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- PDB-4zsf: Crystal structure of pre-specific restriction endonuclease BsaWI-... -

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Basic information

Entry
Database: PDB / ID: 4zsf
TitleCrystal structure of pre-specific restriction endonuclease BsaWI-DNA complex
Components
  • BsaWI endonuclease
  • DNA
KeywordsPROTEIN/DNA / restriction endonuclease / PD-(D/E)XK nuclease / protein-DNA complex
Function / homologyBsaWI restriction endonuclease type 2 / BsaWI restriction endonuclease type 2 / endonuclease activity / metal ion binding / SUCCINIC ACID / DNA / DNA (> 10) / BsaWI endonuclease
Function and homology information
Biological speciesGeobacillus stearothermophilus (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SIRAS / Resolution: 1.8 Å
AuthorsTamulaitiene, G. / Rutkauskas, M. / Grazulis, S. / Siksnys, V.
Funding supportLithuania, 1items
OrganizationGrant numberCountry
Research Council of LithuaniaMIP-41/2013Lithuania
CitationJournal: Nucleic Acids Res. / Year: 2015
Title: Functional significance of protein assemblies predicted by the crystal structure of the restriction endonuclease BsaWI.
Authors: Tamulaitis, G. / Rutkauskas, M. / Zaremba, M. / Grazulis, S. / Tamulaitiene, G. / Siksnys, V.
History
DepositionMay 13, 2015Deposition site: RCSB / Processing site: PDBE
Revision 1.0Oct 7, 2015Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2019Group: Data collection / Category: diffrn_source / Item: _diffrn_source.pdbx_synchrotron_site
Revision 1.2May 8, 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_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.ptnr1_symmetry / _pdbx_struct_conn_angle.ptnr2_symmetry / _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.ptnr3_symmetry / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _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_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_symmetry

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: BsaWI endonuclease
B: DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)36,5616
Polymers36,2442
Non-polymers3164
Water4,450247
1
A: BsaWI endonuclease
B: DNA
hetero molecules

A: BsaWI endonuclease
B: DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)73,12112
Polymers72,4884
Non-polymers6338
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation3_555-x,y,-z+1/21
Buried area9650 Å2
ΔGint-94 kcal/mol
Surface area28150 Å2
MethodPISA
Unit cell
Length a, b, c (Å)89.562, 136.336, 73.482
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221

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Components

#1: Protein BsaWI endonuclease


Mass: 31962.451 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacillus stearothermophilus (bacteria)
Gene: bsaWIR / Plasmid: pBAD24 / Production host: Escherichia coli (E. coli) / Strain (production host): DH10B / References: UniProt: Q6UQ65
#2: DNA chain DNA


Mass: 4281.780 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Chemical ChemComp-SIN / SUCCINIC ACID


Mass: 118.088 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H6O4
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 247 / 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: 3.09 Å3/Da / Density % sol: 60.25 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 5
Details: Crystallization buffer was 0.02 M Calcium chloride, 0.1 M Na-succinate (pH 5.0), 30% (v/v) (+/-)-2-Methyl-2,4-pentanediol

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: EMBL/DESY, HAMBURG / Beamline: X12 / Wavelength: 0.9887, 0.97622
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Nov 15, 2011 / Details: mirrors
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
10.98871
20.976221
ReflectionResolution: 1.8→29.163 Å / Num. all: 41718 / Num. obs: 41718 / % possible obs: 99.4 % / Redundancy: 14.4 % / Biso Wilson estimate: 25.21 Å2 / Rpim(I) all: 0.019 / Rrim(I) all: 0.071 / Rsym value: 0.067 / Net I/av σ(I): 6.27 / Net I/σ(I): 24.3 / Num. measured all: 600277
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allRpim(I) allRsym valueNet I/σ(I) obs% possible all
1.8-1.912.10.5411.46992157820.1650.5414.295.9
1.9-2.0114.90.3452.28480857100.0950.3457.999.9
2.01-2.1514.80.1953.98087854530.0540.19513.6100
2.15-2.3214.90.1315.87452650180.0360.13119.3100
2.32-2.5514.80.0898.16912446560.0240.08926.3100
2.55-2.8514.80.06410.56248742190.0180.06433.7100
2.85-3.2914.80.05211.45554437500.0140.05241.5100
3.29-4.0214.70.069.34692831890.0160.0648100
4.02-5.6914.50.04412.43638225070.0120.04451100
5.69-29.16313.70.04210.91967914340.0120.04249.799

