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- PDB-6yex: VcaM4I restriction endonuclease in the absence of DNA -

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Basic information

Entry
Database: PDB / ID: 6yex
TitleVcaM4I restriction endonuclease in the absence of DNA
ComponentsHNH endonuclease
KeywordsHYDROLASE / PUA superfamily / EVE domain / DNA endonuclease / modification-dependent restriction endonuclease / MDRE / single stranded DNA
Function / homologyHNH endonuclease / HNH nuclease / endonuclease activity / HNH endonuclease
Function and homology information
Biological speciesVibrio campbellii (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 1.5 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
DepositionMar 25, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 16, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 23, 2020Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year
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 1.4May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
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: HNH endonuclease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)71,87921
Polymers70,7812
Non-polymers1,09819
Water18,4471024
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6830 Å2
ΔGint-183 kcal/mol
Surface area27880 Å2
MethodPISA
Unit cell
Length a, b, c (Å)56.970, 93.532, 126.261
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
DetailsThe modification specific VcaM4I restriction endonuclease is active as a dimer (each protomer cleaves one strand of the double stranded DNA substrate). The C-terminal HNH nuclease domains mediate the dimerization. The N-terminal EVE domains are responsible for the modification specificity.

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Components

#1: Protein HNH endonuclease


Mass: 35390.543 Da / Num. of mol.: 2
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: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: SO4
#3: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 12 / Source method: isolated from a natural source / Formula: Cl
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1024 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 2.38 Å3/Da / Density % sol: 48.24 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: Protein (7.2 mg/ml) in 0.27 M NaCl, 13.5 mM Tris-HCl pH 8.5, 0.2 M sodium malonate and 0.9 mM TCEP was mixed in 1:1 ratio with reservoir solution (1.8 M ammonium sulfate, 0.1 M MES pH 5.25). ...Details: Protein (7.2 mg/ml) in 0.27 M NaCl, 13.5 mM Tris-HCl pH 8.5, 0.2 M sodium malonate and 0.9 mM TCEP was mixed in 1:1 ratio with reservoir solution (1.8 M ammonium sulfate, 0.1 M MES pH 5.25). Perfluoropolyether oil was used as a cryoprotectant.

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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 PILATUS 6M-F / Detector: PIXEL / Date: Oct 5, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0064 Å / Relative weight: 1
ReflectionResolution: 1.5→30 Å / Num. obs: 107966 / % possible obs: 99.2 % / Redundancy: 13.1 % / Biso Wilson estimate: 32.7 Å2 / CC1/2: 1 / Rrim(I) all: 0.046 / Rsym value: 0.044 / Net I/σ(I): 26.06
Reflection shellResolution: 1.5→1.59 Å / Redundancy: 13.4 % / Mean I/σ(I) obs: 2.27 / Num. unique obs: 17067 / CC1/2: 0.851 / Rrim(I) all: 1.104 / Rsym value: 1.062 / % possible all: 98.2

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
XDSdata reduction
XDSdata scaling
SHELXDEphasing
ARP/wARPmodel building
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: SAD / Resolution: 1.5→28.48 Å / Cor.coef. Fo:Fc: 0.97 / Cor.coef. Fo:Fc free: 0.965 / SU B: 5.305 / SU ML: 0.082 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.083 / ESU R Free: 0.08
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED THE IDENTITY OF THE SOLVENT MOLECULES HAS BEEN ASSIGNED TENTATIVELY.
RfactorNum. reflection% reflectionSelection details
Rfree0.2132 1978 1.8 %RANDOM
Rwork0.1933 ---
obs0.1936 105907 99.2 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 109.92 Å2 / Biso mean: 33.633 Å2 / Biso min: 18.07 Å2
Baniso -1Baniso -2Baniso -3
1--2.14 Å2-0 Å20 Å2
2--2.66 Å2-0 Å2
3----0.52 Å2
Refinement stepCycle: final / Resolution: 1.5→28.48 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4957 0 47 1025 6029
Biso mean--57.33 46.12 -
Num. residues----612
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0060.0195641
X-RAY DIFFRACTIONr_bond_other_d0.0010.024936
X-RAY DIFFRACTIONr_angle_refined_deg1.1311.9337698
X-RAY DIFFRACTIONr_angle_other_deg0.726311569
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.8375714
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.03524.95299
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.02515989
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.7251527
X-RAY DIFFRACTIONr_chiral_restr0.0730.2782
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.026588
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021195
LS refinement shellResolution: 1.5→1.58 Å / Rfactor Rfree error: 0
RfactorNum. reflection% reflection
Rfree0.328 283 -
Rwork0.305 15090 -
obs--98.15 %
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)
10.82310.02840.09040.3046-0.23380.4844-0.01390.0385-0.050.0544-0.06790.0483-0.02530.00720.08180.137-0.01650.03340.0341-0.05720.126918.554230.528628.8684
21.007-0.47610.05721.2118-0.23330.61060.0066-0.158-0.04110.2282-0.0071-0.0041-0.07610.0460.00050.1907-0.05510.01240.0424-0.00010.080836.772319.122351.4107
30.352-0.4967-0.80091.97460.94672.3580.05550.2628-0.1629-0.113-0.37950.1703-0.1901-0.3320.3240.03490.0237-0.04030.3626-0.18550.126513.059427.2833-1.5962
47.9945-3.45414.28232.2048-2.53142.94540.02980.30720.019-0.1345-0.1179-0.08470.13150.1470.0880.10590.0764-0.04250.1533-0.11950.110233.322824.971218.5595
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A157 - 309
2X-RAY DIFFRACTION1B182 - 309
3X-RAY DIFFRACTION1C1 - 2000
4X-RAY DIFFRACTION1D1 - 2000
5X-RAY DIFFRACTION2A1 - 156
6X-RAY DIFFRACTION2E1 - 2000
7X-RAY DIFFRACTION3B1 - 156
8X-RAY DIFFRACTION3F1 - 2000
9X-RAY DIFFRACTION4B157 - 181
10X-RAY DIFFRACTION4G1 - 2000

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