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- PDB-6yjb: VcaM4I restriction endonuclease 5hmC-ssDNA complex -

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Basic information

Entry
Database: PDB / ID: 6yjb
TitleVcaM4I restriction endonuclease 5hmC-ssDNA complex
Components
  • DNA (5'-D(*CP*AP*(5HC)P*AP*G)-3')
  • HNH endonuclease
KeywordsHYDROLASE / PUA superfamily / EVE domain / DNA endonuclease / modification-dependent restriction endonuclease / MDRE / 5-methylcytosineE / 5-hydroxymethylcytosine / 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: 1.55 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 2, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 23, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 30, 2020Group: Experimental preparation / Category: exptl_crystal_grow / Item: _exptl_crystal_grow.pdbx_details
Revision 1.2Jan 27, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.3Mar 3, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Jan 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*(5HC)P*AP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)38,86624
Polymers36,9102
Non-polymers1,95622
Water10,503583
1
A: HNH endonuclease
B: DNA (5'-D(*CP*AP*(5HC)P*AP*G)-3')
hetero molecules

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


Theoretical massNumber of molelcules
Total (without water)77,73248
Polymers73,8194
Non-polymers3,91344
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation10_444-y-1,-x-1,-z-1/31
Buried area11870 Å2
ΔGint-297 kcal/mol
Surface area30490 Å2
MethodPISA
Unit cell
Length a, b, c (Å)128.685, 128.685, 110.895
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-656-

HOH

31A-701-

HOH

41A-930-

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*(5HC)P*AP*G)-3')


Mass: 1519.052 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, 605 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.: 9 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 583 / 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: 3.59 Å3/Da / Density % sol: 65.74 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: Reservoir solution: 1.6 M (NH4)2SO4, 0.1 M MES pH 5.25; protein:DNA solution: 300 mM NaCl, 15 mM Tris-HCl pH 8.5 and 1mM 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: 300 mM NaCl, 15 mM Tris-HCl pH 8.5 and 1mM TCEP. For cryo-protection the reservoir solution was diluted with glycerol to achieve 30% concentration. 0.1 M spermine tetrahydrochloride was used as an additive.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: EMBL/DESY, HAMBURG / Beamline: X13 / Wavelength: 1.0064 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 28, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0064 Å / Relative weight: 1
ReflectionResolution: 1.55→29.77 Å / Num. obs: 78576 / % possible obs: 99.9 % / Redundancy: 39.2 % / Biso Wilson estimate: 30.8 Å2 / CC1/2: 1 / Rrim(I) all: 0.103 / Rsym value: 0.101 / Net I/σ(I): 26.96
Reflection shellResolution: 1.55→1.64 Å / Redundancy: 39.9 % / Mean I/σ(I) obs: 1.96 / Num. unique obs: 12449 / CC1/2: 0.752 / Rrim(I) all: 2.364 / Rsym value: 2.334 / % possible all: 99.7

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Processing

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

6yex


Resolution: 1.55→29.77 Å / Cor.coef. Fo:Fc: 0.98 / Cor.coef. Fo:Fc free: 0.974 / SU B: 2.724 / SU ML: 0.042 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.055 / ESU R Free: 0.057
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
Rfree0.1636 3912 5 %RANDOM
Rwork0.1425 ---
obs0.1436 74619 99.93 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 90.49 Å2 / Biso mean: 28.835 Å2 / Biso min: 16.71 Å2
Baniso -1Baniso -2Baniso -3
1-0.38 Å20.19 Å20 Å2
2--0.38 Å2-0 Å2
3----1.22 Å2
Refinement stepCycle: final / Resolution: 1.55→29.77 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2499 101 121 585 3306
Biso mean--49.43 51.74 -
Num. residues----314
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0080.0133109
X-RAY DIFFRACTIONr_bond_other_d0.0010.0182688
X-RAY DIFFRACTIONr_angle_refined_deg1.3581.6264257
X-RAY DIFFRACTIONr_angle_other_deg1.451.6146306
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.5345376
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.5123.659164
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.66415519
X-RAY DIFFRACTIONr_dihedral_angle_4_deg24.4571514
X-RAY DIFFRACTIONr_chiral_restr0.0710.2372
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.023506
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02658
LS refinement shellResolution: 1.55→1.59 Å / Rfactor Rfree error: 0
RfactorNum. reflection% reflection
Rfree0.276 294 -
Rwork0.258 5386 -
obs--99.44 %
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)
10.73560.1132-0.02680.0325-0.04680.420.00770.01490.07270.0008-0.00520.036-0.01980.0235-0.00250.0287-0.00730.00370.0473-0.00290.06935.6304-56.5878-19.4686
21.54150.1689-0.36160.2603-0.08540.53040.1291-0.10730.26390.0335-0.13230.0185-0.0109-0.0030.00310.0145-0.01890.01950.0806-0.03830.093734.1806-43.0633-6.1848
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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