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

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Basic information

Entry
Database: PDB / ID: 6uwh
TitleIntermediate engineered variant of I-OnuI meganuclease with improved thermostability and partially altered specificity
Components
  • (DNA (26-MER)) x 2
  • I-OnuI-e-Therm-hChr11v1
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: 2.299 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.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-hChr11v1
B: DNA (26-MER)
C: DNA (26-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,3098
Polymers50,0873
Non-polymers2225
Water64936
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9110 Å2
ΔGint-107 kcal/mol
Surface area18200 Å2
MethodPISA
Unit cell
Length a, b, c (Å)38.890, 72.985, 163.574
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-hChr11v1


Mass: 34113.660 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: 7935.110 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (26-MER)


Mass: 8038.234 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 41 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.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 36 / 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: 46.92 %
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: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.5418 Å
DetectorType: RIGAKU SATURN 944+ / Detector: CCD / Date: May 17, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.299→50 Å / Num. obs: 21003 / % possible obs: 97.4 % / Redundancy: 8.6 % / Biso Wilson estimate: 40.54 Å2 / Rmerge(I) obs: 0.058 / Rpim(I) all: 0.02 / Rrim(I) all: 0.061 / Χ2: 0.996 / Net I/σ(I): 12.9
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.3-2.387.20.42820530.9080.1660.4610.85599.3
2.38-2.488.60.37821020.9510.1310.4020.999
2.48-2.598.80.28321020.9710.0970.30.95298.5
2.59-2.738.80.22520500.9820.0770.2390.9698.2
2.73-2.98.50.17220560.9880.060.1831.00396.6
2.9-3.128.10.11420370.9950.040.1211.11195.5
3.12-3.447.70.06220500.9980.0220.0661.00794.6
3.44-3.937.60.05120410.9990.0190.0551.02494
3.93-4.958.60.03821860.9990.0140.0411.04699.5
4.95-5011.50.03232610.0090.0321.05599.1

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation2.3 Å27.23 Å
Translation2.3 Å27.23 Å

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Processing

Software
NameVersionClassification
HKL-2000data scaling
PHASER2.8.1phasing
PHENIX1.13_2998refinement
PDB_EXTRACT3.25data extraction
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3QQY

3qqy


Resolution: 2.299→27.228 Å / SU ML: 0.3 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 25.36
RfactorNum. reflection% reflection
Rfree0.2502 1991 9.52 %
Rwork0.2038 --
obs0.2082 20908 97.2 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 83.25 Å2 / Biso mean: 41.0903 Å2 / Biso min: 19.74 Å2
Refinement stepCycle: final / Resolution: 2.299→27.228 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2280 1060 14 36 3390
Biso mean--52.83 31.72 -
Num. residues----346
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.299-2.35630.33791380.2535130597
2.3563-2.420.32511440.2415137199
2.42-2.49120.3461400.2399132399
2.4912-2.57150.28621420.2281134798
2.5715-2.66330.26961430.234135498
2.6633-2.76990.27221410.2373133697
2.7699-2.89580.31591380.2379130997
2.8958-3.04830.28491410.2373133295
3.0483-3.2390.26231350.2345129594
3.239-3.48860.26471380.2126130694
3.4886-3.83880.22591390.2004130994
3.8388-4.39230.21771410.1707138298
4.3923-5.52620.21681520.17261438100
5.5262-27.2280.21731590.18221510100

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