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- PDB-7rcc: First stage engineered variant of I-OnuI after initial reassembly -

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Basic information

Entry
Database: PDB / ID: 7rcc
TitleFirst stage engineered variant of I-OnuI after initial reassembly
Components
  • (DNA (26-MER)) x 2
  • I-OnuI_e-hPD1-b
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: 2.45 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)R01 GM 139752 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH R01 GM105691 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 ...chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id ..._struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_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-hPD1-b
B: DNA (26-MER)
C: DNA (26-MER)
D: I-OnuI_e-hPD1-b
E: DNA (26-MER)
F: DNA (26-MER)
G: I-OnuI_e-hPD1-b
H: DNA (26-MER)
I: DNA (26-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)153,40720
Polymers152,9669
Non-polymers44111
Water23413
1
A: I-OnuI_e-hPD1-b
E: DNA (26-MER)
F: DNA (26-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,1497
Polymers50,9893
Non-polymers1604
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8310 Å2
ΔGint-106 kcal/mol
Surface area18260 Å2
MethodPISA
2
B: DNA (26-MER)
C: DNA (26-MER)
D: I-OnuI_e-hPD1-b
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,1096
Polymers50,9893
Non-polymers1203
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8190 Å2
ΔGint-90 kcal/mol
Surface area17820 Å2
MethodPISA
3
G: I-OnuI_e-hPD1-b
H: DNA (26-MER)
I: DNA (26-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,1497
Polymers50,9893
Non-polymers1604
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8270 Å2
ΔGint-104 kcal/mol
Surface area18010 Å2
MethodPISA
Unit cell
Length a, b, c (Å)63.841, 119.741, 165.101
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and ((resid 6 through 10 and (name N...
21(chain D and ((resid 6 through 10 and (name N...
31(chain G and (resid 6 through 51 or (resid 52...
12chain B
22chain E
32chain H
13chain C
23chain F
33chain I

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
111ARGARGILEILE(chain A and ((resid 6 through 10 and (name N...AA6 - 105 - 9
121SERSERGLYGLY(chain A and ((resid 6 through 10 and (name N...AA5 - 3004 - 299
131SERSERGLYGLY(chain A and ((resid 6 through 10 and (name N...AA5 - 3004 - 299
141SERSERGLYGLY(chain A and ((resid 6 through 10 and (name N...AA5 - 3004 - 299
151SERSERGLYGLY(chain A and ((resid 6 through 10 and (name N...AA5 - 3004 - 299
211ARGARGILEILE(chain D and ((resid 6 through 10 and (name N...DD6 - 105 - 9
221SERSERARGARG(chain D and ((resid 6 through 10 and (name N...DD4 - 3013 - 300
231SERSERARGARG(chain D and ((resid 6 through 10 and (name N...DD4 - 3013 - 300
241SERSERARGARG(chain D and ((resid 6 through 10 and (name N...DD4 - 3013 - 300
251SERSERARGARG(chain D and ((resid 6 through 10 and (name N...DD4 - 3013 - 300
311ARGARGASNASN(chain G and (resid 6 through 51 or (resid 52...GG6 - 515 - 50
321LYSLYSLYSLYS(chain G and (resid 6 through 51 or (resid 52...GG5251
331ARGARGARGARG(chain G and (resid 6 through 51 or (resid 52...GG6 - 3015 - 300
341ARGARGARGARG(chain G and (resid 6 through 51 or (resid 52...GG6 - 3015 - 300
351ARGARGARGARG(chain G and (resid 6 through 51 or (resid 52...GG6 - 3015 - 300
361ARGARGARGARG(chain G and (resid 6 through 51 or (resid 52...GG6 - 3015 - 300
112DGDGDGDGchain BBB-1 - 241 - 26
212DGDGDGDGchain EEE-1 - 241 - 26
312DGDGDGDGchain HHH-1 - 241 - 26
113DCDCDCDCchain CCC1 - 261 - 26
213DCDCDCDCchain FFF1 - 261 - 26
313DCDCDCDCchain III1 - 261 - 26

NCS ensembles :
ID
1
2
3

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Components

#1: Protein I-OnuI_e-hPD1-b


Mass: 35007.395 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synthetic construct (others) / Production host: Escherichia coli (E. coli)
#2: DNA chain DNA (26-MER)


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


Mass: 7917.066 Da / Num. of mol.: 3 / Source method: obtained synthetically / Source: (synth.) Synthetic construct (others)
#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: Ca
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 13 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Sequence detailsThe wild type homing endonuclease was extensively engineered to generate I-OnuI_e-hPD1-b.

