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- PDB-4kyw: Restriction endonuclease DPNI in complex with two DNA molecules -

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Basic information

Entry
Database: PDB / ID: 4kyw
TitleRestriction endonuclease DPNI in complex with two DNA molecules
Components
  • 5'-(*DC*DTP*DGP*DGP*6MAP*DTP*DCP*DCP*DAP*DG)-3'
  • Type-2 restriction enzyme DpnI
KeywordsHYDROLASE/DNA / RESTRICTION ENDONUCLEASE-DNA COMPLEX / TYPE IIM / TYPE IIE / RESTRICTION ENZYME / DPNI / METHYLATION DEPENDENT / N6-METHYLADENINE / PD-(D/E)XK TYPE ENDONUCLEASE / WINGED HELIX DOMAIN / RESTRICTION ENDONUCLEASE / DNA BINDING / HYDROLASE-DNA COMPLEX
Function / homology
Function and homology information


type II site-specific deoxyribonuclease / type II site-specific deoxyribonuclease activity / DNA restriction-modification system
Similarity search - Function
Dam replacing family, catalytic PD-(D/E)XK domain / Dam-replacing / Dam-replacing protein, HTH domain / Dam replacing family, catalytic PD-(D/E)XK domain / Dam-replacing family / Dam-replacing HTH domain / PvuII Endonuclease; Chain A / Winged helix-like DNA-binding domain superfamily/Winged helix DNA-binding domain / Arc Repressor Mutant, subunit A / Winged helix-like DNA-binding domain superfamily ...Dam replacing family, catalytic PD-(D/E)XK domain / Dam-replacing / Dam-replacing protein, HTH domain / Dam replacing family, catalytic PD-(D/E)XK domain / Dam-replacing family / Dam-replacing HTH domain / PvuII Endonuclease; Chain A / Winged helix-like DNA-binding domain superfamily/Winged helix DNA-binding domain / Arc Repressor Mutant, subunit A / Winged helix-like DNA-binding domain superfamily / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
DNA / Type-2 restriction enzyme DpnI
Similarity search - Component
Biological speciesStreptococcus pneumoniae (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.35 Å
AuthorsMierzejewska, K. / Siwek, W. / Czapinska, H. / Skowronek, K. / Bujnicki, J.M. / Bochtler, M.
CitationJournal: Nucleic Acids Res. / Year: 2014
Title: Structural basis of the methylation specificity of R.DpnI.
Authors: Mierzejewska, K. / Siwek, W. / Czapinska, H. / Kaus-Drobek, M. / Radlinska, M. / Skowronek, K. / Bujnicki, J.M. / Dadlez, M. / Bochtler, M.
History
DepositionMay 29, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 4, 2014Provider: repository / Type: Initial release
Revision 1.1Jul 2, 2014Group: Database references
Revision 1.2Jul 9, 2014Group: Database references
Revision 1.3Aug 20, 2014Group: Database references
Revision 1.4Mar 7, 2018Group: Data collection / Category: diffrn_source / Item: _diffrn_source.pdbx_synchrotron_site
Revision 1.5Sep 20, 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 / 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 / _struct_conn.pdbx_leaving_atom_flag / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Type-2 restriction enzyme DpnI
C: 5'-(*DC*DTP*DGP*DGP*6MAP*DTP*DCP*DCP*DAP*DG)-3'
D: 5'-(*DC*DTP*DGP*DGP*6MAP*DTP*DCP*DCP*DAP*DG)-3'
E: 5'-(*DC*DTP*DGP*DGP*6MAP*DTP*DCP*DCP*DAP*DG)-3'
F: 5'-(*DC*DTP*DGP*DGP*6MAP*DTP*DCP*DCP*DAP*DG)-3'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)42,4438
Polymers42,3145
Non-polymers1283
Water1,69394
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9680 Å2
ΔGint-42 kcal/mol
Surface area16660 Å2
MethodPISA
Unit cell
Length a, b, c (Å)95.700, 101.050, 114.850
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221
DetailsBIOLOGICAL UNIT CONTAINS PROTEIN CHAIN A AND TWO DOUBLE STRANDED DNA FRAGMENTS BOUND TO ITS N- AND C-TERMINAL DOMAINS. TO THE BEST OF OUR KNOWLEDGE THE OBSERVED COMPLEX CORRESPONDS TO THE ENZYME SUBSTRATE COMPLEX FORMED BY TYPE IIE R.DPNI RESTRICTION ENDONUCLEASE AND DOUBLE STRANDED DNA IN SOLUTION. THE PENTAMER (ACCORDING TO PDB CONVENTIONS) IS A COMPLEX OF THE ENZYME WITH TWO DOUBLE STRANDED DNA FRAGMENTS PRESENT IN THE CRYSTAL.

