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- PDB-7rcf: Fourth stage reengineered variant of I-OnuI with stability enhanc... -

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Basic information

Entry
Database: PDB / ID: 7rcf
TitleFourth stage reengineered variant of I-OnuI with stability enhancing substitutions
Components
  • (DNA (26-MER)) x 2
  • I-OnuI_e-hPD1-e
KeywordsDNA BINDING PROTEIN/DNA / meganuclease / gene editing / protein engineering / DNA BINDING PROTEIN-DNA complex
Function / homologyDNA / DNA (> 10)
Function and homology information
Biological speciesSynthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.233 Å
AuthorsUbilla-Rodriguez, N.C. / Werther, R.A. / 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
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH R01 GM 139752 United States
Other privateFunding from bluebird bio United States
CitationJournal: To Be Published
Title: Characterization of the stepwise engineering and optimization of a retargeted DNA binding protein and gene-editing meganuclease
Authors: Werther, R.A. / Ubilla-Rodriguez, N.C. / Smiley, A.T. / Havens, K. / Lambert, A.R. / Stoddard, B.L.
History
DepositionJul 7, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 29, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: I-OnuI_e-hPD1-e
B: DNA (26-MER)
C: DNA (26-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,37311
Polymers51,0083
Non-polymers3658
Water1,49583
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9360 Å2
ΔGint-106 kcal/mol
Surface area18580 Å2
MethodPISA
Unit cell
Length a, b, c (Å)41.550, 64.055, 171.296
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-hPD1-e


Mass: 35026.426 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: 8064.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Synthetic construct (others)
#3: DNA chain DNA (26-MER)


Mass: 7917.066 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Synthetic construct (others)

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Non-polymers , 5 types, 91 molecules

#4: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H6O2
#5: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca
#6: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#7: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 83 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Sequence detailsThe wild type homing endonuclease was extensively engineered to generate I-OnuI_e-hPD1-e.

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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: 2.23 Å3/Da / Density % sol: 44.95 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 100mM HEPES pH 7.5, 200mM ammonium sulfate, 35% PEG 3350

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Data collection

DiffractionMean temperature: 108 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 0.999993 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Oct 17, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.999993 Å / Relative weight: 1
ReflectionResolution: 2.23→50 Å / Num. obs: 23064 / % possible obs: 100 % / Redundancy: 12.2 % / Biso Wilson estimate: 27.04 Å2 / Rmerge(I) obs: 0.06 / Rpim(I) all: 0.018 / Rrim(I) all: 0.063 / Χ2: 0.814 / Net I/σ(I): 13.6 / Num. measured all: 282390
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.23-2.3111.70.17422680.9920.0530.1820.624100
2.31-2.412.90.14122280.9950.0410.1470.606100
2.4-2.5112.70.11922470.9960.0350.1240.631100
2.51-2.64120.09622850.9970.0290.10.734100
2.64-2.8112.60.08422730.9970.0250.0870.852100
2.81-3.0312.70.0722930.9980.0210.0730.998100
3.03-3.3311.80.05922920.9990.0180.0610.931100
3.33-3.8112.60.05123360.9990.0150.0530.882100
3.81-4.812.10.04723400.9990.0140.0490.895100
4.8-5011.40.04925020.9990.0150.0510.96899.8

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation6.32 Å37.38 Å
Translation6.32 Å37.38 Å

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Processing

Software
NameVersionClassification
HKL-2000data scaling
PHASER2.6.0phasing
PHENIX1.10_2155refinement
HKL-2000data processing
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3QQY

3qqy


Resolution: 2.233→37.383 Å / SU ML: 0.22 / Cross valid method: THROUGHOUT / σ(F): 1.37 / Phase error: 22.45 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2294 1997 8.69 %
Rwork0.1898 20985 -
obs0.1933 22982 99.8 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 77.61 Å2 / Biso mean: 28.0081 Å2 / Biso min: 10.34 Å2
Refinement stepCycle: final / Resolution: 2.233→37.383 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2270 1060 35 83 3448
Biso mean--35.92 23.19 -
Num. residues----342
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0053539
X-RAY DIFFRACTIONf_angle_d0.7245004
X-RAY DIFFRACTIONf_chiral_restr0.043558
X-RAY DIFFRACTIONf_plane_restr0.004457
X-RAY DIFFRACTIONf_dihedral_angle_d20.7751911
LS refinement shell

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

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.2333-2.28910.28241400.2104147099
2.2891-2.3510.28171370.20741440100
2.351-2.42020.26481410.20781481100
2.4202-2.49830.27611400.20891468100
2.4983-2.58760.24321410.20581480100
2.5876-2.69110.31681410.20961491100
2.6911-2.81360.24731410.21351474100
2.8136-2.96190.27441430.2151495100
2.9619-3.14730.24511400.21731482100
3.1473-3.39020.21721420.1899149699
3.3902-3.73110.21731430.18721502100
3.7311-4.27030.19241460.1641532100
4.2703-5.37760.19081460.15781536100
5.3776-37.30.19891560.18071638100

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