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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: 2.06 Å3/Da / Density % sol: 40.37 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: 100mM TRIS pH 7.5, 50mM ammonium sulfate, 30% pentaerythritol ethoxylate 15/4 EO/OH

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Data collection

DiffractionMean temperature: 108 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.1 / Wavelength: 0.977408 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Dec 11, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.977408 Å / Relative weight: 1
ReflectionResolution: 2.45→50.01 Å / Num. obs: 47055 / % possible obs: 99.9 % / Redundancy: 8.3 % / Rmerge(I) obs: 0.087 / Rpim(I) all: 0.032 / Rrim(I) all: 0.093 / Χ2: 0.989 / Net I/σ(I): 7.9
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.45-2.546.40.53145760.8560.2190.5770.93799.2
2.54-2.647.70.4446380.9050.1670.4720.978100
2.64-2.768.10.34646520.9460.1280.3690.988100
2.76-2.98.60.25846590.970.0930.2751.028100
2.9-3.0990.19446360.9850.0680.2061.04100
3.09-3.328.60.11846830.9960.0440.1261.056100
3.32-3.668.70.08547020.9970.0310.0911.043100
3.66-4.1990.06847240.9980.0240.0730.994100
4.19-5.288.60.05347830.9980.0190.0560.9100
5.28-508.30.04150020.9990.0150.0440.913100

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation4.86 Å50.01 Å
Translation4.86 Å50.01 Å

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Processing

Software
NameVersionClassification
HKL-2000data scaling
PHASER2.8.3phasing
PHENIX1.17.1_3660refinement
HKL-2000data processing
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3QQY

3qqy


Resolution: 2.45→50.01 Å / SU ML: 0.34 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 28.15 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2722 1995 4.25 %
Rwork0.2219 44913 -
obs0.224 46908 99.26 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 92.64 Å2 / Biso mean: 41.771 Å2 / Biso min: 14.25 Å2
Refinement stepCycle: final / Resolution: 2.45→50.01 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6436 3180 11 13 9640
Biso mean--36.54 26.37 -
Num. residues----997
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00410123
X-RAY DIFFRACTIONf_angle_d0.64814364
X-RAY DIFFRACTIONf_dihedral_angle_d27.3992419
X-RAY DIFFRACTIONf_chiral_restr0.0411625
X-RAY DIFFRACTIONf_plane_restr0.0031289
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A2469X-RAY DIFFRACTION5.902TORSIONAL
12D2469X-RAY DIFFRACTION5.902TORSIONAL
13G2469X-RAY DIFFRACTION5.902TORSIONAL
21B768X-RAY DIFFRACTION5.902TORSIONAL
22E768X-RAY DIFFRACTION5.902TORSIONAL
23H768X-RAY DIFFRACTION5.902TORSIONAL
31C768X-RAY DIFFRACTION5.902TORSIONAL
32F768X-RAY DIFFRACTION5.902TORSIONAL
33I768X-RAY DIFFRACTION5.902TORSIONAL
LS refinement shell

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

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.45-2.510.34871300.2943291092
2.51-2.580.37161410.28263183100
2.58-2.660.31741410.27063190100
2.66-2.740.30591420.25983179100
2.74-2.840.34891420.26013185100
2.84-2.950.29241430.26033205100
2.95-3.090.34741410.26983207100
3.09-3.250.33191440.24933202100
3.25-3.460.23831410.21643205100
3.46-3.720.26641440.21523229100
3.72-4.10.28361450.19893243100
4.1-4.690.21461430.17683252100
4.69-5.910.21961480.19543317100
5.91-500.24251500.2084340699

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