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- PDB-6uw0: Engineered variant of I-OnuI meganuclease with improved thermosta... -

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Basic information

Entry
Database: PDB / ID: 6uw0
TitleEngineered variant of I-OnuI meganuclease with improved thermostability and fully altered specificity targeting cholera toxin A subunit
Components
  • (DNA (27-MER)) x 2
  • I-OnuI-e-Therm-bCtxA
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: 2.72 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-bCtxA
B: I-OnuI-e-Therm-bCtxA
C: DNA (27-MER)
D: DNA (27-MER)
E: DNA (27-MER)
F: DNA (27-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,86414
Polymers101,4776
Non-polymers3878
Water18010
1
A: I-OnuI-e-Therm-bCtxA
E: DNA (27-MER)
F: DNA (27-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,0058
Polymers50,7393
Non-polymers2665
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9280 Å2
ΔGint-70 kcal/mol
Surface area18080 Å2
MethodPISA
2
B: I-OnuI-e-Therm-bCtxA
C: DNA (27-MER)
D: DNA (27-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,8596
Polymers50,7393
Non-polymers1203
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8760 Å2
ΔGint-88 kcal/mol
Surface area18390 Å2
MethodPISA
Unit cell
Length a, b, c (Å)75.246, 79.679, 168.941
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 8 through 51 or (resid 52...
21(chain B and (resid 8 through 32 or (resid 33...
12(chain C and ((resid -1 and (name O5 or name...
22chain E
13(chain D and ((resid 0 and (name C5 or name...
23chain F

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
111SERSERASNASN(chain A and (resid 8 through 51 or (resid 52...AA8 - 513 - 46
121LYSLYSLYSLYS(chain A and (resid 8 through 51 or (resid 52...AA5247
131SERSERARGARG(chain A and (resid 8 through 51 or (resid 52...AA8 - 3013 - 296
141SERSERARGARG(chain A and (resid 8 through 51 or (resid 52...AA8 - 3013 - 296
151SERSERARGARG(chain A and (resid 8 through 51 or (resid 52...AA8 - 3013 - 296
161SERSERARGARG(chain A and (resid 8 through 51 or (resid 52...AA8 - 3013 - 296
211SERSERARGARG(chain B and (resid 8 through 32 or (resid 33...BB8 - 323 - 27
221ASNASNSERSER(chain B and (resid 8 through 32 or (resid 33...BB33 - 3628 - 31
231ALAALAARGARG(chain B and (resid 8 through 32 or (resid 33...BB7 - 3012 - 296
241ALAALAARGARG(chain B and (resid 8 through 32 or (resid 33...BB7 - 3012 - 296
251ALAALAARGARG(chain B and (resid 8 through 32 or (resid 33...BB7 - 3012 - 296
261ALAALAARGARG(chain B and (resid 8 through 32 or (resid 33...BB7 - 3012 - 296
112DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-11
122DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
132DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
142DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
152DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
162DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
172DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
182DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
192DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
1102DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
1112DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
1122DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
1132DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
1142DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
1152DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
1162DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
1172DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
1182DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
1192DGDGDGDG(chain C and ((resid -1 and (name O5 or name...CC-1 - 251 - 27
212DGDGDGDGchain EEE-1 - 251 - 27
113DCDCDCDC(chain D and ((resid 0 and (name C5 or name...DD01
123DCDCDCDC(chain D and ((resid 0 and (name C5 or name...DD0 - 261 - 27
133DCDCDCDC(chain D and ((resid 0 and (name C5 or name...DD0 - 261 - 27
143DCDCDCDC(chain D and ((resid 0 and (name C5 or name...DD0 - 261 - 27
153DCDCDCDC(chain D and ((resid 0 and (name C5 or name...DD0 - 261 - 27
163DCDCDCDC(chain D and ((resid 0 and (name C5 or name...DD0 - 261 - 27
173DCDCDCDC(chain D and ((resid 0 and (name C5 or name...DD0 - 261 - 27
183DCDCDCDC(chain D and ((resid 0 and (name C5 or name...DD0 - 261 - 27
193DCDCDCDC(chain D and ((resid 0 and (name C5 or name...DD0 - 261 - 27
1103DCDCDCDC(chain D and ((resid 0 and (name C5 or name...DD0 - 261 - 27
1113DCDCDCDC(chain D and ((resid 0 and (name C5 or name...DD0 - 261 - 27
1123DCDCDCDC(chain D and ((resid 0 and (name C5 or name...DD0 - 261 - 27
1133DCDCDCDC(chain D and ((resid 0 and (name C5 or name...DD0 - 261 - 27
1143DCDCDCDC(chain D and ((resid 0 and (name C5 or name...DD0 - 261 - 27
1153DCDCDCDC(chain D and ((resid 0 and (name C5 or name...DD0 - 261 - 27
213DCDCDCDCchain FFF0 - 261 - 27

