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- PDB-6uwk: Engineered variant of I-OnuI meganuclease with improved stability... -

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Basic information

Entry
Database: PDB / ID: 6uwk
TitleEngineered variant of I-OnuI meganuclease with improved stability and fully altered specificity targeting human chromosome 11 trans integration site
Components
  • (DNA (27-MER)) x 2
  • I-OnuI-e-Therm-hChr11v3
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: 2.533 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 / _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-hChr11v3
B: DNA (27-MER)
C: DNA (27-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,25412
Polymers50,8723
Non-polymers3839
Water55831
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9780 Å2
ΔGint-146 kcal/mol
Surface area17320 Å2
MethodPISA
Unit cell
Length a, b, c (Å)38.370, 62.635, 160.199
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-hChr11v3


Mass: 34275.969 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 (27-MER)


Mass: 8204.271 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (27-MER)


Mass: 8391.412 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 40 molecules

#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca
#5: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 31 / 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: 1.89 Å3/Da / Density % sol: 34.99 %
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: Feb 14, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.53→50 Å / Num. obs: 13421 / % possible obs: 99 % / Redundancy: 11.2 % / Biso Wilson estimate: 37.22 Å2 / Rmerge(I) obs: 0.071 / Rpim(I) all: 0.022 / Rrim(I) all: 0.075 / Χ2: 1.056 / Net I/σ(I): 10.8 / Num. measured all: 150302
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.53-2.628.70.33512120.9610.1160.3561.02191.7
2.62-2.73110.31113230.9720.0980.3261.047100
2.73-2.8511.30.25813060.9830.080.2711.044100
2.85-311.30.1913320.9910.0580.1991.076100
3-3.1911.40.13313620.9970.0410.141.086100
3.19-3.4311.30.07613140.9990.0230.0791.1100
3.43-3.7811.30.06113480.9990.0190.0641.06699.9
3.78-4.3311.40.04813680.9990.0150.051.02999.9
4.33-5.4511.80.04413810.9990.0130.0461.041100
5.45-50120.037147510.0110.0391.04598.9

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation3.52 Å28.52 Å
Translation3.52 Å28.52 Å

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Processing

Software
NameVersionClassification
HKL-2000data scaling
PHASER2.8.1phasing
PHENIX1.13_2998refinement
PDB_EXTRACT3.24data extraction
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3QQY

3qqy


Resolution: 2.533→28.524 Å / SU ML: 0.36 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 28.86
RfactorNum. reflection% reflection
Rfree0.2881 1336 10 %
Rwork0.2334 --
obs0.2389 13362 98.96 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 53.7 Å2 / Biso mean: 33.2777 Å2 / Biso min: 24.77 Å2
Refinement stepCycle: final / Resolution: 2.533→28.524 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2273 1022 18 31 3344
Biso mean--35.18 31.15 -
Num. residues----344
LS refinement shell

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

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.5333-2.62310.33411200.2866108090
2.6231-2.72810.37451320.27931190100
2.7281-2.85210.38361310.28591174100
2.8521-3.00230.35951320.28071194100
3.0023-3.19020.32891370.27091222100
3.1902-3.43610.25911290.22311176100
3.4361-3.78120.27011340.2131210100
3.7812-4.32670.23831370.20081230100
4.3267-5.4450.26531370.21031232100
5.445-28.5240.28011470.23181318100

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