100
2.76-3.1612.50.03833580.9990.0110.0391.075100
3.16-3.9911.90.032336310.010.0330.895100
3.99-5011.60.02735780.9990.0080.0280.98499.9

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation3.32 Å48.99 Å
Translation3.32 Å48.99 Å

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Processing

Software
NameVersionClassification
SCALEPACKdata scaling
PHASER2.8.1phasing
PHENIX1.13refinement
PDB_EXTRACT3.25data extraction
DENZOdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3QQY

3qqy


Resolution: 1.852→48.985 Å / SU ML: 0.2 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 23.49
RfactorNum. reflection% reflection
Rfree0.2342 1998 6.05 %
Rwork0.1909 --
obs0.1935 33033 99.55 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 79.58 Å2 / Biso mean: 29.35 Å2 / Biso min: 14.75 Å2
Refinement stepCycle: final / Resolution: 1.852→48.985 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2283 1025 4 139 3451
Biso mean--23.05 28.65 -
Num. residues----343
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.013477
X-RAY DIFFRACTIONf_angle_d1.1364917
X-RAY DIFFRACTIONf_dihedral_angle_d17.0221874
X-RAY DIFFRACTIONf_chiral_restr0.062560
X-RAY DIFFRACTIONf_plane_restr0.008446
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8516-1.89790.29861350.2092093X-RAY DIFFRACTION95
1.8979-1.94930.28311400.20032177X-RAY DIFFRACTION100
1.9493-2.00660.23411420.19152189X-RAY DIFFRACTION100
2.0066-2.07140.25671390.1872171X-RAY DIFFRACTION100
2.0714-2.14540.23541430.18622223X-RAY DIFFRACTION100
2.1454-2.23130.2671410.19282184X-RAY DIFFRACTION100
2.2313-2.33290.25661400.21122185X-RAY DIFFRACTION100
2.3329-2.45590.26781430.20852207X-RAY DIFFRACTION100
2.4559-2.60970.27461430.21992236X-RAY DIFFRACTION100
2.6097-2.81120.24931430.2222203X-RAY DIFFRACTION100
2.8112-3.09410.27761440.21762237X-RAY DIFFRACTION100
3.0941-3.54170.241440.18832242X-RAY DIFFRACTION99
3.5417-4.46170.17551470.16622279X-RAY DIFFRACTION100
4.4617-49.00250.21281540.17442409X-RAY DIFFRACTION100

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