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

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Basic information

Entry
Database: PDB / ID: 6uvw
TitleEngineered variant of I-OnuI meganuclease with improved thermostability
Components
  • (DNA (27-MER)) x 2
  • I-OnuI-e-Therm
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 / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.551 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 4, 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 / struct_ncs_dom_lim
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_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-Therm
B: DNA (27-MER)
C: DNA (27-MER)
D: I-OnuI-e-Therm
E: DNA (27-MER)
F: DNA (27-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)103,19111
Polymers102,9916
Non-polymers2005
Water41423
1
A: I-OnuI-e-Therm
B: DNA (27-MER)
C: DNA (27-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,6166
Polymers51,4953
Non-polymers1203
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8930 Å2
ΔGint-99 kcal/mol
Surface area18320 Å2
MethodPISA
2
D: I-OnuI-e-Therm
E: DNA (27-MER)
F: DNA (27-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,5755
Polymers51,4953
Non-polymers802
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8460 Å2
ΔGint-89 kcal/mol
Surface area18330 Å2
MethodPISA
Unit cell
Length a, b, c (Å)75.426, 82.259, 167.398
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 9 through 30 or (resid 31...
21(chain D and (resid 9 through 58 or (resid 59...
12chain C
22chain F
13chain B
23(chain E and ((resid -1 and (name C4 or name...

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
111SERSERARGARG(chain A and (resid 9 through 30 or (resid 31...AA9 - 308 - 29
121LYSLYSLYSLYS(chain A and (resid 9 through 30 or (resid 31...AA3130
131SERSERPHEPHE(chain A and (resid 9 through 30 or (resid 31...AA9 - 3038 - 302
141SERSERPHEPHE(chain A and (resid 9 through 30 or (resid 31...AA9 - 3038 - 302
151SERSERPHEPHE(chain A and (resid 9 through 30 or (resid 31...AA9 - 3038 - 302
161SERSERPHEPHE(chain A and (resid 9 through 30 or (resid 31...AA9 - 3038 - 302
211SERSERGLUGLU(chain D and (resid 9 through 58 or (resid 59...DD9 - 588 - 57
221ASNASNASNASN(chain D and (resid 9 through 58 or (resid 59...DD5958
231ARGARGPHEPHE(chain D and (resid 9 through 58 or (resid 59...DD7 - 3036 - 302
241ARGARGPHEPHE(chain D and (resid 9 through 58 or (resid 59...DD7 - 3036 - 302
251ARGARGPHEPHE(chain D and (resid 9 through 58 or (resid 59...DD7 - 3036 - 302
261ARGARGPHEPHE(chain D and (resid 9 through 58 or (resid 59...DD7 - 3036 - 302
112DCDCDCDCchain CCC0 - 261 - 27
212DCDCDCDCchain FFF0 - 261 - 27
113DGDGDGDGchain BBB-1 - 251 - 27
213DGDGDGDG(chain E and ((resid -1 and (name C4 or name...EE-11
223DGDGDGDG(chain E and ((resid -1 and (name C4 or name...EE-1 - 251 - 27
233DGDGDGDG(chain E and ((resid -1 and (name C4 or name...EE-1 - 251 - 27
243DGDGDGDG(chain E and ((resid -1 and (name C4 or name...EE-1 - 251 - 27
253DGDGDGDG(chain E and ((resid -1 and (name C4 or name...EE-1 - 251 - 27
263DGDGDGDG(chain E and ((resid -1 and (name C4 or name...EE-1 - 251 - 27
273DGDGDGDG(chain E and ((resid -1 and (name C4 or name...EE-1 - 251 - 27
283DGDGDGDG(chain E and ((resid -1 and (name C4 or name...EE-1 - 251 - 27
293DGDGDGDG(chain E and ((resid -1 and (name C4 or name...EE-1 - 251 - 27
2103DGDGDGDG(chain E and ((resid -1 and (name C4 or name...EE-1 - 251 - 27
2113DGDGDGDG(chain E and ((resid -1 and (name C4 or name...EE-1 - 251 - 27
2123DGDGDGDG(chain E and ((resid -1 and (name C4 or name...EE-1 - 251 - 27
2133DGDGDGDG(chain E and ((resid -1 and (name C4 or name...EE-1 - 251 - 27
2143DGDGDGDG(chain E and ((resid -1 and (name C4 or name...EE-1 - 251 - 27
2153DGDGDGDG(chain E and ((resid -1 and (name C4 or name...EE-1 - 251 - 27
2163DGDGDGDG(chain E and ((resid -1 and (name C4 or name...EE-1 - 251 - 27
2173DGDGDGDG(chain E and ((resid -1 and (name C4 or name...EE-1 - 251 - 27

