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- PDB-6ykf: VcaM4I restriction endonuclease in the presence of 5mC-modified ssDNA -

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Basic information

Entry
Database: PDB / ID: 6ykf
TitleVcaM4I restriction endonuclease in the presence of 5mC-modified ssDNA
Components
  • DNA (5'-D(*CP*AP*(5CM)P*AP*G)-3')
  • HNH endonuclease
KeywordsHYDROLASE / PUA superfamily / EVE domain / DNA endonuclease / modification-dependent restriction endonuclease / MDRE / 5-hydroxymethylcytosine / 5-methylcytosine / single stranded DNA
Function / homologyHNH endonuclease / HNH nuclease / endonuclease activity / DNA / HNH endonuclease
Function and homology information
Biological speciesVibrio campbellii (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.48 Å
AuthorsPastor, M. / Czapinska, H. / Lutz, T. / Helbrecht, I. / Xu, S. / Bochtler, M.
Citation
Journal: Nucleic Acids Res. / Year: 2021
Title: Crystal structures of the EVE-HNH endonuclease VcaM4I in the presence and absence of DNA.
Authors: Pastor, M. / Czapinska, H. / Helbrecht, I. / Krakowska, K. / Lutz, T. / Xu, S.Y. / Bochtler, M.
#1: Journal: Nucleic Acids Res. / Year: 2019
Title: A protein architecture guided screen for modification dependent restriction endonucleases.
Authors: Lutz, T. / Flodman, K. / Copelas, A. / Czapinska, H. / Mabuchi, M. / Fomenkov, A. / He, X. / Bochtler, M. / Xu, S.Y.
History
DepositionApr 6, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 23, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 27, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Mar 3, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: HNH endonuclease
B: DNA (5'-D(*CP*AP*(5CM)P*AP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)38,90724
Polymers36,8942
Non-polymers2,01322
Water10,557586
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5500 Å2
ΔGint-131 kcal/mol
Surface area16260 Å2
MethodPISA
Unit cell
Length a, b, c (Å)129.000, 129.000, 110.911
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number180
Space group name H-MP6222
Components on special symmetry positions
IDModelComponents
11A-410-

GOL

21A-618-

HOH

31A-718-

HOH

41A-919-

HOH

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Components

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Protein / DNA chain , 2 types, 2 molecules AB

#1: Protein HNH endonuclease


Mass: 35390.543 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio campbellii (bacteria) / Gene: DSB67_20905 / Plasmid: pTXB1 (modified) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta / References: UniProt: A0A344KQF3
#2: DNA chain DNA (5'-D(*CP*AP*(5CM)P*AP*G)-3')


Mass: 1503.053 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)

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Non-polymers , 4 types, 608 molecules

#3: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: SO4
#4: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 586 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: 3.61 Å3/Da / Density % sol: 65.93 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 5.25
Details: RESERVOIR SOLUTION: 1.6 M (NH4)2SO4 , 0.1 M MES PH 5.25; PROTEIN:DNA SOLUTION: 0.3 M NACL, 15 MM TRIS-HCL PH 8.5 AND 1 MM TCEP. FOR CRYO-PROTECTION THE RESERVOIR SOLUTION WAS DILUTED WITH ...Details: RESERVOIR SOLUTION: 1.6 M (NH4)2SO4 , 0.1 M MES PH 5.25; PROTEIN:DNA SOLUTION: 0.3 M NACL, 15 MM TRIS-HCL PH 8.5 AND 1 MM TCEP. FOR CRYO-PROTECTION THE RESERVOIR SOLUTION WAS DILUTED WITH GLYCEROL TO ACHIEVE 30% CONCENTRATION.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P13 (MX1) / Wavelength: 0.97626 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Dec 15, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97626 Å / Relative weight: 1
ReflectionResolution: 1.48→42.23 Å / Num. obs: 90208 / % possible obs: 98.9 % / Redundancy: 10.6 % / Biso Wilson estimate: 26.7 Å2 / CC1/2: 0.999 / Rrim(I) all: 0.096 / Rsym value: 0.091 / Net I/σ(I): 14.95
Reflection shellResolution: 1.48→1.56 Å / Redundancy: 10.1 % / Mean I/σ(I) obs: 1.91 / Num. unique obs: 14319 / CC1/2: 0.742 / Rrim(I) all: 1.019 / Rsym value: 0.967 / % possible all: 98.5

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
XDSdata reduction
XDSdata scaling
PDB_EXTRACT3.25data extraction
REFMACphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6YJB

6yjb


Resolution: 1.48→42.23 Å / Cor.coef. Fo:Fc: 0.974 / Cor.coef. Fo:Fc free: 0.967 / SU B: 2.674 / SU ML: 0.043 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.058 / ESU R Free: 0.059
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED. THE CONFORMATION OF TWO RESIDUES AT THE 5' END OF THE OLIGONUCLEOTIDE IS UNCERTAIN THE IDENTITY OF THE SOLVENT ...Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED. THE CONFORMATION OF TWO RESIDUES AT THE 5' END OF THE OLIGONUCLEOTIDE IS UNCERTAIN THE IDENTITY OF THE SOLVENT MOLECULES HAS BEEN ASSIGNED TENTATIVELY.
RfactorNum. reflection% reflectionSelection details
Rfree0.1881 4481 5 %RANDOM
Rwork0.1693 ---
obs0.1702 85724 98.87 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 85.22 Å2 / Biso mean: 23.433 Å2 / Biso min: 12.39 Å2
Baniso -1Baniso -2Baniso -3
1-0.72 Å20.36 Å20 Å2
2--0.72 Å20 Å2
3----2.34 Å2
Refinement stepCycle: final / Resolution: 1.48→42.23 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2499 100 121 589 3309
Biso mean--45.42 44.69 -
Num. residues----314
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0060.0133160
X-RAY DIFFRACTIONr_bond_other_d0.0010.0182742
X-RAY DIFFRACTIONr_angle_refined_deg1.2881.6264329
X-RAY DIFFRACTIONr_angle_other_deg1.3841.6136440
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.4365386
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.1123.772167
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.6315534
X-RAY DIFFRACTIONr_dihedral_angle_4_deg20.0851514
X-RAY DIFFRACTIONr_chiral_restr0.0650.2377
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.023590
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02672
LS refinement shellResolution: 1.48→1.51 Å / Rfactor Rfree error: 0
RfactorNum. reflection% reflection
Rfree0.408 327 -
Rwork0.366 6103 -
obs--96.84 %
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)
10.61750.09680.01220.022-0.03890.38320.00670.00050.0727-0.0002-0.0040.0292-0.02010.0285-0.00260.0268-0.00920.00530.0466-0.00530.17185.558-56.7997-19.4123
21.45680.1502-0.29250.15040.07190.31990.1167-0.10610.21090.0244-0.12860.0146-0.0097-0.0330.0120.0117-0.01650.01490.1134-0.03350.192434.3401-43.1612-6.3165
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 181
2X-RAY DIFFRACTION1B1 - 5
3X-RAY DIFFRACTION1C1 - 2000
4X-RAY DIFFRACTION1D1 - 2000
5X-RAY DIFFRACTION2A182 - 310
6X-RAY DIFFRACTION2E1 - 2000

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