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- PDB-3ko2: I-MsoI re-designed for altered DNA cleavage specificity (-7C) -

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Basic information

Entry
Database: PDB / ID: 3ko2
TitleI-MsoI re-designed for altered DNA cleavage specificity (-7C)
Components
  • 5'-D(*CP*GP*GP*AP*AP*CP*TP*GP*TP*CP*TP*CP*AP*CP*GP*AP*CP*GP*GP*TP*CP*TP*GP*C)-3'
  • 5'-D(*GP*CP*AP*GP*AP*CP*CP*GP*TP*CP*GP*TP*GP*AP*GP*AP*CP*AP*GP*TP*TP*CP*CP*G)-3'
  • Site-specific DNA endonuclease I-MsoI
KeywordsHYDROLASE/DNA / redesign / protein-DNA complex / Chloroplast / Endonuclease / HYDROLASE-DNA complex
Function / homology
Function and homology information


chloroplast / endonuclease activity / metal ion binding
Similarity search - Function
LAGLIDADG endonuclease / Homing endonucleases / Endonuclease I-creI / Homing endonuclease, LAGLIDADG / Homing endonuclease / Roll / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / Site-specific DNA endonuclease I-MsoI
Similarity search - Component
Biological speciesMonomastix sp. (plant)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.9 Å
AuthorsTaylor, G.K. / Stoddard, B.L.
CitationJournal: Nucleic Acids Res. / Year: 2010
Title: Computational reprogramming of homing endonuclease specificity at multiple adjacent base pairs.
Authors: Ashworth, J. / Taylor, G.K. / Havranek, J.J. / Quadri, S.A. / Stoddard, B.L. / Baker, D.
History
DepositionNov 13, 2009Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 19, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Source and taxonomy / Version format compliance
Revision 1.2Oct 13, 2021Group: Database references / Derived calculations
Category: database_2 / pdbx_struct_conn_angle ...database_2 / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
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.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_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_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.3Sep 6, 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: Site-specific DNA endonuclease I-MsoI
B: Site-specific DNA endonuclease I-MsoI
C: 5'-D(*GP*CP*AP*GP*AP*CP*CP*GP*TP*CP*GP*TP*GP*AP*GP*AP*CP*AP*GP*TP*TP*CP*CP*G)-3'
D: 5'-D(*CP*GP*GP*AP*AP*CP*TP*GP*TP*CP*TP*CP*AP*CP*GP*AP*CP*GP*GP*TP*CP*TP*GP*C)-3'
F: Site-specific DNA endonuclease I-MsoI
G: Site-specific DNA endonuclease I-MsoI
H: 5'-D(*GP*CP*AP*GP*AP*CP*CP*GP*TP*CP*GP*TP*GP*AP*GP*AP*CP*AP*GP*TP*TP*CP*CP*G)-3'
I: 5'-D(*CP*GP*GP*AP*AP*CP*TP*GP*TP*CP*TP*CP*AP*CP*GP*AP*CP*GP*GP*TP*CP*TP*GP*C)-3'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)108,13214
Polymers107,8918
Non-polymers2406
Water1086
1
A: Site-specific DNA endonuclease I-MsoI
B: Site-specific DNA endonuclease I-MsoI
C: 5'-D(*GP*CP*AP*GP*AP*CP*CP*GP*TP*CP*GP*TP*GP*AP*GP*AP*CP*AP*GP*TP*TP*CP*CP*G)-3'
D: 5'-D(*CP*GP*GP*AP*AP*CP*TP*GP*TP*CP*TP*CP*AP*CP*GP*AP*CP*GP*GP*TP*CP*TP*GP*C)-3'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)54,0667
Polymers53,9464
Non-polymers1203
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9910 Å2
ΔGint-48 kcal/mol
Surface area20250 Å2
MethodPISA
2
F: Site-specific DNA endonuclease I-MsoI
G: Site-specific DNA endonuclease I-MsoI
H: 5'-D(*GP*CP*AP*GP*AP*CP*CP*GP*TP*CP*GP*TP*GP*AP*GP*AP*CP*AP*GP*TP*TP*CP*CP*G)-3'
I: 5'-D(*CP*GP*GP*AP*AP*CP*TP*GP*TP*CP*TP*CP*AP*CP*GP*AP*CP*GP*GP*TP*CP*TP*GP*C)-3'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)54,0667
Polymers53,9464
Non-polymers1203
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10210 Å2
ΔGint-52 kcal/mol
Surface area19840 Å2
MethodPISA
Unit cell
Length a, b, c (Å)49.031, 70.006, 72.258
Angle α, β, γ (deg.)81.660, 70.000, 89.480
Int Tables number1
Space group name H-MP1
DetailsThe biological unit is a protein-dimer bound to DNA. There are two copies of this complex in the asymmetric unit.