NCS ensembles :
ID
1
2
3

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein I-OnuI-e-Therm-bCtxA


Mass: 34146.824 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)

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DNA chain , 2 types, 4 molecules CEDF

#2: DNA chain DNA (27-MER)


Mass: 8344.363 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (27-MER)


Mass: 8247.366 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 18 molecules

#4: Chemical
ChemComp-CA / CALCIUM ION


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


Mass: 62.068 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H6O2
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 10 / 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: 2.5 Å3/Da / Density % sol: 50.71 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: 100mM HEPES pH 8.5, 200mM ammonium sulfate, 30% PEG 3350

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Data collection

DiffractionMean temperature: 108 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.1 / Wavelength: 0.977 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Jun 20, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.977 Å / Relative weight: 1
ReflectionResolution: 2.7→50 Å / Num. obs: 27693 / % possible obs: 98.7 % / Redundancy: 11.4 % / Rmerge(I) obs: 0.075 / Rpim(I) all: 0.023 / Rrim(I) all: 0.078 / Χ2: 0.931 / Net I/σ(I): 8.4 / Num. measured all: 315424
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.7-2.89.10.41624930.9650.1380.4390.90890.5
2.8-2.9110.40.32826520.9850.1040.3450.89496.6
2.91-3.0410.70.23427550.9950.0730.2450.90499.6
3.04-3.2120.17327710.9980.0520.1810.898100
3.2-3.411.70.09327760.9990.0280.0980.896100
3.4-3.6612.10.09727800.9990.0290.1010.925100
3.66-4.0312.40.07627970.9990.0220.0790.934100
4.03-4.6211.90.055280710.0170.0570.985100
4.62-5.8112.10.05228580.9990.0150.0540.952100
5.81-5011.30.035300410.0110.0370.989100

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation2.92 Å45.99 Å
Translation2.92 Å45.99 Å

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Processing

Software
NameVersionClassification
PHENIX1.17_3644refinement
HKL-2000data scaling
PHASER2.8.1phasing
PDB_EXTRACT3.25data extraction
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3QQY

3qqy


Resolution: 2.72→45.99 Å / SU ML: 0.36 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 34.14 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2809 2006 7.29 %
Rwork0.2234 25502 -
obs0.2276 27508 97.65 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 104.14 Å2 / Biso mean: 53.8399 Å2 / Biso min: 35.86 Å2
Refinement stepCycle: final / Resolution: 2.72→45.99 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4396 2204 35 10 6645
Biso mean--63.67 46.47 -
Num. residues----696
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A1766X-RAY DIFFRACTION9.666TORSIONAL
12B1766X-RAY DIFFRACTION9.666TORSIONAL
21C532X-RAY DIFFRACTION9.666TORSIONAL
22E532X-RAY DIFFRACTION9.666TORSIONAL
31D530X-RAY DIFFRACTION9.666TORSIONAL
32F530X-RAY DIFFRACTION9.666TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.72-2.780.36441170.30691470158781
2.78-2.860.36871330.3041725185894
2.86-2.940.39161470.31803195098
2.94-3.040.33931400.29321824196499
3.04-3.150.4021400.27218501990100
3.15-3.270.28941420.23961803194599
3.27-3.420.26671490.22231819196898
3.42-3.60.26821440.21541837198199
3.6-3.830.28391430.23218341977100
3.83-4.120.24571430.209618802023100
4.12-4.540.26911500.191818652015100
4.54-5.190.24661500.205118872037100
5.19-6.540.28051470.229619042051100
6.54-45.990.24711610.18852001216299

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