NCS ensembles :
ID
1
2
3

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Components

#1: Protein I-OnuI-e-Therm


Mass: 34904.559 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#2: DNA chain DNA (27-MER)


Mass: 8248.316 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (27-MER)


Mass: 8342.427 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical
ChemComp-CA / CALCIUM ION


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


Mass: 18.015 Da / Num. of mol.: 23 / Source method: isolated from a natural source / Formula: H2O
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: 2.52 Å3/Da / Density % sol: 51.21 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop
Details: 100mM HEPES pH 8.5, 200mM ammonium sulfate, 28% PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.1 / Wavelength: 0.977 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Apr 6, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.977 Å / Relative weight: 1
ReflectionResolution: 2.55→50 Å / Num. obs: 34285 / % possible obs: 98.6 % / Redundancy: 11.9 % / Biso Wilson estimate: 51.3 Å2 / Rmerge(I) obs: 0.073 / Rpim(I) all: 0.022 / Rrim(I) all: 0.077 / Χ2: 1.022 / Net I/σ(I): 10 / Num. measured all: 409080
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.55-2.648.50.37756250.9550.1290.40.9190
2.64-2.7510.10.3562480.9690.1110.3680.94396.8
2.75-2.8711.50.29665120.9860.090.310.92999.4
2.87-3.0212.10.21465350.9930.0630.2230.974100
3.02-3.2112.80.14465530.9970.0420.151.057100
3.21-3.4613.20.09565540.9980.0270.0991.196100
3.46-3.8112.80.07665750.9990.0220.0791.106100
3.81-4.36130.06265360.9990.0180.0641.034100
4.36-5.4912.50.05465730.9990.0160.0560.968100
5.49-5012.30.04765770.9980.0140.0491.009100

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation5.46 Å46.31 Å
Translation5.46 Å46.31 Å

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Processing

Software
NameVersionClassification
HKL-2000data scaling
PHASER2.7.16phasing
PHENIX1.11.1_2575refinement
PDB_EXTRACT3.22data extraction
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3QQY

3qqy


Resolution: 2.551→46.309 Å / SU ML: 0.31 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 32.59
RfactorNum. reflection% reflection
Rfree0.2607 1988 5.81 %
Rwork0.2226 --
obs0.2249 34193 98.54 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 93.57 Å2 / Biso mean: 55.8565 Å2 / Biso min: 34.34 Å2
Refinement stepCycle: final / Resolution: 2.551→46.309 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4522 2200 5 23 6750
Biso mean--47.96 48.51 -
Num. residues----699
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0057081
X-RAY DIFFRACTIONf_angle_d0.74310057
X-RAY DIFFRACTIONf_chiral_restr0.0451148
X-RAY DIFFRACTIONf_plane_restr0.005899
X-RAY DIFFRACTIONf_dihedral_angle_d17.5593765
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A2594X-RAY DIFFRACTION9.114TORSIONAL
12D2594X-RAY DIFFRACTION9.114TORSIONAL
21C532X-RAY DIFFRACTION9.114TORSIONAL
22F532X-RAY DIFFRACTION9.114TORSIONAL
31B526X-RAY DIFFRACTION9.114TORSIONAL
32E526X-RAY DIFFRACTION9.114TORSIONAL
LS refinement shell

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

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.551-2.61460.3781260.3014203889
2.6146-2.68520.35621290.3045214494
2.6852-2.76430.31471460.2944226398
2.7643-2.85350.3711310.2925229899
2.8535-2.95540.35381450.29322294100
2.9554-3.07370.31531480.28822298100
3.0737-3.21360.36581370.27062317100
3.2136-3.3830.26961400.242319100
3.383-3.59490.27971510.23552332100
3.5949-3.87230.26991490.22742302100
3.8723-4.26170.2611420.20372342100
4.2617-4.87780.23261360.18352372100
4.8778-6.14330.21771500.19992389100
6.1433-46.3090.17811580.17272497100

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