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- PDB-6uwg: Engineered variant of I-OnuI meganuclease with improved thermosta... -

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Basic information

Entry
Database: PDB / ID: 6uwg
TitleEngineered variant of I-OnuI meganuclease with improved thermostability and E178D mutation at catalytic site
Components
  • (DNA (26-MER)) x 2
  • I-OnuI-e-Therm-E178D
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 / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.22 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_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-Therm-E178D
B: DNA (26-MER)
C: DNA (26-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,20910
Polymers50,8633
Non-polymers3477
Water1,58588
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9860 Å2
ΔGint-95 kcal/mol
Surface area18090 Å2
MethodPISA
Unit cell
Length a, b, c (Å)39.324, 73.516, 163.526
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 1 types, 1 molecules A

#1: Protein I-OnuI-e-Therm-E178D


Mass: 34890.535 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)

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DNA chain , 2 types, 2 molecules BC

#2: DNA chain DNA (26-MER)


Mass: 7919.111 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (26-MER)


Mass: 8053.245 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 95 molecules

#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
#5: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H6O2
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 88 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 2.32 Å3/Da / Density % sol: 47.07 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 200mM Calcium acetate, 100mM sodium acetate pH 5.5, 100mM sodium acetate pH 6.0, 35% PEG 400

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Data collection

DiffractionMean temperature: 108 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.5418 Å
DetectorType: RIGAKU SATURN 944+ / Detector: CCD / Date: Oct 4, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.22→50 Å / Num. obs: 24263 / % possible obs: 99.5 % / Redundancy: 6.5 % / Rmerge(I) obs: 0.057 / Rpim(I) all: 0.024 / Rrim(I) all: 0.062 / Χ2: 0.933 / Net I/σ(I): 11.4 / Num. measured all: 158867
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.22-2.350.34123070.9090.1660.3810.82595.8
2.3-2.396.20.2923660.9520.1250.3160.848100
2.39-2.56.40.23823860.9670.1010.2590.894100
2.5-2.636.50.18324280.9830.0770.1980.941100
2.63-2.86.70.14523920.9890.060.1570.92100
2.8-3.016.80.10424210.9950.0430.1130.959100
3.01-3.3270.06224190.9990.0250.0671.011100
3.32-3.870.04424400.9990.0180.0480.926100
3.8-4.7870.03624850.9990.0150.0390.977100
4.78-506.70.03326190.9990.0140.0360.96998.9

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation2.77 Å24.43 Å
Translation2.77 Å24.43 Å

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Processing

Software
NameVersionClassification
HKL-2000data scaling
PHASER2.8.1phasing
PHENIX1.17_3644refinement
PDB_EXTRACT3.25data extraction
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3QQY

3qqy


Resolution: 2.22→24.43 Å / SU ML: 0.25 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 22.97
RfactorNum. reflection% reflection
Rfree0.2406 2000 8.27 %
Rwork0.1932 --
obs0.197 24196 99.86 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 70.88 Å2 / Biso mean: 34.1765 Å2 / Biso min: 18.48 Å2
Refinement stepCycle: final / Resolution: 2.22→24.43 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2338 1060 34 88 3520
Biso mean--37.24 30.44 -
Num. residues----350
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.22-2.280.27811380.2141539167799
2.28-2.340.28971420.21215641706100
2.34-2.410.2761390.214215391678100
2.41-2.490.28211410.218615831724100
2.49-2.570.2811400.215715521692100
2.57-2.680.26221410.217315631704100
2.68-2.80.28261430.218615861729100
2.8-2.950.26991410.225515601701100
2.95-3.130.2691400.223715631703100
3.13-3.370.22381450.202715911736100
3.37-3.710.23541430.18815861729100
3.71-4.240.24091440.169916041748100
4.24-5.340.19041470.165116361783100
5.34-24.430.22141560.176317301886100

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