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- PDB-5e67: K103A/K262A double mutant of I-SmaMI -

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Basic information

Entry
Database: PDB / ID: 5.0E+67
TitleK103A/K262A double mutant of I-SmaMI
Components
  • DNA bottom strand
  • DNA top strand
  • I-SmaMI LAGLIDADG meganuclease
KeywordsHydrolase/DNA / LAGLIDADG / I-SmaMI / K103A/K262A / Hydrolase-DNA complex
Function / homology
Function and homology information


endonuclease activity / mitochondrion / 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
2-METHOXYETHANOL / DNA / DNA (> 10) / Homing endonuclease LAGLIDADG domain-containing protein
Similarity search - Component
Biological speciesSordaria macrospora (fungus)
Synthetic construct (others)
MethodX-RAY DIFFRACTION / Resolution: 2.2 Å
AuthorsShen, B.W. / Stoddard, B.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)RO1 GM United States
CitationJournal: J.Mol.Biol. / Year: 2016
Title: The Structural Basis of Asymmetry in DNA Binding and Cleavage as Exhibited by the I-SmaMI LAGLIDADG Meganuclease.
Authors: Shen, B.W. / Lambert, A. / Walker, B.C. / Stoddard, B.L. / Kaiser, B.K.
History
DepositionOct 9, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 13, 2016Provider: repository / Type: Initial release
Revision 1.1Feb 10, 2016Group: Database references
Revision 1.2Sep 13, 2017Group: Author supporting evidence / Database references / Derived calculations
Category: citation / pdbx_audit_support / pdbx_struct_oper_list
Item: _citation.journal_id_CSD / _pdbx_audit_support.funding_organization / _pdbx_struct_oper_list.symmetry_operation
Revision 1.3Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Mar 6, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_conn_angle / struct_conn
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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: I-SmaMI LAGLIDADG meganuclease
B: DNA bottom strand
C: DNA top strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,6387
Polymers49,3933
Non-polymers2454
Water3,387188
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8490 Å2
ΔGint-57 kcal/mol
Surface area18790 Å2
MethodPISA
Unit cell
Length a, b, c (Å)60.518, 67.326, 98.572
Angle α, β, γ (deg.)90.00, 90.00, 90.00
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-SmaMI LAGLIDADG meganuclease


Mass: 34036.438 Da / Num. of mol.: 1 / Fragment: UNP residues 114-415 / Mutation: K103A, L165N, K262A, M268Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sordaria macrospora (strain ATCC MYA-333 / DSM 997 / K(L3346) / K-hell) (fungus)
Strain: ATCC MYA-333 / DSM 997 / K(L3346) / K-hell / Gene: SMAC_12671 / Plasmid: pET24d(+) / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3)RIL+ / References: UniProt: F7WD42

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DNA chain , 2 types, 2 molecules BC

#2: DNA chain DNA bottom strand


Mass: 7691.988 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Synthetic construct (others)
#3: DNA chain DNA top strand


Mass: 7664.945 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Synthetic construct (others)

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Non-polymers , 4 types, 192 molecules

#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-MXE / 2-METHOXYETHANOL


Mass: 76.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O2
#6: Chemical ChemComp-PG0 / 2-(2-METHOXYETHOXY)ETHANOL / PEG 6000


Mass: 120.147 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H12O3 / Comment: inhibitor, precipitant*YM
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 188 / Source method: isolated from a natural source / Formula: H2O

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Details

Sequence detailsMutation of L165N and M267Q were made to improve expression/purification.

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION

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Sample preparation

CrystalDensity Matthews: 2.22 Å3/Da / Density % sol: 39.49 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 7.5 / Details: 28 - 32% PEGMME550, HEPES, Mg++ / PH range: 7.5 - 8.0

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.54 Å
DetectorType: RIGAKU SATURN 70 / Detector: CCD / Date: Apr 15, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 2.2→25 Å / Num. obs: 19836 / % possible obs: 92 % / Observed criterion σ(F): 1 / Observed criterion σ(I): 1 / Redundancy: 6.7 % / Rsym value: 0.088 / Net I/σ(I): 17
Reflection shellResolution: 2.2→2.28 Å / Redundancy: 4.4 % / Rmerge(I) obs: 0.619 / Mean I/σ(I) obs: 2.3 / % possible all: 59.2

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Processing

Software
NameVersionClassification
REFMAC5.8.0124refinement
HKL-2000data reduction
SCALEPACKdata scaling
PHASERphasing
RefinementResolution: 2.2→25 Å / Cor.coef. Fo:Fc: 0.961 / Cor.coef. Fo:Fc free: 0.913 / SU B: 15.168 / SU ML: 0.189 / Cross valid method: THROUGHOUT / ESU R: 0.376 / ESU R Free: 0.256 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.25448 957 4.9 %RANDOM
Rwork0.17909 ---
obs0.18276 18593 92.73 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 34.571 Å2
Baniso -1Baniso -2Baniso -3
1-0.68 Å20 Å20 Å2
2---0.66 Å20 Å2
3----0.02 Å2
Refinement stepCycle: 1 / Resolution: 2.2→25 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2339 1019 15 188 3561
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0180.0173563
X-RAY DIFFRACTIONr_bond_other_d0.0020.022958
X-RAY DIFFRACTIONr_angle_refined_deg2.0641.6925011
X-RAY DIFFRACTIONr_angle_other_deg1.30436869
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.9915295
X-RAY DIFFRACTIONr_dihedral_angle_2_deg37.32224.38898
X-RAY DIFFRACTIONr_dihedral_angle_3_deg17.60115449
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.627158
X-RAY DIFFRACTIONr_chiral_restr0.1170.2505
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.023255
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02792
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.3792.0341180
X-RAY DIFFRACTIONr_mcbond_other1.382.0311179
X-RAY DIFFRACTIONr_mcangle_it2.3033.0481475
X-RAY DIFFRACTIONr_mcangle_other2.3023.0511476
X-RAY DIFFRACTIONr_scbond_it1.5062.2192383
X-RAY DIFFRACTIONr_scbond_other1.5032.2192383
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other2.463.2713537
X-RAY DIFFRACTIONr_long_range_B_refined4.6718.3134422
X-RAY DIFFRACTIONr_long_range_B_other4.6718.3214423
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.2→2.257 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.307 45 -
Rwork0.233 861 -
obs--59.64 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.47830.37850.18752.48691.50051.7188-0.0292-0.01630.0686-0.0770.01730.1646-0.1456-0.07660.01190.02120.01520.00150.05110.02030.02935.8802-1.16-0.9494
20.3319-0.199-0.48462.81472.89813.80310.0882-0.11530.07360.0688-0.0620.117-0.0931-0.2736-0.02620.15490.0391-0.02450.2252-0.01410.1613-4.2619-0.8369-0.6502
30.09710.32170.58951.74892.51134.0991-0.0009-0.0664-0.0088-0.2351-0.11990.2436-0.2977-0.35790.12080.23170.0242-0.01540.2246-0.0320.2245-4.3520.3497-0.8656
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A7 - 300
2X-RAY DIFFRACTION2B1 - 25
3X-RAY DIFFRACTION3C1 - 25

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