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Components

#1: Protein
Site-specific DNA endonuclease I-MsoI


Mass: 19601.520 Da / Num. of mol.: 4 / Mutation: L28R, S43E, N70T, I85Y
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Monomastix sp. (plant) / Strain: OKE-1 / Gene: I-MsoI, orf170 / Plasmid: pET24d / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pLysS / References: UniProt: C0JWR6
#2: DNA chain 5'-D(*GP*CP*AP*GP*AP*CP*CP*GP*TP*CP*GP*TP*GP*AP*GP*AP*CP*AP*GP*TP*TP*CP*CP*G)-3'


Mass: 7395.767 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: Synthetic DNA
#3: DNA chain 5'-D(*CP*GP*GP*AP*AP*CP*TP*GP*TP*CP*TP*CP*AP*CP*GP*AP*CP*GP*GP*TP*CP*TP*GP*C)-3'


Mass: 7346.729 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: Synthetic DNA
#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O

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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: 2.14 Å3/Da / Density % sol: 42.4 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: 22% PEG 400, 100mM Tris-HCl, 5mM CaCl2, 5mM NaCl, pH 8.5, VAPOR DIFFUSION, HANGING DROP, temperature 291K
Components of the solutions
IDNameCrystal-IDSol-ID
1PEG 40011
2Tris-HCl11
3CaCl211
4NaCl11
5PEG 40012
6Tris-HCl12
7CaCl212
8NaCl12

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Data collection

DiffractionMean temperature: 95 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.54 Å
DetectorType: RIGAKU SATURN 944+ / Detector: CCD / Date: Apr 30, 2009
RadiationMonochromator: Confocal Varimax Optics Systems / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 2.9→33.56 Å / Num. all: 19742 / Num. obs: 19127 / % possible obs: 96.9 % / Observed criterion σ(I): -3 / Redundancy: 3.7 % / Rmerge(I) obs: 0.078 / Χ2: 0.97 / Net I/σ(I): 10.6 / Scaling rejects: 535
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allΧ2% possible all
2.9-33.580.3293.2661418331.1994
3-3.123.640.2623.8701119081.1296.2
3.12-3.273.660.1984.8703419091.0896.2
3.27-3.443.690.1336.6708719071.0196.4
3.44-3.653.690.1128.2699818780.9796.9
3.65-3.933.720.09510.2717619150.9497.1
3.93-4.333.740.06812.9733519490.8797.3
4.33-4.953.750.05217730619420.8498
4.95-6.243.770.06115.2737419480.8798.3
6.24-33.563.750.03722.6735419380.8898.3

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Phasing

PhasingMethod: molecular replacement
Phasing MRRfactor: 40.46
Highest resolutionLowest resolution
Rotation2.9 Å33.56 Å
Translation2.9 Å33.56 Å

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Processing

Software
NameVersionClassificationNB
d*TREK9.7 W8RSSIdata scaling
PHASERphasing
PHENIX1.5_2refinement
PDB_EXTRACT3.005data extraction
CrystalCleardata collection
d*TREKdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 1M5X

1m5x


Resolution: 2.9→33.56 Å / Occupancy max: 1 / Occupancy min: 0 / FOM work R set: 0.744 / SU ML: 0.47 / σ(F): 0.05 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.286 1894 10 %selected by Phenix following molecular replacement
Rwork0.248 ---
obs0.252 18940 95.93 %-
all-19127 --
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 40 Å2 / ksol: 0.304 e/Å3
Displacement parametersBiso max: 190.08 Å2 / Biso mean: 60.667 Å2 / Biso min: 10.72 Å2
Baniso -1Baniso -2Baniso -3
1-9.499 Å2-6.929 Å22.054 Å2
2--9.278 Å2-5.147 Å2
3----21.442 Å2
Refinement stepCycle: LAST / Resolution: 2.9→33.56 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5264 1956 6 6 7232
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0067560
X-RAY DIFFRACTIONf_angle_d0.93610624
X-RAY DIFFRACTIONf_chiral_restr0.0551180
X-RAY DIFFRACTIONf_plane_restr0.0041024
X-RAY DIFFRACTIONf_dihedral_angle_d22.0172988
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.9-2.9730.3691250.3441128125390
2.973-3.0530.4111340.3371196133094
3.053-3.1430.3761320.3311193132594
3.143-3.2440.3181340.3021204133895
3.244-3.360.3041330.2711196132995
3.36-3.4940.2981380.2431231136996
3.494-3.6530.2821310.2511198132996
3.653-3.8460.311390.2411253139297
3.846-4.0860.2631380.2381240137897
4.086-4.4010.2491370.2141228136597
4.401-4.8430.2451390.1941250138998
4.843-5.5410.2551380.2261258139698
5.541-6.970.2931380.2351238137698
6.97-33.560.2361380.2181233137197

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