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Phasing

PhasingMethod: SIRAS
Phasing dmFOM : 0.67 / FOM acentric: 0.67 / FOM centric: 0.64 / Reflection: 12653 / Reflection acentric: 11145 / Reflection centric: 1508
Phasing dm shell
Resolution (Å)FOM FOM acentricFOM centricReflectionReflection acentricReflection centric
7.7-19.8890.880.890.87557404153
4.8-7.70.810.830.7517291434295
3.9-4.80.840.850.7721411869272
3.4-3.90.770.790.6621361902234
2.9-3.40.590.60.5237593402357
2.7-2.90.390.40.3223312134197

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Processing

Software
NameVersionClassification
MAR345dtbdata collection
MOSFLM7.0.6data reduction
SCALA3.3.16data scaling
SHELXphasing
Cootmodel building
PHENIX1.8.3refinement
PDB_EXTRACT3.15data extraction
RefinementMethod to determine structure: SIRAS / Resolution: 1.8→28.404 Å / SU ML: 0.18 / Cross valid method: FREE R-VALUE / σ(F): 1.39 / Phase error: 17.25 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.1879 8024 10.03 %Random selection
Rwork0.1524 72004 --
obs0.1559 41703 99.34 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 99.77 Å2 / Biso mean: 30.4588 Å2 / Biso min: 13.78 Å2
Refinement stepCycle: final / Resolution: 1.8→28.404 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2186 284 18 247 2735
Biso mean--62.1 38.11 -
Num. residues----286
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0192698
X-RAY DIFFRACTIONf_angle_d1.6593728
X-RAY DIFFRACTIONf_chiral_restr0.096388
X-RAY DIFFRACTIONf_plane_restr0.01446
X-RAY DIFFRACTIONf_dihedral_angle_d17.0851017
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.8-1.82050.27162590.26322098235788
1.8205-1.84190.22872740.24742275254994
1.8419-1.86440.27132480.22462443269199
1.8644-1.8880.25552510.20923952646100
1.888-1.91280.22292860.196523592645100
1.9128-1.9390.21332560.180324382694100
1.939-1.96670.21882690.170524202689100
1.9667-1.9960.18863100.174624172727100
1.996-2.02720.20862830.165623832666100
2.0272-2.06040.21792900.165823532643100
2.0604-2.0960.19192610.15424772738100
2.096-2.13410.21182450.151623952640100
2.1341-2.17510.17792700.156824172687100
2.1751-2.21950.18182610.144124192680100
2.2195-2.26770.18862480.152524652713100
2.2677-2.32040.19612770.151123972674100
2.3204-2.37840.17772520.141324102662100
2.3784-2.44270.18712920.140124172709100
2.4427-2.51460.20242770.15724022679100
2.5146-2.59570.18412470.151424142661100
2.5957-2.68840.21592740.157224342708100
2.6884-2.79590.19272660.158724322698100
2.7959-2.9230.20442510.15124182669100
2.923-3.0770.21012920.169723852677100
3.077-3.26950.1832740.154224172691100
3.2695-3.52150.20672700.145824252695100
3.5215-3.87510.17312360.136224682704100
3.8751-4.4340.13542720.123123902662100
4.434-5.57940.15322460.128124502696100
5.5794-28.40780.18042870.161723912678100
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)DetailsOrigin x (Å)Origin y (Å)Origin z (Å)
11.22880.3425-0.68550.5005-0.38440.7744-0.1194-0.1244-0.3005-0.02550.0147-0.0110.14640.0191-0.00010.1832-0.00080.01490.14360.0180.1838N domain and helix H4-10.5723-42.266724.3115
20.7592-1.00790.0472.3954-0.71371.0568-0.0635-0.0567-0.07330.4184-0.0441-0.2105-0.14040.1054-0.00080.224-0.004-0.01340.1890.02480.1875part2 of C domain21.5731-16.32929.5681
30.65990.01350.20972.1030.52742.26850.00890.1710.0048-0.0257-0.07920.25620.0367-0.2038-0.0020.14090.02380.01460.19490.00790.1837part2 of C domain11.9666-17.2967-3.3925
40.2067-0.28480.32330.391-0.42271.0696-0.42890.7115-0.3514-0.56910.57570.0569-0.54410.33130.58350.3693-0.2552-0.05250.5910.05430.31565'-end of DNA-10.2413-26.11334.2822
50.5701-0.1857-0.4490.6258-0.44210.9531-0.3224-0.46640.20920.07440.1628-0.1042-0.30760.1352-0.00430.21520.0055-0.03810.2432-0.02820.1972target site and 3'-end of DNA6.2603-29.716223.5578
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1(chain B and resid 5:14)B5 - 14
2X-RAY DIFFRACTION2(chain A and resid 95:172)A95 - 172
3X-RAY DIFFRACTION3(chain A and resid 173:272)A173 - 272
4X-RAY DIFFRACTION4(chain B and resid 1:4)B1 - 4
5X-RAY DIFFRACTION5(chain B and resid 5:14)B5 - 14

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