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- PDB-6ymg: VcaM4I restriction endonuclease in complex with 5mC-modified dsDNA -

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Basic information

Entry
Database: PDB / ID: 6ymg
TitleVcaM4I restriction endonuclease in complex with 5mC-modified dsDNA
Components
  • DNA (5'-D(*CP*CP*AP*TP*GP*(5CM)P*GP*CP*TP*GP*A)-3')
  • DNA (5'-D(P*CP*AP*GP*CP*GP*CP*AP*TP*GP*G)-3')
  • HNH endonuclease
KeywordsHYDROLASE / PUA superfamily / EVE domain / DNA endonuclease / modification-dependent restriction endonuclease / MDRE / 5-hydroxymethylcytosine / 5-methylcytosine
Function / homologyHNH endonuclease / HNH nuclease / endonuclease activity / DNA / DNA (> 10) / HNH endonuclease
Function and homology information
Biological speciesVibrio campbellii (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.14 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 8, 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 / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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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
C: DNA (5'-D(*CP*CP*AP*TP*GP*(5CM)P*GP*CP*TP*GP*A)-3')
D: DNA (5'-D(P*CP*AP*GP*CP*GP*CP*AP*TP*GP*G)-3')
E: DNA (5'-D(*CP*CP*AP*TP*GP*(5CM)P*GP*CP*TP*GP*A)-3')
F: DNA (5'-D(P*CP*AP*GP*CP*GP*CP*AP*TP*GP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)85,12616
Polymers84,2266
Non-polymers90010
Water1,54986
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area13250 Å2
ΔGint-144 kcal/mol
Surface area31500 Å2
MethodPISA
Unit cell
Length a, b, c (Å)81.111, 81.111, 617.307
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number178
Space group name H-MP6122
Components on special symmetry positions
IDModelComponents
11A-404-

SO4

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12C
22E
13D
23F

NCS domain segments:

Component-ID: _ / Refine code: _

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11METMETLEULEUAA1 - 3091 - 309
21METMETLEULEUBB1 - 3091 - 309
12DCDCDADACC1 - 111 - 11
22DCDCDADAEE1 - 111 - 11
13DTDTDGDGDD1 - 111 - 11
23DTDTDGDGFF1 - 111 - 11

NCS ensembles :
ID
1
2
3

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Components

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Protein , 1 types, 2 molecules AB

#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

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DNA chain , 2 types, 4 molecules CEDF

#2: DNA chain DNA (5'-D(*CP*CP*AP*TP*GP*(5CM)P*GP*CP*TP*GP*A)-3')


Mass: 3348.213 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#3: DNA chain DNA (5'-D(P*CP*AP*GP*CP*GP*CP*AP*TP*GP*G)-3')


Mass: 3374.210 Da / Num. of mol.: 2
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 , 3 types, 96 molecules

#4: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#5: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: SO4
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 86 / 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: 3.48 Å3/Da / Density % sol: 64.65 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 5.25
Details: Reservoir solution: 1.2 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: 1.2 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: BESSY / Beamline: 14.1 / Wavelength: 0.9184 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jan 30, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9184 Å / Relative weight: 1
ReflectionResolution: 3.14→49.1 Å / Num. obs: 20874 / % possible obs: 92.8 % / Redundancy: 9.6 % / Biso Wilson estimate: 54.3 Å2 / CC1/2: 0.991 / Rrim(I) all: 0.227 / Rsym value: 0.215 / Net I/σ(I): 8.6
Reflection shellResolution: 3.14→3.33 Å / Redundancy: 5.2 % / Mean I/σ(I) obs: 0.89 / Num. unique obs: 2591 / CC1/2: 0.306 / Rrim(I) all: 1.641 / Rsym value: 1.521 / % possible all: 74.4

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Processing

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

6yex


Resolution: 3.14→49.1 Å / Cor.coef. Fo:Fc: 0.904 / Cor.coef. Fo:Fc free: 0.881 / SU B: 65.356 / SU ML: 0.464 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 0.482
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
Rfree0.2662 1031 4.9 %RANDOM
Rwork0.2415 ---
obs0.2428 19843 92.79 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 182.33 Å2 / Biso mean: 71.579 Å2 / Biso min: 22.18 Å2
Baniso -1Baniso -2Baniso -3
1--0.76 Å2-0.38 Å2-0 Å2
2---0.76 Å20 Å2
3---2.47 Å2
Refinement stepCycle: final / Resolution: 3.14→49.1 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4998 892 46 86 6022
Biso mean--119.71 46.55 -
Num. residues----662
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0030.0126203
X-RAY DIFFRACTIONr_bond_other_d0.0010.0185057
X-RAY DIFFRACTIONr_angle_refined_deg1.1931.568587
X-RAY DIFFRACTIONr_angle_other_deg1.1731.71711807
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.1375621
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.54323.805297
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.92415881
X-RAY DIFFRACTIONr_dihedral_angle_4_deg11.3941522
X-RAY DIFFRACTIONr_chiral_restr0.0460.2772
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.026397
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021335
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A102950.07
12B102950.07
21C7480.13
22E7480.13
31D8790.1
32F8790.1
LS refinement shellResolution: 3.143→3.312 Å / Rfactor Rfree error: 0
RfactorNum. reflection% reflection
Rfree0.37 104 -
Rwork0.387 2214 -
obs--73.85 %
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)
10.67480.30550.06530.1630.15680.7030.02280.0476-0.1072-0.020.0195-0.0471-0.180.075-0.04230.1986-0.00540.02790.1648-0.0760.0536.3098-32.183719.9641
21.2566-0.5159-0.01940.57550.62491.1066-0.0543-0.0561-0.09030.02080.04880.0243-0.00820.08360.00550.1573-0.00250.02710.0949-0.0130.0388-1.8412-24.92853.1742
30.66920.2309-0.28552.2556-0.99011.22990.01060.0693-0.06870.1326-0.12450.0319-0.08760.05640.11390.10960.0264-0.01770.2042-0.01920.02647.807-18.4713-12.0967
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A157 - 310
2X-RAY DIFFRACTION1B157 - 310
3X-RAY DIFFRACTION2A1 - 156
4X-RAY DIFFRACTION2C1 - 11
5X-RAY DIFFRACTION2D1 - 11
6X-RAY DIFFRACTION3B1 - 156
7X-RAY DIFFRACTION3E1 - 11
8X-RAY DIFFRACTION3F1 - 11

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