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- PDB-3bm3: Restriction endonuclease PspGI-substrate DNA complex -

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Basic information

Entry
Database: PDB / ID: 3bm3
TitleRestriction endonuclease PspGI-substrate DNA complex
Components
  • DNA (5'-D(*CP*AP*TP*CP*CP*AP*GP*GP*TP*AP*C)-3')
  • DNA (5'-D(*GP*GP*TP*AP*CP*CP*TP*GP*GP*AP*T)-3')
  • PspGI restriction endonuclease
KeywordsHYDROLASE/DNA / ENDONUCLEASE-DNA COMPLEX / RESTRICTION ENZYME / PSPGI / BASE FLIPPING / HYDROLASE-DNA COMPLEX
Function / homology
Function and homology information


type II site-specific deoxyribonuclease activity / DNA restriction-modification system / DNA binding
Similarity search - Function
Restriction Endonuclease - #80 / Restriction endonuclease, type II, EcoRII, C-terminal / EcoRII, C-terminal domain superfamily / EcoRII C terminal / Restriction Endonuclease / Restriction endonuclease type II-like / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
CITRIC ACID / DNA / DNA (> 10) / PspGI restriction endonuclease
Similarity search - Component
Biological speciesPyrococcus sp. GI-H (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / Resolution: 1.7 Å
AuthorsSzczepanowski, R.H. / Carpenter, M. / Czapinska, H. / Tamulaitis, G. / Siksnys, V. / Bhagwat, A. / Bochtler, M.
Citation
Journal: To be Published
Title: A direct crystallographic demonstration that Type II restriction endonuclease PspGI flips nucleotides
Authors: Szczepanowski, R.H. / Carpenter, M. / Czapinska, H. / Tamulaitis, G. / Siksnys, V. / Bhagwat, A. / Bochtler, M.
#1: Journal: Nucleic Acids Res. / Year: 2007
Title: Nucleotide flipping by restriction enzymes analyzed by 2-aminopurine steady-state fluorescence.
Authors: Tamulaitis, G. / Zaremba, M. / Szczepanowski, R.H. / Bochtler, M. / Siksnys, V.
#2: Journal: Nucleic Acids Res. / Year: 2006
Title: Sequence-dependent enhancement of hydrolytic deamination of cytosines in DNA by the restriction enzyme PspGI.
Authors: Carpenter, M. / Divvela, P. / Pingoud, V. / Bujnicki, J. / Bhagwat, A.S.
#3: Journal: J.Mol.Biol. / Year: 2003
Title: PspGI, a type II restriction endonuclease from the extreme thermophile Pyrococcus sp.: structural and functional studies to investigate an evolutionary relationship with several mesophilic restriction enzymes.
Authors: Pingoud, V. / Conzelmann, C. / Kinzebach, S. / Sudina, A. / Metelev, V. / Kubareva, E. / Bujnicki, J.M. / Lurz, R. / Luder, G. / Xu, S.Y. / Pingoud, A.
#4: Journal: Appl.Environ.Microbiol. / Year: 1998
Title: Characterization of an extremely thermostable restriction enzyme, PspGI, from a Pyrococcus strain and cloning of the PspGI restriction-modification system in Escherichia coli.
Authors: Morgan, R. / Xiao, J. / Xu, S.
History
DepositionDec 12, 2007Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 16, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Advisory / Refinement description / Version format compliance
Revision 1.2Oct 25, 2017Group: Advisory / Refinement description / Category: pdbx_unobs_or_zero_occ_atoms / software / Item: _software.name
Revision 1.3Oct 20, 2021Group: Advisory / Database references / Derived calculations
Category: database_2 / pdbx_unobs_or_zero_occ_atoms ...database_2 / pdbx_unobs_or_zero_occ_atoms / 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
C: DNA (5'-D(*CP*AP*TP*CP*CP*AP*GP*GP*TP*AP*C)-3')
D: DNA (5'-D(*GP*GP*TP*AP*CP*CP*TP*GP*GP*AP*T)-3')
A: PspGI restriction endonuclease
B: PspGI restriction endonuclease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)71,3305
Polymers71,1384
Non-polymers1921
Water5,278293
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10070 Å2
MethodPISA
Unit cell
Length a, b, c (Å)46.884, 96.060, 127.069
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121
DetailsTHE TETRAMER (ACCORDING TO PDB CONVENTIONS) IS A COMPLEX OF THE DIMERIC RESTRICTION ENZYME WITH ITS SUBSTRATE, DOUBLE STRANDED DNA.

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Components

#1: DNA chain DNA (5'-D(*CP*AP*TP*CP*CP*AP*GP*GP*TP*AP*C)-3')


Mass: 3318.187 Da / Num. of mol.: 1 / Source method: obtained synthetically
#2: DNA chain DNA (5'-D(*GP*GP*TP*AP*CP*CP*TP*GP*GP*AP*T)-3')


Mass: 3389.221 Da / Num. of mol.: 1 / Source method: obtained synthetically
#3: Protein PspGI restriction endonuclease


Mass: 32215.279 Da / Num. of mol.: 2 / Mutation: D138A, A146T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus sp. GI-H (archaea) / Plasmid: pET21a / Production host: Escherichia coli (E. coli) / Strain (production host): B834 / References: UniProt: O93646
#4: Chemical ChemComp-CIT / CITRIC ACID / Citric acid


Mass: 192.124 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H8O7
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 293 / 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: 2.01 Å3/Da / Density % sol: 38.84 %
Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop / pH: 4
Details: 20% MPD, 0.1 M citric acid, 0.1 M cobalt (II) chloride as an additive, pH 4.0, VAPOR DIFFUSION, SITTING DROP, temperature 294K
Components of the solutions
IDNameCrystal-IDSol-ID
1MPD11
2citric acid11
3cobalt (II) chloride11
4MPD12
5citric acid12
6cobalt (II) chloride12