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Components

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Protein / DNA chain , 2 types, 5 molecules ACDEF

#1: Protein Type-2 restriction enzyme DpnI / R.DpnI / Endonuclease DpnI / Type II restriction enzyme DpnI


Mass: 30078.355 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pneumoniae (bacteria) / Strain: TIGR4 / Gene: dpnC, spr1665, SP_1850 / Plasmid: PET28 / Production host: Escherichia coli (E. coli) / Strain (production host): ER2925
References: UniProt: P0A459, type II site-specific deoxyribonuclease
#2: DNA chain
5'-(*DC*DTP*DGP*DGP*6MAP*DTP*DCP*DCP*DAP*DG)-3'


Mass: 3059.031 Da / Num. of mol.: 4 / Source method: obtained synthetically / Details: SYNTHETIC OLIGONUCLEOTIDE

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Non-polymers , 4 types, 97 molecules

#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#5: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 94 / 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: 3.28 Å3/Da / Density % sol: 62.51 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: 160 mM CALCIUM ACETATE, 80 mM SODIUM CACODYLATE PH 6.5, 14.4% W/V PEG 8000, 20% V/V GLYCEROL, 10 mM SPERMIDINE., VAPOR DIFFUSION, SITTING DROP, temperature 277K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P14 (MX2) / Wavelength: 1.23953 / Wavelength: 1.23953 Å
DetectorType: PSI PILATUS 6M / Detector: PIXEL / Date: Oct 2, 2012
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.23953 Å / Relative weight: 1
ReflectionResolution: 2.35→50 Å / Num. all: 23502 / Num. obs: 23502 / % possible obs: 99.7 % / Redundancy: 6.37 % / Rmerge(I) obs: 0.049 / Rsym value: 0.049 / Net I/σ(I): 20.51
Reflection shellResolution: 2.35→2.41 Å / Redundancy: 6.68 % / Rmerge(I) obs: 0.44 / Mean I/σ(I) obs: 4.14 / Num. unique all: 1720 / Rsym value: 0.44 / % possible all: 99.9

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Processing

Software
NameVersionClassification
MOLREPphasing
ARP/wARPmodel building
REFMAC5.2.0019refinement
XDSdata reduction
XSCALEdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 4ESJ

