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- PDB-6eko: Crystal structure of Type IIP restriction endonuclease PfoI with ... -

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Basic information

Entry
Database: PDB / ID: 6eko
TitleCrystal structure of Type IIP restriction endonuclease PfoI with cognate DNA
Components
  • DNA (5'-D(*CP*GP*CP*TP*CP*CP*CP*GP*GP*AP*GP*CP*GP*T)-3')
  • Restriction endonuclease PfoI
KeywordsHYDROLASE / Restriction endonuclease / PD-(D/E)xK nuclease
Function / homologytype II site-specific deoxyribonuclease / type II site-specific deoxyribonuclease activity / metal ion binding / DNA / DNA (> 10) / Restriction endonuclease PfoI
Function and homology information
Biological speciesPseudomonas fluorescens (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2.284 Å
AuthorsTamulaitiene, G. / Manakova, E. / Jovaisaite, V. / Grazulis, S. / Siksnys, V.
Funding supportLithuania, 2items
OrganizationGrant numberCountry
Research Council of LithuaniaMIP-41/2013Lithuania
European UnionBioStruct-X (grant agreement 283570)
CitationJournal: Nucleic Acids Res. / Year: 2019
Title: Unique mechanism of target recognition by PfoI restriction endonuclease of the CCGG-family.
Authors: Tamulaitiene, G. / Manakova, E. / Jovaisaite, V. / Tamulaitis, G. / Grazulis, S. / Bochtler, M. / Siksnys, V.
History
DepositionSep 26, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 10, 2018Provider: repository / Type: Initial release
Revision 1.1Apr 24, 2019Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / entity / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _entity.formula_weight / _pdbx_seq_map_depositor_info.one_letter_code_mod
Revision 1.2Oct 16, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature / 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.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _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.ptnr1_symmetry / _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_conn.ptnr2_symmetry

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Restriction endonuclease PfoI
B: Restriction endonuclease PfoI
F: DNA (5'-D(*CP*GP*CP*TP*CP*CP*CP*GP*GP*AP*GP*CP*GP*T)-3')
E: DNA (5'-D(*CP*GP*CP*TP*CP*CP*CP*GP*GP*AP*GP*CP*GP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)79,8518
Polymers79,6914
Non-polymers1604
Water4,738263
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, The dimeric assembly is evidenced by gel filtration, dynamic light scattering, crystal structure and from the homology to other related restriction endonucleases
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area13620 Å2
ΔGint-133 kcal/mol
Surface area25820 Å2
MethodPISA
Unit cell
Length a, b, c (Å)56.279, 91.663, 152.742
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Restriction endonuclease PfoI


Mass: 35587.695 Da / Num. of mol.: 2 / Mutation: K187A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas fluorescens (bacteria) / Variant: biovar 126 / Plasmid: pBAD24 / Production host: Escherichia coli (E. coli) / Strain (production host): ER2566
References: UniProt: A0A452CST9*PLUS, type II site-specific deoxyribonuclease
#2: DNA chain DNA (5'-D(*CP*GP*CP*TP*CP*CP*CP*GP*GP*AP*GP*CP*GP*T)-3')


Mass: 4257.754 Da / Num. of mol.: 2 / Source method: obtained synthetically
Details: Cognate oligoduplex with 3'-T-overhang and unpaired central recognition sequence base pair (C-C)
Source: (synth.) Pseudomonas fluorescens (bacteria)
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 263 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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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: 2.8 Å3/Da / Density % sol: 56.09 % / Description: needles
Crystal growTemperature: 292 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: Reservoir: 20% PEG8000, 0.1M TrisHCl pH8.5, LiCl 0.2M and glycerol 10%. Protein-DNA complex concentration 5.3 mg/ml