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: MPG/DESY, HAMBURG / Beamline: BW6 / Wavelength: 0.976 Å
DetectorType: MARRESEARCH / Detector: CCD / Date: Aug 16, 2007 / Details: BENT MIRROR
RadiationMonochromator: TRIANGULAR MONOCHROMATOR / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976 Å / Relative weight: 1
ReflectionResolution: 1.7→20 Å / Num. all: 63138 / Num. obs: 63138 / % possible obs: 98.7 % / Redundancy: 3.3 % / Biso Wilson estimate: 20.7 Å2 / Rsym value: 0.065 / Net I/σ(I): 21.3
Reflection shellResolution: 1.7→1.72 Å / Redundancy: 3.1 % / Mean I/σ(I) obs: 3.2 / Num. unique all: 2460 / Rsym value: 0.254 / % possible all: 98

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Processing

Software
NameVersionClassification
SHELXDphasing
SHELXEmodel building
DMmodel building
ARP/wARPmodel building
REFMAC5.2.0019refinement
MAR345data collection
DENZOdata reduction
SCALEPACKdata scaling
DMphasing
RefinementResolution: 1.7→19.38 Å / Cor.coef. Fo:Fc: 0.962 / Cor.coef. Fo:Fc free: 0.955 / TLS residual ADP flag: LIKELY RESIDUAL / Cross valid method: THROUGHOUT / ESU R: 0.109 / ESU R Free: 0.099 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: CNS HAS BEEN USED FOR DNA REFINEMENT. NO CONSTRAINTS FOR SUGAR PUCKER WERE APPLIED. RESIDUES AT THE ENDS OF THE DNA DUPLEX ( 5 AND 5 IN CHAIN C AND -6 AND -5 IN CHAIN D) HAVE POOR DENSITY. ...Details: CNS HAS BEEN USED FOR DNA REFINEMENT. NO CONSTRAINTS FOR SUGAR PUCKER WERE APPLIED. RESIDUES AT THE ENDS OF THE DNA DUPLEX ( 5 AND 5 IN CHAIN C AND -6 AND -5 IN CHAIN D) HAVE POOR DENSITY. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. TLS REFINEMENT HAS BEEN USED.
RfactorNum. reflection% reflectionSelection details
Rfree0.1924 3230 5.1 %RANDOM
Rwork0.1698 ---
all0.171 63135 --
obs0.171 63135 98.7 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 17.9 Å2
Baniso -1Baniso -2Baniso -3
1--0.63 Å20 Å20 Å2
2---1.11 Å20 Å2
3---1.74 Å2
Refinement stepCycle: LAST / Resolution: 1.7→19.38 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4319 445 13 293 5070
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0130.0225167
X-RAY DIFFRACTIONr_bond_other_d00.023538
X-RAY DIFFRACTIONr_angle_refined_deg1.3692.0777083
X-RAY DIFFRACTIONr_angle_other_deg3.85238607
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.0865567
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.03724.024251
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.78615898
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.0831542
X-RAY DIFFRACTIONr_chiral_restr0.0790.2754
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.025511
X-RAY DIFFRACTIONr_gen_planes_other0.010.021058
X-RAY DIFFRACTIONr_nbd_refined0.2220.21308
X-RAY DIFFRACTIONr_nbd_other0.2520.24044
X-RAY DIFFRACTIONr_nbtor_refined0.1990.22669
X-RAY DIFFRACTIONr_nbtor_other0.1030.22557
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1490.2361
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.2230.233
X-RAY DIFFRACTIONr_symmetry_vdw_other0.2570.270
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1380.210
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it0.7851.52739
X-RAY DIFFRACTIONr_mcbond_other01.51092
X-RAY DIFFRACTIONr_mcangle_it1.41724496
X-RAY DIFFRACTIONr_scbond_it2.34432428
X-RAY DIFFRACTIONr_scangle_it3.6864.52587
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.7→1.744 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.222 236 -
Rwork0.202 4290 -
obs-4290 97.5 %
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)
10.33690.11640.11812.61921.16570.68450.0236-0.1015-0.00250.1321-0.07480.16230.1026-0.03260.0512-0.023-0.02210.021-0.0076-0.0091-0.026410.6244.30126.535
20.823-0.0817-0.27440.163-0.14080.85650.0373-0.07060.1169-0.0013-0.00980.0106-0.04630.0266-0.0275-0.0079-0.00780.0011-0.0378-0.0241-0.01325.14664.23821.342
30.16890.2793-0.29220.548-0.41170.7019-0.0153-0.012-0.0669-0.05470.0478-0.06480.06940.0104-0.03250.02330.01150.0194-0.0263-0.006-0.020936.14831.1285.715
40.4988-0.1151-0.04340.5720.27780.7173-0.01430.0350.0697-0.05980.0386-0.0603-0.02360.0405-0.0243-0.0018-0.02050.0139-0.02620.0073-0.022837.29755.8661.265
50.5332-0.2184-0.45520.7940.01420.4988-0.0244-0.01620.04210.01620.0122-0.05090.02010.03640.01220.0051-0.00240.0033-0.0158-0.0006-0.017926.443.83514.765
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A2 - 116
2X-RAY DIFFRACTION2A117 - 260
3X-RAY DIFFRACTION3B3 - 116
4X-RAY DIFFRACTION4B117 - 260
5X-RAY DIFFRACTION5C-5 - 5
6X-RAY DIFFRACTION5D-6 - 4

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