4esj


Resolution: 2.35→46.23 Å / Cor.coef. Fo:Fc: 0.954 / Cor.coef. Fo:Fc free: 0.949 / Cross valid method: THROUGHOUT / ESU R: 0.263 / ESU R Free: 0.193 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: AT THE FLANK OF THE RECOGNITION SEQUENCE SHALLOW INTERCALATION OF AN UNKNOWN MOLECULE IS SUGGESTED BY ELECTRON DENSITY MAPS. WE HAVE NOT BEEN ABLE TO DISTINGUISH WHETHER THIS DENSITY SHOULD ...Details: AT THE FLANK OF THE RECOGNITION SEQUENCE SHALLOW INTERCALATION OF AN UNKNOWN MOLECULE IS SUGGESTED BY ELECTRON DENSITY MAPS. WE HAVE NOT BEEN ABLE TO DISTINGUISH WHETHER THIS DENSITY SHOULD BE ACCOUNTED FOR BY A SECOND CONFORMER OF ASN77 OR EITHER B-MERCAPTOETHANOL, ACETATE OR AZIDE FROM THE BUFFER. THE DESIGNATION OF ONE OF THE IONS AS NA+ IS TENTATIVE ONLY. PROGRAM CNS HAS BEEN USED FOR DNA REFINEMENT. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. TLS REFINEMENT HAS BEEN USED.
RfactorNum. reflection% reflectionSelection details
Rfree0.21904 1204 5.1 %RANDOM
Rwork0.20125 ---
all0.20216 23477 --
obs0.20216 23477 99.72 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 43.385 Å2
Baniso -1Baniso -2Baniso -3
1--0.77 Å20 Å20 Å2
2---0.42 Å20 Å2
3---1.19 Å2
Refinement stepCycle: LAST / Resolution: 2.35→46.23 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2096 812 3 94 3005
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0110.0223070
X-RAY DIFFRACTIONr_bond_other_d00.021910
X-RAY DIFFRACTIONr_angle_refined_deg1.342.2884302
X-RAY DIFFRACTIONr_angle_other_deg3.80434674
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.2175256
X-RAY DIFFRACTIONr_dihedral_angle_2_deg37.88724.435115
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.49515405
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.5891515
X-RAY DIFFRACTIONr_chiral_restr0.0830.2461
X-RAY DIFFRACTIONr_gen_planes_refined0.0120.022828
X-RAY DIFFRACTIONr_gen_planes_other0.010.02540
X-RAY DIFFRACTIONr_nbd_refined0.1970.2539
X-RAY DIFFRACTIONr_nbd_other0.2730.22004
X-RAY DIFFRACTIONr_nbtor_refined0.2010.21368
X-RAY DIFFRACTIONr_nbtor_other0.1090.21332
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1890.2135
X-RAY DIFFRACTIONr_xyhbond_nbd_other0.0710.21
X-RAY DIFFRACTIONr_metal_ion_refined0.2180.210
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.140.218
X-RAY DIFFRACTIONr_symmetry_vdw_other0.2220.218
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.2120.25
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it0.6751.51279
X-RAY DIFFRACTIONr_mcbond_other01.5513
X-RAY DIFFRACTIONr_mcangle_it1.28222076
X-RAY DIFFRACTIONr_scbond_it1.79431791
X-RAY DIFFRACTIONr_scangle_it2.8484.52226
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.35→2.411 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.319 90 -
Rwork0.309 1626 -
obs-1716 99.83 %
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)
10.9863-0.60520.23951.6433-0.01952.5057-0.02480.06540.1812-0.10730.0293-0.14410.16090.3614-0.00450.0605-0.01360.01630.1110.0115-0.0444-35.89861.716-44.524
25.279-0.4907-1.1323.2687-0.57516.9367-0.13190.02920.23130.00680.1811-0.234-0.21140.0806-0.0492-0.06260.12510.07490.03330.04260.134-12.91749.885-23.039
30.9907-1.0393-0.9891.31540.54382.78030.0059-0.07710.1352-0.3531-0.16220.2124-0.0782-0.18830.15630.0762-0.0555-0.07440.1011-0.0210.0684-50.84368.498-47.62
42.3527-2.2427-0.23424.5193-0.31012.3223-0.2948-0.03610.08660.61670.1417-0.11880.043-0.46440.15310.08740.18140.07560.23240.03280.1759-20.54643.36-12.176
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 182
2X-RAY DIFFRACTION1A301 - 302
3X-RAY DIFFRACTION1A303
4X-RAY DIFFRACTION2A183 - 254
5X-RAY DIFFRACTION3C1 - 10
6X-RAY DIFFRACTION3D1 - 10
7X-RAY DIFFRACTION4E1 - 10
8X-RAY DIFFRACTION4F1 - 10

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