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Data collection

DiffractionMean temperature: 100 K
Ambient temp details: cryo-protected by passing through mineral oil
Diffraction sourceSource: SYNCHROTRON / Site: MAX II / Beamline: I911-3 / Wavelength: 0.96112,0.98010
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Dec 3, 2015
Details: Rh-coated Si mirrors: M1 collimating mirror, M2 toroidal focusing mirror
RadiationMonochromator: Water-cooled double-crystal monochromator, Si(111)
Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
10.961121
20.98011
ReflectionResolution: 2.28→47.96 Å / Num. obs: 36181 / % possible obs: 98.5 % / Redundancy: 13.5 % / Biso Wilson estimate: 27.3 Å2 / Rmerge(I) obs: 0.198 / Rpim(I) all: 0.058 / Rrim(I) all: 0.216 / Rsym value: 0.198 / Net I/σ(I): 11.6
Reflection shellResolution: 2.28→2.41 Å / Redundancy: 6.1 % / Mean I/σ(I) obs: 1.8 / Num. unique obs: 4683 / Rpim(I) all: 0.442 / % possible all: 89.8

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Processing

Software
NameVersionClassification
XDSVERSION Jun 17, 2015 BUILT=20150617data reduction
SCALACCP4 6.2: Scala version 3.3.20 : 23/06/11data scaling
Auto-Rickshawphasing
Coot0.6.1 Miramar (revision 2740)model building
PHENIX1.8.3_1479refinement
RefinementMethod to determine structure: MAD / Resolution: 2.284→47.96 Å / SU ML: 0.28 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 27.99
RfactorNum. reflection% reflection
Rfree0.2478 6650 9.77 %
Rwork0.2051 --
obs0.2094 36082 98.31 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.284→47.96 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4919 564 4 263 5750
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0095652
X-RAY DIFFRACTIONf_angle_d1.0857739
X-RAY DIFFRACTIONf_dihedral_angle_d18.1472129
X-RAY DIFFRACTIONf_chiral_restr0.062868
X-RAY DIFFRACTIONf_plane_restr0.005895
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.2842-2.31010.36021520.34941526X-RAY DIFFRACTION73
2.3101-2.33730.39852000.32281831X-RAY DIFFRACTION86
2.3373-2.36580.30611780.3041945X-RAY DIFFRACTION94
2.3658-2.39580.34692170.31132058X-RAY DIFFRACTION98
2.3958-2.42730.35952090.27832110X-RAY DIFFRACTION100
2.4273-2.46050.29612180.28212079X-RAY DIFFRACTION100
2.4605-2.49570.33432480.26392056X-RAY DIFFRACTION100
2.4957-2.53290.32761820.25852106X-RAY DIFFRACTION100
2.5329-2.57250.28961910.25522093X-RAY DIFFRACTION100
2.5725-2.61470.2992210.2712095X-RAY DIFFRACTION100
2.6147-2.65980.25372430.27992052X-RAY DIFFRACTION100
2.6598-2.70810.3262070.25742138X-RAY DIFFRACTION100
2.7081-2.76020.34042270.24532048X-RAY DIFFRACTION100
2.7602-2.81660.29812120.24782111X-RAY DIFFRACTION100
2.8166-2.87780.28052260.24722062X-RAY DIFFRACTION100
2.8778-2.94470.32882570.23932054X-RAY DIFFRACTION100
2.9447-3.01840.28662350.23952079X-RAY DIFFRACTION100
3.0184-3.10.29032130.2362110X-RAY DIFFRACTION100
3.1-3.19120.3092170.22952070X-RAY DIFFRACTION100
3.1912-3.29410.26982460.22142082X-RAY DIFFRACTION100
3.2941-3.41180.26542540.20012036X-RAY DIFFRACTION100
3.4118-3.54840.25162270.17632104X-RAY DIFFRACTION100
3.5484-3.70980.21562070.17962097X-RAY DIFFRACTION100
3.7098-3.90530.20082410.16252057X-RAY DIFFRACTION100
3.9053-4.14990.17982660.15152063X-RAY DIFFRACTION100
4.1499-4.47010.17562130.13962063X-RAY DIFFRACTION100
4.4701-4.91950.15052200.1342100X-RAY DIFFRACTION100
4.9195-5.63050.1972690.15172049X-RAY DIFFRACTION100
5.6305-7.09010.20972280.16092080X-RAY DIFFRACTION100
7.0901-47.97110.16952260.15982051X-RAY DIFFRACTION99

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